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Sample records for lymphocyte perforin expression

  1. Cytometric analysis of perforin expression in NK cells, CD8+, and CD4+ lymphocytes in children with autoimmune Hashimoto's thyroiditis--a preliminary study.

    Science.gov (United States)

    Popko, Katarzyna; Osińska, Iwona; Kucharska, Anna; Demkow, Urszula

    2015-07-01

    Perforin plays an essential role in cytotoxicity of natural killers (NK) and CD8+ lymphocytes. Cytotoxicity of T and NK cells is one of the mechanisms of destruction of cells in Hashimoto's disease (HD). The aim of this study was analysis of the expression of perforin in CD8+, CD4+, and NK cells and cytotoxic abilities of these cells in children with HD compared to healthy controls. The expression of perforin and surface antigens, as well as cytotoxicity were analyzed with a flow cytometry. Lower expression of perforin in CD8+ and NK was found in HD compared to controls (p=0.01; p=0.004). A significant correlation between perforin expression in CD8+ lymphocytes and in NK was observed (p=0.05). The spontaneous cytotoxicity of NK was significantly higher in HD compared to controls (p=0.04). Our results suggest that perforin plays an important role in the pathogenesis of autoimmune Hashimoto's thyroiditis.

  2. Perforin expression in feline epitheliotropic cutaneous lymphoma.

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    Neta, Michal; Naigamwalla, Dinaz; Bienzle, Dorothee

    2008-11-01

    Cutaneous lymphomas are uncommon in people and companion animals. The tumors can be broadly categorized into epitheliotropic and nonepitheliotropic forms, which appear to have different biological behaviors. The present case describes a feline cutaneous epitheliotropic lymphoma. Masses in a 9-year-old cat were first identified on the tail. The cat was treated with chemotherapy, but additional skin masses developed on the flank, face, and ears. Local radiation induced transient tumor regression, but eventual dissemination prompted euthanasia 13 months after initial tumor appearance. Granular lymphocytes were consistently detected on blood smears, and histologically, the tumor involved the skin and superficial subcutis. Tumor lymphocytes expressed cluster of differentiation 3 (CD3) and perforin molecules, suggestive of a cytotoxic phenotype. Location, histopathological features, and perforin expression were similar to a distinct entity in human medicine designated primary cutaneous, CD8-positive, epidermotropic, cytotoxic, T-cell lymphoma.

  3. AKT/SGK-sensitive phosphorylation of GSK3 in the regulation of L-selectin and perforin expression as well as activation induced cell death of T-lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Bhavsar, Shefalee K.; Merches, Katja; Bobbala, Diwakar [Department of Physiology, University of Tuebingen (Germany); Lang, Florian, E-mail: florian.lang@uni-tuebingen.de [Department of Physiology, University of Tuebingen (Germany)

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer Akt/SGK dependent phosphorylation of GSK3{alpha},{beta} regulates T lymphocytes. Black-Right-Pointing-Pointer T cells from mice expressing Akt/SGK insensitive GSK3{alpha},{beta} (gsk3{sup KI}) release less IL-2. Black-Right-Pointing-Pointer CD4{sup +} cells from gsk3{sup KI} mice express less CD62L. Black-Right-Pointing-Pointer CD8{sup +} cells from gsk3{sup KI} mice are relatively resistant to activation induced cell death. Black-Right-Pointing-Pointer Perforin expression is enhanced in gsk3{sup KI} T cells. -- Abstract: Survival and function of T-lymphocytes critically depends on phosphoinositide (PI) 3 kinase. PI3 kinase signaling includes the PKB/Akt and SGK dependent phosphorylation and thus inhibition of glycogen synthase kinase GSK3{alpha},{beta}. Lithium, a known unspecific GSK3 inhibitor protects against experimental autoimmune encephalomyelitis. The present study explored, whether Akt/SGK-dependent regulation of GSK3 activity is a determinant of T cell survival and function. Experiments were performed in mutant mice in which Akt/SGK-dependent GSK3{alpha},{beta} inhibition was disrupted by replacement of the serine residue in the respective SGK/Akt-phosphorylation consensus sequence by alanine (gsk3{sup KI}). T cells from gsk3{sup KI} mice were compared to T cells from corresponding wild type mice (gsk3{sup WT}). As a result, in gsk3{sup KI} CD4{sup +} cells surface CD62L (L-selectin) was significantly less abundant than in gsk3{sup WT} CD4{sup +} cells. Upon activation in vitro T cells from gsk3{sup KI} mice reacted with enhanced perforin production and reduced activation induced cell death. Cytokine production was rather reduced in gsk3{sup KI} T cells, suggesting that GSK3 induces effector function in CD8{sup +} T cells. In conclusion, PKB/Akt and SGK sensitive phosphorylation of GSK3{alpha},{beta} is a potent regulator of perforin expression and activation induced cell death in T lymphocytes.

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

    DEFF Research Database (Denmark)

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

    2006-01-01

    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...

  5. Correlation between expression of granzyme B and perforin and apoptosis in epithelial cells in oral lichen planus with local lymphocyte infiltration%口腔扁平苔藓局部浸润淋巴细胞颗粒酶B和穿孔素蛋白表达与上皮细胞凋亡的关系

    Institute of Scientific and Technical Information of China (English)

    雷蕾; 詹丽华; 谭为霞; 周序珑

    2011-01-01

    目的 检测口腔扁平苔藓(oral lichen planus,OLP)上皮细胞凋亡状况,观察颗粒酶B、穿孔素在OLP固有层浸润淋巴细胞中的蛋白表达,探讨细胞毒作用对OLP上皮细胞凋亡的影响。方法采用原位缺口末端DNA标记技术,定位检测OLP上皮细胞凋亡情况;分别使用免疫组化法检测颗粒酶B和穿孔素在组织总淋巴细胞以及CD8+细胞中的表达,以健康口腔黏膜组织(NOM)作对照。结果与NOM相比,OLP上皮细胞凋亡显著增加(P=0.037);OLP淋巴细胞中颗粒酶B、穿孔素表达明显增强(P值分别为0. 016和0.012),其部分阳性细胞接近上皮基底层。OLP中CD8+/颗粒酶B+细胞双阳性表达和CD8+/穿孔素+细胞双阳性表达较NOM明显升高(P值分别为0.043和0.032)。OLP上皮细胞凋亡与淋巴细胞颗粒酶B和穿孔素表达成正相关(r=0.56,P=0. 038;r =0.42,P=0.041)。结论OLP浸润淋巴细胞包括CD8+T细胞中颗粒酶B、穿孔素表达增强,提示OLP淋巴细胞可能以颗粒酶B、穿孔素作为细胞毒介质攻击口腔上皮细胞引起其凋亡。%Objective To study the expression of granzyme B and perform and the effect of cytotoxin on apoptosis of epithelial cells in oral lichen planus( OLP) with lymphocyte infiltration by detecting the apoptosis of epithelial cells. Methods Apoptosis of epithelial cells in OLP with lymphocyte infiltration was detected with the terminal deoxymuleotigyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) technique. Expression of granzyme B and perfoin in lymphocytes and CD8+ cells was detected with the immunohistochemcial method. Normal oral mucosa( NOM) was used as a control. Results The apoptosis level of epithelial cells and the expression level of granzyme B and perforin were significantly higher in epithelium of OLP than in control (P <0. 05). Some positive cells were observed in the basal lamina of epithelium. The double positive expression levels of CD8

  6. Compromised virus control and augmented perforin-mediated immunopathology in IFN-gamma-deficient mice infected with lymphocytic choriomeningitis virus

    DEFF Research Database (Denmark)

    Nansen, A; Jensen, Teis; Christensen, Jan Pravsgaard

    1999-01-01

    -specific TCR are adoptively transferred before virus challenge, indicating that the disease is the result of an unfortunate balance between virus replication in internal organs, e.g., liver and spleen, and the host response; resetting this balance by increasing host responsiveness will again lead to a rapidly...... in wild-type mice. Our results reveal that IFN-gamma is pivotal to T cell-mediated control of a rapidly invasive stain, whereas it is less important in the acute elimination of a slowly invasive strain. Moreover, the majority of mice infected with the rapidly invasive strain succumb to a wasting syndrome...... mediated by CD8+ effector cells. The primary effector mechanism underlying this disease is perforin-dependent lysis, but other mechanisms are also involved. Wasting disease can be prevented if naive CD8+ cells from mice transgenic for an MHC class I-restricted lymphocytic choriomeningitis virus...

  7. Co-expression of perforin and granzyme B genes induces apoptosis and inhibits the tumorigenicity of laryngeal cancer cell line Hep-2.

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    Li, Xiu-Ying; Li, Zhi; An, Gui-Jie; Liu, Sha; Lai, Yan-Dong

    2014-01-01

    Granzyme B and perforin, two of the most important components, have shown anticancer properties in various cancers, but their effects in laryngeal cancer remain unexplored. Here we decided to examine the effects of Granzyme B and perforin in Hep-2 cells and clarify the role of perforin and granzyme B in the tumorigenicity of laryngeal cancer cell line. Hep-2 cells were transfected with pVAX1-PIG co-expression vector (comprising perforin and granzyme B genes), and then the growth and apoptosis of these Hep-2 cells were evaluated. The tumorigenicity of Hep-2 cell line co-expressing perforin and granzyme B genes was tested in BALB/c nu/nu mice. We found that the co-expression of perforin and granzyme B genes could obviously inhibit cell focus formation and induce cell apoptosis in Hep-2 cells. Furthermore, after subcutaneous injection of Hep-2 cells transfected with pVAX1-PIG, an extensive delay in tumor growth was observed in BALB/c-nu/nu mice. Moreover, our studies demonstrated that the anticancer activity of perforin and granzyme B was sustainable in vivo as tumor development by inducing cell apoptosis. Taken together, our data indicate that the co-expression of perforin and granzyme B genes exhibits anticancer potential, and hopefully provide potential therapeutic applications in laryngeal cancer.

  8. Cloning and characterising an unusual perforin from chicken (Gallus gallus).

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    D'Angelo, Michael E; Arjomand, Arash; Trapani, Joseph A; Bird, Phillip I

    2013-10-01

    In mammals the 67 kDa pore-forming protein perforin is essential to the granule exocytosis pathway used by cytotoxic lymphocytes to eliminate virally infected and malignant cells. There is indirect evidence that this pathway exists in lower vertebrates such as teleost fish and birds, although in genome databases for the chicken and other bird species the perforin gene is incomplete and no full length expressed sequence tag has been reported. We present here the full gene and transcript sequence of chicken perforin. The inferred protein product contains an extended C-terminus that is at least 90 amino acids longer than any mammalian perforin, which is also evident in partial genomic sequences from other birds. To determine whether this extension is present in the translated protein, we raised two polyclonal antisera. The antisera identified a protein of just less than 80 kDa in both transfected COS-1 cells and concanavalin A stimulated chicken splenocytes, indicating that the extended C-terminus is present in the mature protein. Our findings confirm that perforin exists in birds, and show that it is considerably longer than perforin of non-avian vertebrates. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Expression, purification and anticancer analysis of GST-tagged human perforin and granzyme B proteins in human laryngeal cancer Hep-2 cells.

    Science.gov (United States)

    Li, Xiuying; Zhang, Guang; An, Guijie; Liu, Sha; Lai, Yandong

    2014-03-01

    Granzyme B and perforin, two major effector molecules in the granule-mediated cytolytic pathway, are thought to be involved in suppression of tumor progression. In this study, the pGEX-4T-1 expression vector was used to express full-length human perforin or granzyme B as a GST-tagged fusion protein in Escherichia coli (E. coli). GST-tagged proteins were induced with IPTG and purified by GSTrap 4B columns. Purified fusion proteins migrated at the predicted molecular mass on SDS-PAGE and were recognized by specific antibodies. Moreover, the fusion proteins can induce apoptosis and directly inhibit the growth of human laryngeal cancer Hep-2 cells in vitro. These results suggest that active perforin and granzyme B fusion proteins can be produced in E. coli and exhibit anticancer potential in laryngeal cancer cells.

  10. Granzyme B-dependent and perforin-independent DNA fragmentation in intestinal epithelial cells induced by anti-CD3 mAb-activated intra-epithelial lymphocytes.

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    Ogata, Masaki; Ota, Yuta; Matsutani, Takaji; Nanno, Masanobu; Suzuki, Ryuji; Itoh, Tsunetoshi

    2013-05-01

    We previously found that an i.p. injection of anti-CD3 monoclonal antibody (mAb) into mice caused DNA fragmentation in the intestinal villous epithelial cells (IVECs) of the duodenum and the jejunum. In this study, in order to elucidate the mechanism of DNA fragmentation in IVECs, we searched for the inducer(s) of DNA fragmentation by using immunohistochemistry. The release of cytoplasmic granules from intraepithelial lymphocytes (IELs) and the formation of large gaps between IELs and IVECs were observed electron microscopically after antibody administration. The presence and distribution pattern of Granzyme B (GrB), a serine protease in cytolytic granules present in cytotoxic T lymphocytes and natural killer cells and considered to be the responsible molecule for DNA fragmentation in target cells, was examined in detail in intestinal villi by immunohistology. GrB was detected in cytoplasmic granules in nearly all IELs. The time-kinetics of granule release from IELs after mAb injection coincided not only with that of the extracellular diffusion of GrB, but also with that of DNA fragmentation in IVECs. On the other hand, perforin (Pfn), assumed to cooperate with GrB in DNA fragmentation, could not be detected in IELs, and its release was not confirmed after the anti-CD3 mAb injection. Anti-CD3 mAb injection also induced DNA fragmentation in IVECs in Pfn-knockout mice. These results support the notion that DNA fragmentation in IVECs by the stimulated IELs in the present study is induced by a mechanism involving GrB, but independent of Pfn.

  11. The proposed mechanism of action during different pain management techniques on expression of cytolytic molecule perforin in patients after colorectal cancer surgery.

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    Golubovic, S; Golubovic, V; Sotosek-Tokmadzic, V; Sustic, A; Petkovic, M; Bacic, D; Mrakovcic-Sutic, I

    2011-03-01

    The postoperative period is accompanied with neuroendocrine, metabolic and immune alteration which is caused by tissue damage, anesthesia, postoperative pain and psychological stress. Postoperative pain contributes to dysfunction of immune response as a result of interaction between central nervous and immune system. The postoperatively activated hypotalamo-pituitary-adrenocortical axis, sympathic and parasympathic nerve systems are important modulators of immune response. According to bidirectional communication of immune and nervous system, appropriate postoperative pain management could affect immune response in postoperative period. Although the postoperative suppression of immune response has been reported, a very little are known about the influences of different pain management techniques on cytotoxic function of immune cells in patients with colorectal cancer in early postoperative period. Perforin is a cytotoxic molecule expressed by activated lymphocytes which has a crucial role in elimination of tumor cells and virus-infected cells, mostly during the effector's phase of immune response. Immune compromise during the postoperative period could affect the healing processes, incidence of postoperative infections and rate and size of tumor metastases disseminated during operation. The pharmacological management of postoperative pain in patients with malignancies uses very different analgesic techniques whose possible influence on cytotoxic functions of immune cells are still understood poor. For decades the most common way of treating postoperative pain after colorectal cancer surgery was intravenous analgesia with opiods. In the last decade many investigations pointed out that opiods can also contribute to postoperative suppression of immune response. Epidural analgesia is a regional anesthesia technique that acts directly on the origin of pain impulses and pain relief can be achieved with small doses of opiods combined with local anesthetics. Local

  12. Eosinophilia of dystrophin-deficient muscle is promoted by perforin-mediated cytotoxicity by T cell effectors

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    Cai, B.; Spencer, M. J.; Nakamura, G.; Tseng-Ong, L.; Tidball, J. G.

    2000-01-01

    Previous investigations have shown that cytotoxic T lymphocytes (CTLs) contribute to muscle pathology in the dystrophin-null mutant mouse (mdx) model of Duchenne muscular dystrophy through perforin-dependent and perforin-independent mechanisms. We have assessed whether the CTL-mediated pathology includes the promotion of eosinophilia in dystrophic muscle, and thereby provides a secondary mechanism through which CTLs contribute to muscular dystrophy. Quantitative immunohistochemistry confirmed that eosinophilia is a component of the mdx dystrophy. In addition, electron microscopic observations show that eosinophils traverse the basement membrane of mdx muscle fibers and display sites of close apposition of eosinophil and muscle membranes. The close membrane apposition is characterized by impingement of eosinophilic rods of major basic protein into the muscle cell membrane. Transfer of mdx splenocytes and mdx muscle extracts to irradiated C57 mice by intraperitoneal injection resulted in muscle eosinophilia in the recipient mice. Double-mutant mice lacking dystrophin and perforin showed less eosinophilia than was displayed by mdx mice that expressed perforin. Finally, administration of prednisolone, which has been shown previously to reduce the concentration of CTLs in dystrophic muscle, produced a significant reduction in eosinophilia. These findings indicate that eosinophilia is a component of the mdx pathology that is promoted by perforin-dependent cytotoxicity of effector T cells. However, some eosinophilia of mdx muscle is independent of perforin-mediated processes.

  13. Perforin expression by CD8 T cells is sufficient to cause fatal brain edema during experimental cerebral malaria.

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    Huggins, Matthew; Johnson, Holly L; Jin, Fang; N'Songo, Aurelie; Hanson, Lisa M; LaFrance, Stephanie J; Butler, Noah S; Harty, John T; Johnson, Aaron J

    2017-03-06

    Human cerebral malaria (HCM) is a serious complication of Plasmodium falciparum infection. The most severe outcomes for patients include coma, permanent neurological deficits, and death. Recently, a large-scale magnetic resonance imaging (MRI) study in humans identified brain swelling as the most prominent predictor of fatal HCM. Therefore, in this study we sought to define the mechanism controlling brain edema through the use of the murine experimental cerebral malaria (ECM) model. Specifically, we investigated the ability of CD8 T cells to initiate brain edema during ECM. We determined that areas of blood-brain barrier (BBB) permeability colocalized with a reduction of the cerebral endothelial cell tight junction proteins claudin-5 and occludin. Furthermore, through small animal MRI we analyzed edema and vascular leakage. Using gadolinium enhanced T1-weighted MRI we determined that vascular permeability is not homogeneous, but rather confined to specific regions of the brain. Our findings show that BBB permeability was localized within the brainstem, olfactory bulb, and lateral ventricle. Concurrently with the initiation of vascular permeability, T2-weighted MRI revealed edema and brain swelling. Importantly, ablation of the cytolytic effector molecule perforin fully protected against vascular permeability and edema. Furthermore, perforin production specifically by CD8 T cells was required to cause fatal edema during ECM. We propose that CD8 T cells initiate BBB breakdown through perforin mediated disruption of tight junctions. In turn, leakage from the vasculature into the parenchyma causes brain swelling and edema. This results in a breakdown of homeostatic maintenance that likely contributes to ECM pathology.

  14. Immune-mediated loss of transgene expression from virally transduced brain cells is irreversible, mediated by IFNγ, perforin, and TNFα, and due to the elimination of transduced cells.

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    Zirger, Jeffrey M; Puntel, Mariana; Bergeron, Josee; Wibowo, Mia; Moridzadeh, Rameen; Bondale, Niyati; Barcia, Carlos; Kroeger, Kurt M; Liu, Chunyan; Castro, Maria G; Lowenstein, Pedro R

    2012-04-01

    The adaptive immune response to viral vectors reduces vector-mediated transgene expression from the brain. It is unknown, however, whether this loss is caused by functional downregulation of transgene expression or death of transduced cells. Herein, we demonstrate that during the elimination of transgene expression, the brain becomes infiltrated with CD4(+) and CD8(+) T cells and that these T cells are necessary for transgene elimination. Further, the loss of transgene-expressing brain cells fails to occur in the absence of IFNγ, perforin, and TNFα receptor. Two methods to induce severe immune suppression in immunized animals also fail to restitute transgene expression, demonstrating the irreversibility of this process. The need for cytotoxic molecules and the irreversibility of the reduction in transgene expression suggested to us that elimination of transduced cells is responsible for the loss of transgene expression. A new experimental paradigm that discriminates between downregulation of transgene expression and the elimination of transduced cells demonstrates that transduced cells are lost from the brain upon the induction of a specific antiviral immune response. We conclude that the anti-adenoviral immune response reduces transgene expression in the brain through loss of transduced cells.

  15. Perforin-2 is essential for intracellular defense of parenchymal cells and phagocytes against pathogenic bacteria.

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    McCormack, Ryan M; de Armas, Lesley R; Shiratsuchi, Motoaki; Fiorentino, Desiree G; Olsson, Melissa L; Lichtenheld, Mathias G; Morales, Alejo; Lyapichev, Kirill; Gonzalez, Louis E; Strbo, Natasa; Sukumar, Neelima; Stojadinovic, Olivera; Plano, Gregory V; Munson, George P; Tomic-Canic, Marjana; Kirsner, Robert S; Russell, David G; Podack, Eckhard R

    2015-09-24

    Perforin-2 (MPEG1) is a pore-forming, antibacterial protein with broad-spectrum activity. Perforin-2 is expressed constitutively in phagocytes and inducibly in parenchymal, tissue-forming cells. In vitro, Perforin-2 prevents the intracellular replication and proliferation of bacterial pathogens in these cells. Perforin-2 knockout mice are unable to control the systemic dissemination of methicillin-resistant Staphylococcus aureus (MRSA) or Salmonella typhimurium and perish shortly after epicutaneous or orogastric infection respectively. In contrast, Perforin-2-sufficient littermates clear the infection. Perforin-2 is a transmembrane protein of cytosolic vesicles -derived from multiple organelles- that translocate to and fuse with bacterium containing vesicles. Subsequently, Perforin-2 polymerizes and forms large clusters of 100 Å pores in the bacterial surface with Perforin-2 cleavage products present in bacteria. Perforin-2 is also required for the bactericidal activity of reactive oxygen and nitrogen species and hydrolytic enzymes. Perforin-2 constitutes a novel and apparently essential bactericidal effector molecule of the innate immune system.

  16. Perforin enhances the granulysin-induced lysis of Listeria innocua in human dendritic cells

    Directory of Open Access Journals (Sweden)

    Wagner Carsten A

    2007-08-01

    Full Text Available Abstract Background Cytotoxic T lymphocytes (CTL and natural killer (NK cells play an essential role in the host defence against intracellular pathogens such as Listeria, and Mycobacteria. The key mediator of bacteria-directed cytotoxicity is granulysin, a 9 kDa protein stored in cytolytic granules together with perforin and granzymes. Granulysin binds to cell membranes and is subsequently taken up via a lipid raft-associated mechanism. In dendritic cells (DC granulysin is further transferred via early endosomes to L. innocua-containing phagosomes were bacteriolysis is induced. In the present study we analysed the role of perforin in granulysin-induced intracellular bacteriolysis in DC. Results We found granulysin-induced lysis of intracellular Listeria significantly increased when perforin was simultaneously present. In pulse-chase experiments enhanced bacteriolysis was observed when perforin was added up to 25 minutes after loading the cells with granulysin demonstrating no ultimate need for simultaneous uptake of granulysin and perforin. The perforin concentration sufficient to enhance granulysin-induced intracellular bacteriolysis did not cause permanent membrane pores in Listeria-challenged DC as shown by dye exclusion test and LDH release. This was in contrast to non challenged DC that were more susceptible to perforin lysis. For Listeria-challenged DC, there was clear evidence for an Ca2+ influx in response to sublytic perforin demonstrating a short-lived change in the plasma membrane permeability. Perforin treatment did not affect granulysin binding, initial uptake or intracellular trafficking to early endosomes. However, enhanced colocalization of granulysin with listerial DNA in presence of perforin was found by confocal laser scanning microscopy. Conclusion The results provide evidence that perforin increases granulysin-mediated killing of intracellular Listeria by enhanced phagosome-endosome fusion triggered by a transient Ca2+ flux.

  17. Protecting a serial killer: pathways for perforin trafficking and self-defence ensure sequential target cell death.

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    Lopez, Jamie A; Brennan, Amelia J; Whisstock, James C; Voskoboinik, Ilia; Trapani, Joseph A

    2012-08-01

    Considerable progress has been made in understanding how cytotoxic lymphocytes use the highly toxic pore-forming protein perforin to eliminate dangerous cells, while remaining refractory to lysis. At least two mechanisms jointly preserve the killer cell: the C-terminal residues of perforin dictate its rapid export from the endoplasmic reticulum (ER), whose milieu otherwise favours pore formation; perforin is then stored in secretory granules whose acidity prevent its oligomerisation. Following exocytosis, perforin delivers the proapoptotic protease, granzyme B, into the target cell by disrupting its plasma membrane. Although the precise mechanism of perforin/granzyme synergy remains controversial, the recently defined crystal structure of the perforin monomer and cryo-electron microscopy (EM) of the entire pore suggest that passive transmembrane granzyme diffusion is the dominant proapoptotic mechanism.

  18. A radio-resistant perforin-expressing lymphoid population controls allogeneic T cell engraftment, activation, and onset of graft-versus-host disease in mice.

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    Davis, Joanne E; Harvey, Michael; Gherardin, Nicholas A; Koldej, Rachel; Huntington, Nicholas; Neeson, Paul; Trapani, Joseph A; Ritchie, David S

    2015-02-01

    Immunosuppressive pretransplantation conditioning is essential for donor cell engraftment in allogeneic bone marrow transplantation (BMT). The role of residual postconditioning recipient immunity in determining engraftment is poorly understood. We examined the role of recipient perforin in the kinetics of donor cell engraftment. MHC-mismatched BMT mouse models demonstrated that both the rate and proportion of donor lymphoid cell engraftment and expansion of effector memory donor T cells in both spleen and BM were significantly increased within 5 to 7 days post-BMT in perforin-deficient (pfn(-/-)) recipients, compared with wild-type. In wild-type recipients, depletion of natural killer (NK) cells before BMT enhanced donor lymphoid cell engraftment to that seen in pfn(-/-) recipients. This demonstrated that a perforin-dependent, NK-mediated, host-versus-graft (HVG) effect limits the rate of donor engraftment and T cell activation. Radiation-resistant natural killer T (NKT) cells survived in the BM of lethally irradiated mice and may drive NK cell activation, resulting in the HVG effect. Furthermore, reduced pretransplant irradiation doses in pfn(-/-) recipients permitted long-term donor lymphoid cell engraftment. These findings suggest that suppression of perforin activity or selective depletion of recipient NK cells before BMT could be used to improve donor stem cell engraftment, in turn allowing for the reduction of pretransplant conditioning.

  19. Perforinopathy: a spectrum of human immune disease caused by defective perforin delivery or function.

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    Voskoboinik, Ilia; Trapani, Joseph A

    2013-12-12

    Congenital perforin deficiency is considered a rare cause of human immunopathology and immune dysregulation, and classically presents as a fatal illness early in infancy. However, we propose that a group of related disorders in which killer lymphocytes deliver only partially active perforin or a reduced quantum of wild-type perforin to the immune synapse should be considered part of an extended syndrome with overlapping but more variable clinical features. Apart from the many rare mutations scattered over the coding sequences, up to 10% of Caucasians carry the severely hypomorphic PRF1 allele C272 > T (leading to A91V mutation) and the overall prevalence of the homozygous state for A91V is around 1 in 600 individuals. We therefore postulate that the partial loss of perforin function and its clinical consequences may be more common then currently suspected. An acute clinical presentation is infrequent in A91V heterozygous individuals, but we postulate that the partial loss of perforin function may potentially be manifested in childhood or early adulthood as "idiopathic" inflammatory disease, or through increased cancer susceptibility - either hematological malignancy or multiple, independent primary cancers. We suggest the new term "perforinopathy" to signify the common functional endpoints of all the known consequences of perforin deficiency and failure to deliver fully functional perforin.

  20. Perforinopathy: a spectrum of human immune disease caused by defective perforin delivery or function

    Directory of Open Access Journals (Sweden)

    Joseph A. Trapani

    2013-12-01

    Full Text Available Congenital perforin deficiency is considered a rare cause of human immunopathology and immune dysregulation, and classically presents as a fatal illness early in infancy. However, we propose that a group of related disorders in which killer lymphocytes deliver only partially active perforin or a reduced quantum of wild-type perforin to the immune synapse should be considered part of an extended syndrome with overlapping but more variable clinical features. Apart from the many rare mutations scattered over the coding sequences, up to 10% of Caucasians carry the severely hypomorphic PRF1 allele C272>T (leading to A91V mutation and the overall prevalence of the homozygous state for A91V is around 1 in 600 individuals. We therefore postulate that the partial loss of perforin function and its clinical consequences may be more common then currently suspected. An acute clinical presentation is infrequent in A91V heterozygous individuals, but we postulate that the partial loss of perforin function may potentially be manifested in childhood or early adulthood as ‘idiopathic’ inflammatory disease, or through increased cancer susceptibility – either hematological malignancy or multiple, independent primary cancers. We suggest the new term ‘perforinopathy’ to signify the common functional endpoints of all the known consequences of perforin-deficiency and failure to deliver fully functional perforin.

  1. Expression of tenascin in lymphocytic autoimmune thyroiditis.

    OpenAIRE

    Back, W; Heubner, C; Winter, J.; Bleyl, U

    1997-01-01

    AIMS: To study the distribution of tenascin by immunocytochemistry in autoimmune diseases of the thyroid. METHODS: Thyroids from patients with inflammatory lesions of the thyroid (lymphocytic thyroiditis Hashimoto, Grave's disease, thyroiditis DeQuervain) were studied by immunocytochemistry using antibodies against tenascin, collagen III, and collagen IV. RESULTS: In autoimmune lymphocytic thyroiditis Hashimoto there was a characteristic corona-like staining pattern of tenascin around all act...

  2. Expression of protooncogenes during lymphocyte activation by growth factors.

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    Bulanova, E G; Budagyan, V M; Yarilin, A A; Mazurenko, N N

    1997-09-01

    Effects of growth factors of non-immune origin including somatotropin (ST) and platelet-derived growth factor (PDGF) on the expression of the proteins encoded by c-fos, c-myc, c-fun, and c-ets family protooncogenes were studied for the first time. The dynamics of the oncoprotein expression in activated CD(3+)-lymphocytes was investigated by immunoblotting. The accumulation of the Fos and Myc proteins was enhanced in T-lymphocytes treated with ST, PDGF, or phytohemagglutinin; the accumulation was maximum at 30-60 min and decreased in 2 h; the data indicate that the oncoproteins participate in the early lymphocyte activation by various growth factors. The Jun protein appears only in 3 h after the onset of lymphocyte activation; this suggests independent participation of Fos in the early stages of lymphocyte activation prior to the appearance of Jun, preceding the joint action of Fos and Jun within the AP-1 transcription complex. The products of the c-ets family are differentially activated by the studied growth factors. Resting lymphocytes actively accumulate the Ets-1 protein; ST and PDGF activation decreases Ets-1 expression in 2 h. The Ets-2 protein is not detected in resting cells and PDGF-activated lymphocytes, whereas lymphocyte activation by ST is associated with accumulation of Ets-2. The data suggest that the product of the c-ets-1 gene is more important in the regulation of resting cells and the product of the c-ets-2 gene is important during activation of lymphocytes by ST. The results indicate that activation of lymphocytes with growth factors of non-immune origin is mediated by several signal transduction pathways.

  3. Signaling lymphocyte activating molecule (SLAM) expression in subacute sclerosing panencephalitis.

    Science.gov (United States)

    Piskin, A Kevser; Akpinar, Pinar; Muftuoglu, Sevda; Anlar, Banu

    2007-08-01

    Signaling lymphocyte activating molecule (SLAM) is a receptor for measles virus which also has immunomodulatory activity. We analyzed SLAM expression in mononuclear cells (MNC) of patients with SSPE (n=7) and control subjects (n=7) from the same population. Native 10% PAGE analysis in cell and brain tissue extracts followed by Western blotting using monoclonal anti-human SLAM showed four types of bands. Differences in the type and amount of SLAM expression were observed between SSPE and control cases. Lymphocytes of SSPE patients showed two types of SLAM bands in comparison to only one in control lymphocytes. Stimulation of cells with lipopolysaccharide (80 u/ml) and concanavalin A (1 microg/ml) in vitro led to the appearance of a second isoform in both groups. Brain homogenates of SSPE patients (n=2) displayed all four types of SLAM isoforms at significantly higher levels than those of control brains (n=2). Our results show native PAGE enables the detection of all SLAM isotypes. The expression of SLAM is increased in lymphocytes, monocytes, and brain tissues of SSPE patients.

  4. Intrinsic Contribution of Perforin to NK-Cell Homeostasis during Mouse Cytomegalovirus Infection

    Directory of Open Access Journals (Sweden)

    Maja eArapovic

    2016-04-01

    Full Text Available In addition to their role as effector cells in virus control, natural killer (NK cells have an immunoregulatory function in shaping the antiviral T-cell response. This function is further pronounced in perforin-deficient mice that show the enhanced NK-cell proliferation and cytokine secretion upon mouse cytomegalovirus (MCMV infection. Here we confirmed that stronger activation and maturation of NK cells in perforin-deficient mice correlates with higher MCMV load. To further characterize the immunoregulatory potential of perforin, we compared the response of NK cells that express or do not express perforin using bone-marrow chimeras. Our results demonstrated that the enhanced proliferation and maturation of NK cells in MCMV-infected bone-marrow chimeras is an intrinsic property of perforin-deficient NK cells. Thus, in addition to confirming that NK-cell proliferation is virus load dependent, our data extend this notion demonstrating that perforin plays an intrinsic role as a feedback mechanism in regulation of NK-cell proliferation during viral infections.

  5. Interference of CD95 expression on human lymphocytes.

    Science.gov (United States)

    Petanova, Jitka; Fucikova, Terezie; Bencko, Vladimir

    2002-02-01

    The study presents the exogenous influence of cadmium in comparison with zinc on the apoptosis of human lymphocytes by CD95 expression and its kinetic changes. The salts of both metals were used in final concentrations of 20 microM in cell cultures with whole blood. The duration of cultivation was 18 and 90 hours. The expression of surface antigens was evaluated by flow cytometry with monoclonal antibodies. In cultures of not stimulated cells we found in average 51.54% CD95 positive lymphocytes. The kinetic study of untreated cells showed elevation after 18 hours of cultivation and a very low expression after 90 hours. The CD95 expression on lymphocytes in cell culture with cadmium and zinc was lower after 18 hours of cultivation than in untreated cells. After 90 hours cultivation we found low levels of CD95 expression on cells treated with cadmium and a great individual variability in the number of positive cells upon the influence of zinc.

  6. CD8+ T-cells expressing interferon gamma or perforin play antagonistic roles in heart injury in experimental Trypanosoma cruzi-elicited cardiomyopathy.

    Directory of Open Access Journals (Sweden)

    Jaline Coutinho Silverio

    Full Text Available In Chagas disease, CD8(+ T-cells are critical for the control of Trypanosoma cruzi during acute infection. Conversely, CD8(+ T-cell accumulation in the myocardium during chronic infection may cause tissue injury leading to chronic chagasic cardiomyopathy (CCC. Here we explored the role of CD8(+ T-cells in T. cruzi-elicited heart injury in C57BL/6 mice infected with the Colombian strain. Cardiomyocyte lesion evaluated by creatine kinase-MB isoenzyme activity levels in the serum and electrical abnormalities revealed by electrocardiogram were not associated with the intensity of heart parasitism and myocarditis in the chronic infection. Further, there was no association between heart injury and systemic anti-T. cruzi CD8(+ T-cell capacity to produce interferon-gamma (IFNγ and to perform specific cytotoxicity. Heart injury, however, paralleled accumulation of anti-T. cruzi cells in the cardiac tissue. In T. cruzi infection, most of the CD8(+ T-cells segregated into IFNγ(+ perforin (Pfn(neg or IFNγ(negPfn(+ cell populations. Colonization of the cardiac tissue by anti-T. cruzi CD8(+Pfn(+ cells paralleled the worsening of CCC. The adoptive cell transfer to T. cruzi-infected cd8(-/- recipients showed that the CD8(+ cells from infected ifnγ(-/-pfn(+/+ donors migrate towards the cardiac tissue to a greater extent and caused a more severe cardiomyocyte lesion than CD8(+ cells from ifnγ(+/+pfn(-/- donors. Moreover, the reconstitution of naïve cd8(-/- mice with CD8(+ cells from naïve ifnγ(+/+pfn(-/- donors ameliorated T. cruzi-elicited heart injury paralleled IFNγ(+ cells accumulation, whereas reconstitution with CD8(+ cells from naïve ifnγ(-/-pfn(+/+ donors led to an aggravation of the cardiomyocyte lesion, which was associated with the accumulation of Pfn(+ cells in the cardiac tissue. Our data support a possible antagonist effect of CD8(+Pfn(+ and CD8(+IFNγ(+ cells during CCC. CD8(+IFNγ(+ cells may exert a beneficial role, whereas CD8(+Pfn

  7. CD8+ T-Cells Expressing Interferon Gamma or Perforin Play Antagonistic Roles in Heart Injury in Experimental Trypanosoma Cruzi-Elicited Cardiomyopathy

    Science.gov (United States)

    Cipitelli, Márcio da Costa; Vinagre, Nathália Ferreira; Rodrigues, Maurício Martins; Gazzinelli, Ricardo Tostes; Lannes-Vieira, Joseli

    2012-01-01

    In Chagas disease, CD8+ T-cells are critical for the control of Trypanosoma cruzi during acute infection. Conversely, CD8+ T-cell accumulation in the myocardium during chronic infection may cause tissue injury leading to chronic chagasic cardiomyopathy (CCC). Here we explored the role of CD8+ T-cells in T. cruzi-elicited heart injury in C57BL/6 mice infected with the Colombian strain. Cardiomyocyte lesion evaluated by creatine kinase-MB isoenzyme activity levels in the serum and electrical abnormalities revealed by electrocardiogram were not associated with the intensity of heart parasitism and myocarditis in the chronic infection. Further, there was no association between heart injury and systemic anti-T. cruzi CD8+ T-cell capacity to produce interferon-gamma (IFNγ) and to perform specific cytotoxicity. Heart injury, however, paralleled accumulation of anti-T. cruzi cells in the cardiac tissue. In T. cruzi infection, most of the CD8+ T-cells segregated into IFNγ+ perforin (Pfn)neg or IFNγnegPfn+ cell populations. Colonization of the cardiac tissue by anti-T. cruzi CD8+Pfn+ cells paralleled the worsening of CCC. The adoptive cell transfer to T. cruzi-infected cd8−/− recipients showed that the CD8+ cells from infected ifnγ−/−pfn+/+ donors migrate towards the cardiac tissue to a greater extent and caused a more severe cardiomyocyte lesion than CD8+ cells from ifnγ+/+pfn−/− donors. Moreover, the reconstitution of naïve cd8−/− mice with CD8+ cells from naïve ifnγ+/+pfn−/− donors ameliorated T. cruzi-elicited heart injury paralleled IFNγ+ cells accumulation, whereas reconstitution with CD8+ cells from naïve ifnγ−/−pfn+/+ donors led to an aggravation of the cardiomyocyte lesion, which was associated with the accumulation of Pfn+ cells in the cardiac tissue. Our data support a possible antagonist effect of CD8+Pfn+ and CD8+IFNγ+ cells during CCC. CD8+IFNγ+ cells may exert a beneficial role, whereas CD8+Pfn+ may play a detrimental role

  8. CD8+ T-cells expressing interferon gamma or perforin play antagonistic roles in heart injury in experimental Trypanosoma cruzi-elicited cardiomyopathy.

    Science.gov (United States)

    Silverio, Jaline Coutinho; Pereira, Isabela Resende; Cipitelli, Márcio da Costa; Vinagre, Nathália Ferreira; Rodrigues, Maurício Martins; Gazzinelli, Ricardo Tostes; Lannes-Vieira, Joseli

    2012-01-01

    In Chagas disease, CD8(+) T-cells are critical for the control of Trypanosoma cruzi during acute infection. Conversely, CD8(+) T-cell accumulation in the myocardium during chronic infection may cause tissue injury leading to chronic chagasic cardiomyopathy (CCC). Here we explored the role of CD8(+) T-cells in T. cruzi-elicited heart injury in C57BL/6 mice infected with the Colombian strain. Cardiomyocyte lesion evaluated by creatine kinase-MB isoenzyme activity levels in the serum and electrical abnormalities revealed by electrocardiogram were not associated with the intensity of heart parasitism and myocarditis in the chronic infection. Further, there was no association between heart injury and systemic anti-T. cruzi CD8(+) T-cell capacity to produce interferon-gamma (IFNγ) and to perform specific cytotoxicity. Heart injury, however, paralleled accumulation of anti-T. cruzi cells in the cardiac tissue. In T. cruzi infection, most of the CD8(+) T-cells segregated into IFNγ(+) perforin (Pfn)(neg) or IFNγ(neg)Pfn(+) cell populations. Colonization of the cardiac tissue by anti-T. cruzi CD8(+)Pfn(+) cells paralleled the worsening of CCC. The adoptive cell transfer to T. cruzi-infected cd8(-/-) recipients showed that the CD8(+) cells from infected ifnγ(-/-)pfn(+/+) donors migrate towards the cardiac tissue to a greater extent and caused a more severe cardiomyocyte lesion than CD8(+) cells from ifnγ(+/+)pfn(-/-) donors. Moreover, the reconstitution of naïve cd8(-/-) mice with CD8(+) cells from naïve ifnγ(+/+)pfn(-/-) donors ameliorated T. cruzi-elicited heart injury paralleled IFNγ(+) cells accumulation, whereas reconstitution with CD8(+) cells from naïve ifnγ(-/-)pfn(+/+) donors led to an aggravation of the cardiomyocyte lesion, which was associated with the accumulation of Pfn(+) cells in the cardiac tissue. Our data support a possible antagonist effect of CD8(+)Pfn(+) and CD8(+)IFNγ(+) cells during CCC. CD8(+)IFNγ(+) cells may exert a beneficial role

  9. FcgammaRIIb expression on human germinal center B lymphocytes.

    Science.gov (United States)

    Macardle, Peter J; Mardell, Carolyn; Bailey, Sheree; Wheatland, Loretta; Ho, Alice; Jessup, Claire; Roberton, Donal M; Zola, Heddy

    2002-12-01

    IgG antibody can specifically suppress the antibody response to antigen. This has been explained by the hypothesis that signaling through the B cell antigen receptor is negatively modulated by the co-ligation of immunoglobulin with the receptor for IgG, FcgammaRIIb. We hypothesized that inhibitory signaling through FcgammaRIIb would be counter-productive in germinal center cells undergoing selection by affinity maturation, since these cells are thought to receive a survival/proliferative signal by interacting with antigen displayed on follicular dendritic cells. We have identified and characterized a population of B lymphocytes with low/negative FcgammaRIIb expression that are present in human tonsil. Phenotypically these cells correspond to germinal center B cells and comprise both centroblast and centrocyte populations. In examining expression at the molecular level we determined that these B cells do not express detectable mRNA for FcgammaRIIb. We examined several culture conditions to induce expression of FcgammaRIIb on germinal center cells but could not determine conditions that altered expression. We then examined the functional consequence of cross-linking membrane immunoglobulin and the receptor for IgG on human B lymphocytes. Our results cast some doubt on the value of anti-IgG as a model for antigen-antibody complexes in studying human B cell regulation.

  10. Lymphocyte integrin expression differences between SIRS and sepsis patients.

    Science.gov (United States)

    Heffernan, D S; Monaghan, S F; Ayala, Alfred

    2016-10-31

    Systemic Inflammatory Response Syndrome (SIRS) and sepsis remain leading causes of death. Despite many similarities, the two entities are very distinct clinically and immunologically. T-Lymphocytes play a key pivotal role in the pathogenesis and ultimately outcome following both SIRS and sepsis. Integrins are essential in the trafficking and migration of lymphocytes. They also serve vital roles in efficient wound healing and clearance of infections. Here, we investigate whether integrin expression, specifically β1 (CD29) and β2 (CD18), are disrupted in SIRS and sepsis, and assess differences in integrin expression between these two critically ill clinical categories. T-Lymphocytes were isolated from whole blood collected from ICU patients exhibiting SIRS or sepsis. Samples were analyzed for CD18 (β2) and CD29 (β1) on CD3(+) T cells through flow cytometry. Septic patients were stratified into either exclusively abdominal or non-abdominal sources of sepsis. CD18 was almost ubiquitously expressed on CD3(+) T cells irrespective of clinical condition. However, CD29 (β1 integrin) was lowest in SIRS patients (20.4% of CD3(+) T cells) when compared with either septic patients (35.5%) or healthy volunteers (54.1%). Furthermore, there was evidence of compartmentalization in septic patients, where abdominal sources had a greater percentage of CD3(+)CD29(+) T cells (41.7%) when compared with those with non-abdominal sources (29.5%). Distinct differences in T-cell integrin expression exists between patients in SIRS versus sepsis, as well as relative to the source of sepsis. Further work is needed to understand cause and effect relative to the progression from SIRS into sepsis.

  11. 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

    2008-01-01

    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.

  12. Perforin is a novel immune regulator of obesity-related insulin resistance.

    Science.gov (United States)

    Revelo, Xavier S; Tsai, Sue; Lei, Helena; Luck, Helen; Ghazarian, Magar; Tsui, Hubert; Shi, Sally Y; Schroer, Stephanie; Luk, Cynthia T; Lin, Gloria H Y; Mak, Tak W; Woo, Minna; Winer, Shawn; Winer, Daniel A

    2015-01-01

    Obesity-related insulin resistance is associated with an influx of pathogenic T cells into visceral adipose tissue (VAT), but the mechanisms regulating lymphocyte balance in such tissues are unknown. Here we describe an important role for the immune cytotoxic effector molecule perforin in regulating this process. Perforin-deficient mice (Prf1(null)) show early increased body weight and adiposity, glucose intolerance, and insulin resistance when placed on high-fat diet (HFD). Regulatory effects of perforin on glucose tolerance are mechanistically linked to the control of T-cell proliferation and cytokine production in inflamed VAT. HFD-fed Prf1(null) mice have increased accumulation of proinflammatory IFN-γ-producing CD4(+) and CD8(+) T cells and M1-polarized macrophages in VAT. CD8(+) T cells from the VAT of Prf1(null) mice have increased proliferation and impaired early apoptosis, suggesting a role for perforin in the regulation of T-cell turnover during HFD feeding. Transfer of CD8(+) T cells from Prf1(null) mice into CD8-deficient mice (CD8(null)) resulted in worsening of metabolic parameters compared with wild-type donors. Improved metabolic parameters in HFD natural killer (NK) cell-deficient mice (NK(null)) ruled out a role for NK cells as a single source of perforin in regulating glucose homeostasis. The findings support the importance of T-cell function in insulin resistance and suggest that modulation of lymphocyte homeostasis in inflamed VAT is one possible avenue for therapeutic intervention. © 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

  13. Syntaxin 8 is required for efficient lytic granule trafficking in cytotoxic T lymphocytes.

    Science.gov (United States)

    Bhat, Shruthi S; Friedmann, Kim S; Knörck, Arne; Hoxha, Cora; Leidinger, Petra; Backes, Christina; Meese, Eckart; Keller, Andreas; Rettig, Jens; Hoth, Markus; Qu, Bin; Schwarz, Eva C

    2016-07-01

    Cytotoxic T lymphocytes (CTL) eliminate pathogen-infected and cancerous cells mainly by polarized secretion of lytic granules (LG, containing cytotoxic molecules like perforin and granzymes) at the immunological synapse (IS). Members of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family are involved in trafficking (generation, transport and fusion) of vesicles at the IS. Syntaxin 8 (Stx8) is expressed in LG and colocalizes with the T cell receptor (TCR) upon IS formation. Here, we report the significance of Stx8 for human CTL cytotoxicity. We found that Stx8 mostly localized in late, recycling endosomal and lysosomal compartments with little expression in early endosomal compartments. Down-regulation of Stx8 by siRNA resulted in reduced cytotoxicity. We found that following perforin release of the pre-existing pool upon target cell contact, Stx8 down-regulated CTL regenerate perforin pools less efficiently and thus release less perforin compared to control CTL. CD107a degranulation, real-time and end-point population cytotoxicity assays, and high resolution microscopy support our conclusion that Stx8 is required for proper and timely sorting and trafficking of cytotoxic molecules to functional LG through the endosomal pathway in human CTL.

  14. TACI Expression and Signaling in Chronic Lymphocytic Leukemia

    Science.gov (United States)

    Mamara, Antigoni; Germenis, Anastasios E.; Kompoti, Maria; Palassopoulou, Maria; Mandala, Eudokia; Banti, Anastasia; Giannakoulas, Nikolaos

    2015-01-01

    TACI is a membrane receptor of BAFF and APRIL, contributing to the differentiation and survival of normal B cells. Although malignant B cells are also subjected on TACI signaling, there is a remarkable intradisease and interindividual variability of TACI expression in B-cell malignancies. The aim of our study was to explore the possible role of TACI signaling in the biology of chronic lymphocytic leukemia (CLL), including its phenotypic and clinical characteristics and prognosis. Ninety-four patients and 19 healthy controls were studied. CLL patients exhibited variable TACI expression, with the majority of cases displaying low to undetectable TACI, along with low to undetectable BAFF and increased APRIL serum levels compared to healthy controls. CLL cells with high TACI expression displayed a better survival capacity in vitro, when cultured with BAFF and/or APRIL. Moreover, TACI expression was positively correlated with the presence of monoclonal gammopathy and inversely with CD11c expression. Therefore, our study provides further evidence for the contribution of BAFF/APRIL signaling to CLL biology, suggesting also that TACI detection might be useful in the selection of patients for novel targeting therapeutic approaches. PMID:25950010

  15. TACI Expression and Signaling in Chronic Lymphocytic Leukemia

    Directory of Open Access Journals (Sweden)

    Antigoni Mamara

    2015-01-01

    Full Text Available TACI is a membrane receptor of BAFF and APRIL, contributing to the differentiation and survival of normal B cells. Although malignant B cells are also subjected on TACI signaling, there is a remarkable intradisease and interindividual variability of TACI expression in B-cell malignancies. The aim of our study was to explore the possible role of TACI signaling in the biology of chronic lymphocytic leukemia (CLL, including its phenotypic and clinical characteristics and prognosis. Ninety-four patients and 19 healthy controls were studied. CLL patients exhibited variable TACI expression, with the majority of cases displaying low to undetectable TACI, along with low to undetectable BAFF and increased APRIL serum levels compared to healthy controls. CLL cells with high TACI expression displayed a better survival capacity in vitro, when cultured with BAFF and/or APRIL. Moreover, TACI expression was positively correlated with the presence of monoclonal gammopathy and inversely with CD11c expression. Therefore, our study provides further evidence for the contribution of BAFF/APRIL signaling to CLL biology, suggesting also that TACI detection might be useful in the selection of patients for novel targeting therapeutic approaches.

  16. Lymphocyte Cc Chemokine Receptor 9 and Epithelial Thymus-Expressed Chemokine (Teck) Expression Distinguish the Small Intestinal Immune Compartment

    OpenAIRE

    2000-01-01

    The immune system has evolved specialized cellular and molecular mechanisms for targeting and regulating immune responses at epithelial surfaces. Here we show that small intestinal intraepithelial lymphocytes and lamina propria lymphocytes migrate to thymus-expressed chemokine (TECK). This attraction is mediated by CC chemokine receptor (CCR)9, a chemoattractant receptor expressed at high levels by essentially all CD4+ and CD8+ T lymphocytes in the small intestine. Only a small subset of lymp...

  17. miRNA expression profiles in chronic lymphocytic and acute lymphocytic leukemia

    Directory of Open Access Journals (Sweden)

    D.L. Zanette

    2007-11-01

    Full Text Available MicroRNAs (miRNAs are a class of small endogenous RNAs that play important regulatory roles by targeting mRNAs for cleavage or translational repression. miRNAs act in diverse biological processes including development, cell growth, apoptosis, and hematopoiesis, suggesting their association with cancer. We determined the miRNA expression profile of chronic and acute lymphocytic leukemias (CLL and ALL using the TaqMan® MicroRNA Assays Human Panel (Applied Biosystems. Pooled leukemia samples were compared to pooled CD19+ samples from healthy individuals (calibrator by the 2-DDCt method. Total RNA input was normalized based on the Ct values obtained for hsa-miR-30b. The five most highly expressed miRNAs were miR-128b, miR-204, miR-218, miR-331, and miR-181b-1 in ALL, and miR-331, miR-29a, miR-195, miR-34a, and miR-29c in CLL. To our knowledge, this is the first report associating miR-128b, miR-204 and miR-331 to hematological malignancies. The miR-17-92 cluster was also found to be up-regulated in ALL, as previously reported for some types of lymphomas. The differences observed in gene expression levels were validated for miR-331 and miR-128b in ALL and CD19+ samples. These miRNAs were up-regulated in ALL, in agreement with our initial results. A brief target analysis was performed for miR-331. One of its putative targets, SOCS1, promotes STAT activation, which is a known mediator of cell proliferation and survival, suggesting the possibility of an association between miR-331 and these processes. This initial screening provided information on miRNA differentially expressed in normal and malignant B-cells that could suggest the potential roles of these miRNAs in hematopoiesis and leukemogenesis.

  18. Expression of an accessory cell phenotype by hairy cells during lymphocyte colony formation in agar culture.

    Science.gov (United States)

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

    1983-01-01

    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.

  19. Expression of activated molecules on CD5(+)B lymphocytes in autoimmune hemolytic anemia.

    Science.gov (United States)

    Zhu, Hongli; Xu, Wenyan; Liu, Hong; Wang, Huaquan; Fu, Rong; Wu, Yuhong; Qu, Wen; Wang, Guojin; Guan, Jing; Song, Jia; Xing, Limin; Shao, Zonghong

    2016-05-01

    To investigate the expression of activation molecules on CD5(+)B lymphocytes in peripheral blood of autoimmune hemolytic anemia (AIHA)/Evans patients. The expression of CD80, CD86, and CD69 on CD5(+)B lymphocytes was detected using flow cytometry in 30 AIHA/Evans patients, 18 normal controls (NC) and nine chronic lymphocytic leukemia (CLL) patients. CD80 on CD5(+)B lymphocytes in untreated patients was higher than that in remission patients (P 0.05), but lower than those of CD5(-)B lymphocytes in remission patients and NC (P 0.05). CD80 and CD86 on CD5(+)B lymphocytes was negatively correlated with hemoglobin (HB), C3, C4 (P < 0.05) and positively correlated with reticulocyte (Ret) (P < 0.05). CD69 on CD5(+) and CD5(-)B lymphocytes of CLL was higher than those of AIHA/Evans patients and NC (P < 0.05). The active molecules on CD5(+)B lymphocytes in peripheral blood of AIHA/Evans patients differ from those on CD5(-) and clonal CD5(+)B lymphocytes.

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

    Science.gov (United States)

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

    2016-02-01

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

  1. PTK2 expression and immunochemotherapy outcome in chronic lymphocytic leukemia

    DEFF Research Database (Denmark)

    Weisser, Martin; Yeh, Ru-Fang; Duchateau-Nguyen, Guillemette

    2014-01-01

    Addition of rituximab (R) to fludarabine and cyclophosphamide (FC) has significantly improved patient outcomes in chronic lymphocytic leukemia (CLL). Whether baseline gene expression can identify patients who will benefit from immunochemotherapy over chemotherapy alone has not been determined. We...

  2. Lymphocyte Cc Chemokine Receptor 9 and Epithelial Thymus-Expressed Chemokine (Teck) Expression Distinguish the Small Intestinal Immune Compartment

    Science.gov (United States)

    Kunkel, Eric J.; Campbell, James J.; Haraldsen, Guttorm; Pan, Junliang; Boisvert, Judie; Roberts, Arthur I.; Ebert, Ellen C.; Vierra, Mark A.; Goodman, Stuart B.; Genovese, Mark C.; Wardlaw, Andy J.; Greenberg, Harry B.; Parker, Christina M.; Butcher, Eugene C.; Andrew, David P.; Agace, William W.

    2000-01-01

    The immune system has evolved specialized cellular and molecular mechanisms for targeting and regulating immune responses at epithelial surfaces. Here we show that small intestinal intraepithelial lymphocytes and lamina propria lymphocytes migrate to thymus-expressed chemokine (TECK). This attraction is mediated by CC chemokine receptor (CCR)9, a chemoattractant receptor expressed at high levels by essentially all CD4+ and CD8+ T lymphocytes in the small intestine. Only a small subset of lymphocytes in the colon are CCR9+, and lymphocytes from other tissues including tonsils, lung, inflamed liver, normal or inflamed skin, inflamed synovium and synovial fluid, breast milk, and seminal fluid are universally CCR9−. TECK expression is also restricted to the small intestine: immunohistochemistry reveals that intense anti-TECK reactivity characterizes crypt epithelium in the jejunum and ileum, but not in other epithelia of the digestive tract (including stomach and colon), skin, lung, or salivary gland. These results imply a restricted role for lymphocyte CCR9 and its ligand TECK in the small intestine, and provide the first evidence for distinctive mechanisms of lymphocyte recruitment that may permit functional specialization of immune responses in different segments of the gastrointestinal tract. Selective expression of chemokines by differentiated epithelium may represent an important mechanism for targeting and specialization of immune responses. PMID:10974041

  3. Perforin rapidly induces plasma membrane phospholipid flip-flop.

    Directory of Open Access Journals (Sweden)

    Sunil S Metkar

    Full Text Available The cytotoxic cell granule secretory pathway is essential for host defense. This pathway is fundamentally a form of intracellular protein delivery where granule proteases (granzymes from cytotoxic lymphocytes are thought to diffuse through barrel stave pores generated in the plasma membrane of the target cell by the pore forming protein perforin (PFN and mediate apoptotic as well as additional biological effects. While recent electron microscopy and structural analyses indicate that recombinant PFN oligomerizes to form pores containing 20 monomers (20 nm when applied to liposomal membranes, these pores are not observed by propidium iodide uptake in target cells. Instead, concentrations of human PFN that encourage granzyme-mediated apoptosis are associated with pore structures that unexpectedly favor phosphatidylserine flip-flop measured by Annexin-V and Lactadherin. Efforts that reduce PFN mediated Ca influx in targets did not reduce Annexin-V reactivity. Antigen specific mouse CD8 cells initiate a similar rapid flip-flop in target cells. A lipid that augments plasma membrane curvature as well as cholesterol depletion in target cells enhance flip-flop. Annexin-V staining highly correlated with apoptosis after Granzyme B (GzmB treatment. We propose the structures that PFN oligomers form in the membrane bilayer may include arcs previously observed by electron microscopy and that these unusual structures represent an incomplete mixture of plasma membrane lipid and PFN oligomers that may act as a flexible gateway for GzmB to translocate across the bilayer to the cytosolic leaflet of target cells.

  4. Expression of CD45RB functionally distinguishes intestinal T lymphocytes in inflammatory bowel disease.

    NARCIS (Netherlands)

    Hove, T. ten; The Olle, F.; Berkhout, M.; Bruggeman, J.P.; Vyth-Dreese, F.A.; Slors, J.F.M.; Deventer, S.J.H. van; Velde, A.A. te

    2004-01-01

    The importance of CD45RB expression on T cells was already shown in mice where CD45RB(high) expression determines pathogenic potential. In this study, we analyzed the expression of CD45RA, CD45RB, and CD45RO on CD4(+) T lymphocytes in the intestinal mucosa and in the circulation of patients with inf

  5. Expression of the Chemokine Receptors CCR4, CCR5, and CXCR3 by Human Tissue-Infiltrating Lymphocytes

    Science.gov (United States)

    Kunkel, Eric J.; Boisvert, Judie; Murphy, Kristine; Vierra, Mark A.; Genovese, Mark C.; Wardlaw, Andrew J.; Greenberg, Harry B.; Hodge, Martin R.; Wu, Lijun; Butcher, Eugene C.; Campbell, James J.

    2002-01-01

    Differential expression of adhesion molecules and chemokine receptors has been useful for identification of peripheral blood memory lymphocyte subsets with distinct tissue and microenvironmental tropisms. Expression of CCR4 by circulating memory CD4+ lymphocytes is associated with cutaneous and other systemic populations while expression of CCR9 is associated with a small intestine-homing subset. CCR5 and CXCR3 are also expressed by discrete memory CD4+ populations in blood, as well as by tissue-infiltrating lymphocytes from a number of sites. To characterize the similarities and differences among tissue-infiltrating lymphocytes, and to shed light on the specialization of lymphocyte subsets that mediate inflammation and immune surveillance in particular tissues, we have examined the expression of CCR4, CXCR3, and CCR5 on CD4+ lymphocytes directly isolated from a wide variety of normal and inflamed tissues. Extra-lymphoid tissues contained only memory lymphocytes, many of which were activated (CD69+). As predicted by classical studies, skin lymphocytes were enriched in CLA expression whereas intestinal lymphocytes were enriched in α4β7 expression. CCR4 was expressed at high levels by skin-infiltrating lymphocytes, at lower levels by lung and synovial fluid lymphocytes, but never by intestinal lymphocytes. Only the high CCR4 levels characteristic of skin lymphocytes were associated with robust chemotactic and adhesive responses to TARC, consistent with a selective role for CCR4 in skin lymphocyte homing. In contrast, CXCR3 and CCR5 were present on the majority of lymphocytes from each non-lymphoid tissue examined, suggesting that these receptors are unlikely to determine tissue specificity, but rather, may play a wider role in tissue inflammation. PMID:11786428

  6. Increased Expression of PI-3K in Asthmatic Rat T Lymphocytes

    Institute of Scientific and Technical Information of China (English)

    LIU Jin; ZHOU Shixin; XIONG Shengdao; XU Yongjian; ZHANG Zhenxiang; XIONG Weining

    2007-01-01

    In order to explore the expression of PI-3K in T lymphocytes of asthmatic rats and the relationship between PI-3K and activation of T lymphocytes, 24 Wistar rats were randomly divided into 4 groups: normal control group, asthmatic one-week group, asthmatic two-week group and asthmatic four-week group. T cells were purified from blood of each rat and the expression of PI-3K was observed by immunocytochemical fluorescence staining, the semiquantitative fluorescence intensity was measured by HPIAS-2000 analytic software, and the expression of IL-4 in supernatants was detected by ELISA. The results showed that the fluorescence intensity of T lymphocytes in asthmatic groups was significantly higher than that in normal control (P<0.001), indicating that the expression of PI-3K in T lymphocytes of asthmatic rats was significantly higher than that in those of normal controls, and the difference between acute and chronic stage asthmatic groups was significant (P<0.05). The expression levels of IL-4 protein in supernatants of asthmatic T lymphocytes were significantly higher than those in the normal controls (P<0.05). There was a significant positive correlation between the expression of PI-3K in T lymphocytes and the IL-4 protein expression in supernatants (r=0.583, P<0.01). It was suggested that PI-3K signal pathway may participate in the processes of activation and other cytological effects of asthmatic T lymphocytes, thus may play an important roles in the pathogenesis of asthma.

  7. Expression of HSP70 in Peripheral Lymphocytes of the Patients with Allergic Rhinitis

    Institute of Scientific and Technical Information of China (English)

    刘立思; 肖成峰; 张明; 程磊; 王鄂芬; 邬堂春

    2003-01-01

    The expression levels of heat shock protein 70 (HSP70) from peripheral lymphocytes ofthe patients with allergic rhinitis (AR) and the clinical implication were investigated. In the morn-ing, 3 ml of fasting venous blood was taken out. The lymphocytes were isolated by using Ficoll-Hypaque and the expression of HSP70 in the lymphocytes was detected by using Western blot. Inthe AR patients the HSP70 level (41.49± 15.77 integrated optical density, IOD) were significantlyhigher than that in the control group (23.89±10. 13 IOD, P<0.05). Western blot demonstratedthat HSP70 bands in AR patients were more intensive than those in the control group. It was con-cluded that the elevated HSP70 level in peripheral lymphocytes of the AR patients might contributeto the development of AR.

  8. Differential expression and function of CD27 in chronic lymphocytic leukemia cells expressing ZAP-70.

    Science.gov (United States)

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

    2015-07-01

    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.

  9. Expression of a single, viral oncoprotein in skin epithelium is sufficient to recruit lymphocytes.

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    Allison Choyce

    Full Text Available Established cancers are frequently associated with a lymphocytic infiltrate that fails to clear the tumour mass. In contrast, the importance of recruited lymphocytes during premalignancy is less well understood. In a mouse model of premalignant skin epithelium, transgenic mice that express the human papillomavirus type 16 (HPV16 E7 oncoprotein under a keratin 14 promoter (K14E7 mice display epidermal hyperplasia and have a predominant infiltrate of lymphocytes consisting of both CD4 and CD8 T cells. Activated, but not naïve T cells, were shown to preferentially traffic to hyperplastic skin with an increased frequency of proliferative CD8+ T cells and CD4+ T cells expressing CCR6 within the tissue. Disruption of the interaction between E7 protein and retinoblastoma tumour suppressor protein (pRb led to reduced epithelial hyperplasia and T cell infiltrate. Finally, while K14E7 donor skin grafts are readily accepted onto syngeneic, non-transgenic recipients, these same skin grafts lacking skin-resident lymphocytes were rejected. Our data suggests that expression of a single oncoprotein in the epidermis is sufficient for lymphocyte trafficking (including immunosuppressive lymphocytes to premalignant skin.

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

    Science.gov (United States)

    2011-01-01

    Background Arctium lappa (Niubang), a Chinese herbal medicine, is used to treat tissue inflammation. This study investigates the effects of arctigenin (AC), isolated from A. lappa, on anti-CD3/CD28 Ab-stimulated cell proliferation and cytokine gene expression in primary human T lymphocytes. Methods Cell proliferation was determined with enzyme immunoassays and the tritiated thymidine uptake method. Cytokine production and gene expression were analyzed with reverse transcription-polymerase chain reaction. Results AC inhibited primary human T lymphocytes proliferation activated by anti-CD3/CD28 Ab. Cell viability test indicated that the inhibitory effects of AC on primary human T lymphocyte proliferation were not due to direct cytotoxicity. AC suppressed interleukin-2 (IL-2) and interferon-γ (IFN-γ) production in a concentration-dependent manner. Furthermore, AC decreased the IL-2 and IFN-γ gene expression in primary human T lymphocytes induced by anti-CD3/CD28 Ab. Reporter gene analyses revealed that AC decreased NF-AT-mediated reporter gene expression. Conclusion AC inhibited T lymphocyte proliferation and decreased the gene expression of IL-2, IFN-γ and NF-AT. PMID:21435270

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

    Science.gov (United States)

    Tsai, Wei-Jern; Chang, Chu-Ting; Wang, Guei-Jane; Lee, Tzong-Huei; Chang, Shwu-Fen; Lu, Shao-Chun; Kuo, Yuh-Chi

    2011-03-25

    Arctium lappa (Niubang), a Chinese herbal medicine, is used to treat tissue inflammation. This study investigates the effects of arctigenin (AC), isolated from A. lappa, on anti-CD3/CD28 Ab-stimulated cell proliferation and cytokine gene expression in primary human T lymphocytes. Cell proliferation was determined with enzyme immunoassays and the tritiated thymidine uptake method. Cytokine production and gene expression were analyzed with reverse transcription-polymerase chain reaction. AC inhibited primary human T lymphocytes proliferation activated by anti-CD3/CD28 Ab. Cell viability test indicated that the inhibitory effects of AC on primary human T lymphocyte proliferation were not due to direct cytotoxicity. AC suppressed interleukin-2 (IL-2) and interferon-γ (IFN-γ) production in a concentration-dependent manner. Furthermore, AC decreased the IL-2 and IFN-γ gene expression in primary human T lymphocytes induced by anti-CD3/CD28 Ab. Reporter gene analyses revealed that AC decreased NF-AT-mediated reporter gene expression. AC inhibited T lymphocyte proliferation and decreased the gene expression of IL-2, IFN-γ and NF-AT.

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

    Directory of Open Access Journals (Sweden)

    Chang Shwu-Fen

    2011-03-01

    Full Text Available Abstract Background Arctium lappa (Niubang, a Chinese herbal medicine, is used to treat tissue inflammation. This study investigates the effects of arctigenin (AC, isolated from A. lappa, on anti-CD3/CD28 Ab-stimulated cell proliferation and cytokine gene expression in primary human T lymphocytes. Methods Cell proliferation was determined with enzyme immunoassays and the tritiated thymidine uptake method. Cytokine production and gene expression were analyzed with reverse transcription-polymerase chain reaction. Results AC inhibited primary human T lymphocytes proliferation activated by anti-CD3/CD28 Ab. Cell viability test indicated that the inhibitory effects of AC on primary human T lymphocyte proliferation were not due to direct cytotoxicity. AC suppressed interleukin-2 (IL-2 and interferon-γ (IFN-γ production in a concentration-dependent manner. Furthermore, AC decreased the IL-2 and IFN-γ gene expression in primary human T lymphocytes induced by anti-CD3/CD28 Ab. Reporter gene analyses revealed that AC decreased NF-AT-mediated reporter gene expression. Conclusion AC inhibited T lymphocyte proliferation and decreased the gene expression of IL-2, IFN-γ and NF-AT.

  13. Familial Hemophagocytic Lymphohistiocytosis with A665 G Perforin Gene Mutation: A Case Report

    Directory of Open Access Journals (Sweden)

    Idil Yenicesu

    2012-09-01

    Full Text Available Familial hemophagocytic lymphohistiocytosis (FHL is a genetically heterogeneous disease. Presentation of the disease such as primarily fever, hepatosplenomegaly, and cytopenia, which are the results of functional degradation in cytotoxic T-lymphocytes and natural killer cells, activation of macrophages and T-lymphocytes, over production of proinflammatory cytokines, and hemophagocytosis. In all, 5 genetic loci have been identified in FHL, and all known affected genes encode critical components of the granule exocytosis pathway, which is essential for the release of cytotoxic granules and proteases that are necessary for targeted cell death. Herein we present an FHL patient with a severe clinical course and a very rare perforin gene mutation. The patient was homozygous for A665G mutation. However, the child died in a short period of time. Prenatal diagnosis was performed in the family and the fetus was found to be heterozygous for the mutation.

  14. Expression of Bcl-2-family proteins in peripheral blood B-lymphocytes in patients with cronic lymphocytic leukemia

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    Brajušković Goran R.

    2004-01-01

    Full Text Available Chronic lymphocytic leukemia (CLL is a neoplastic disease characterized by the accumulation of morphologically mature monoclonal CD 5+ B cells in the early phase (G0/G1 of the cell cycle. It is considered that the accumulation of neoplastically transformed lymphocytes B (CLL cells is primarily the consequence of the disturbance, i.e., blockade of these cells' apoptosis process. Apoptosis is the specific process of programmed cell death regulated by numerous extracellular and intracellular mechanisms. The Bcl-2 proteins are well-known modulators of this process. Some of these proteins (such as Bcl-2, and Bcl-Xl are anti-apoptotic, while others (such as Bad or Bax are pro-apoptotic. Our study included the analysis of 20 peripheral blood specimens from 20 patients with CLL, and 20 peripheral blood specimens of healthy persons who represented the control group. Using Western blotting analysis, we quantitatively examined the protein expression of Bcl-2 family (Bcl-2, Bax, Bad, and Bcl-Xl. The level of Bcl-2 (p=3,68´10-10, Bax (p=0,019, and Bad (p=0,073 proteins expression was significantly increased in all the analyzed peripheral blood samples of patients, while the level of Bcl-Xl protein (p=0,75 did not significantly differ in peripheral blood samples of patients, compared to the controls. The results of this study showed that the increased level of expression of Bcl-2, Bax, and Bad protein represented the most striking feature of CLL cells. Moreover, the variations in the expression of only one protein of the Bcl-2 family could not represent the prognostic parameter in the treatment of this disease.

  15. Effect of carbamate pesticides on perforin, granzymes A-B-3/K, and granulysin in human natural killer cells.

    Science.gov (United States)

    Li, Qing; Kobayashi, Maiko; Kawada, Tomoyuki

    2015-09-01

    We previously found that ziram, a carbamate pesticide, significantly reduced perforin, granzyme A (GrA), granzyme B (GrB), granzyme 3/K (Gr3/K), and granulysin (GRN) levels in NK-92MI cells, a human natural killer (NK) cell line. To investigate whether other carbamate pesticides also show similar toxicity on human NK cells, we conducted further experiments with NK-92CI cells, a human NK cell line, using a more sensitive assay. We previously confirmed that NK-92CI cells express CD56, perforin, GrA, GrB, Gr3/K, and GRN and are highly cytotoxic to K562 cells in a chromium release assay, which are more sensitive to organophosphorus pesticides and ziram than the NK-92MI cell line. NK-92CI cells were treated with ziram, thiram, maneb, or carbaryl at various concentrations for 4-24 h at 37°C in vitro. Thereafter, intracellular levels of perforin, GrA, GrB, Gr3/K, and GRN were determined by flow cytometry. It was found that all carbamate pesticides significantly reduced the intracellular levels of perforin, GrA, GrB, Gr3/K, and GRN in NK-92CI cells in a dose-dependent manner. However, the strength of the effect differed among the pesticides, and the order was thiram > ziram > maneb > carbaryl. In addition, it was also found that the degree of the reductions differed among the five proteins, with perforin more sensitive to pesticides than GRN, GrA, GrB, and Gr3/K, and the order was perforin > GRN > Gr3/K ≒ GrA ≒ GrB. © The Author(s) 2015.

  16. 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.

    Science.gov (United States)

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

    1980-06-01

    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.

  17. Expression of molecules involved in B lymphocyte survival and differentiation by synovial fibroblasts.

    Science.gov (United States)

    Edwards, J C; Leigh, R D; Cambridge, G

    1997-06-01

    The synovitis of rheumatoid arthritis (RA) is one of few pathological lesions in which B lymphocyte accumulation progresses to the extent of germinal centre formation. The present study was designed to assess the ability of synovial fibroblasts to express molecules implicated in B lymphocyte survival and differentiation, both in vivo, and in response to cytokines in vitro. Normal and diseased synovia were examined by indirect immunofluorescence. In all tissues synovial intimal fibroblasts showed co-expression of vascular cell adhesion molecule-1 (VCAM-1) and complement decay-accelerating factor (DAF) comparable to that of follicular dendritic cells (FDC), but not complement receptor 2 (CR2). In rheumatoid synovia, subintimal cells showed variable expression of VCAM-1 and DAF, with bright co-expression of VCAM-1, DAF and CR2 in lymphoid follicle centres. B lymphocytes, some of which were proliferating cell nuclear antigen-positive, were present in contact with subintimal cells expressing VCAM-1 with or without DAF or CR2. B lymphocytes were rarely present in the intimal layer, and, where present, showed fragmentation. In vitro, synovial fibroblasts exposed to tumour necrosis factor-alpha (TNF-alpha) in combination with interferon-gamma (IFN-gamma) showed enhanced expression of VCAM-1, in comparison with fibroblasts from skin and lung and, unlike skin and lung fibroblasts, also expressed DAF and CR2. These findings support the hypothesis that synovial targeting in RA involves an enhanced ability of synovial fibroblasts to support B lymphocyte survival. This appears to be dependent, not on the constitutive expression of VCAM-1 and DAF on intimal cells, but on the increased ability of subintimal cells to respond to proinflammatory cytokines, perhaps critically in the expression of VCAM-1.

  18. Lymphocytes from wasted mice express enhanced spontaneous and {gamma}-ray-induced apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Woloschak, G.E. [Argonne National Lab., IL (United States)]|[Loyola Univ. Medical Center, Maywood, IL (United States); Chang-Liu, Chin-Mei [Argonne National Lab., IL (United States); Chung, Jen; Libertin, C.R. [Loyola Univ. Medical Center, Maywood, IL (United States)

    1993-09-01

    Mice bearing the autosomal recessive mutation wasted (wst/wst) display a disease pattern including faulty repair of DNA damage in lymphocytes after radiation exposure, neurologic abnormalities, and immunodeficiency. Many of the features of this mouse model have suggested a premature or increased spontaneous frequency of apoptosis in thymocytes; past work has shown an inability to establish cultured T cell lines, an abnormally high death rate of stimulated T cells in culture, and an increased sensitivity of T cells to the killing effects of ionizing radiations in wst/wst mice relative to controls. The experiments reported here were designed to examine splenic and thymic lymphocytes from wasted and control mice for signs of early apoptosis. Our results revealed enhanced expression of Rp-8 mRNA (associated with apoptosis) in thymic lymphocytes and reduced expression in splenic lymphocytes of wst/wst mice relative to controls; expression of Rp-2 and Td-30 mRNA (induced during apoptosis) were not detectable in spleen or thymus. Higher spontaneous DNA fragmentation was observed in wasted mice than in controls; however, {gamma}-ray-induced DNA fragmentation peaked at a lower dose and occurred to a greater extent in wasted mice relative to controls. These results provide evidence for high spontaneous and {gamma}-ray-induced apoptosis in T cells of wasted mice as a mechanism underlying the observed lymphocyte and DNA repair abnormalities.

  19. Fas and Bcl-2 Expression on T Lymphocyte Subsets in the Peripheral Blood of Relapsing Patients with Condyloma Acuminatum

    Institute of Scientific and Technical Information of China (English)

    顾军; 范清源; 高春芳; 代夫; 郑茂荣

    2003-01-01

    Objective: To study the expression of Fas and Bcl-2 proteins on T lymphocyte subsets in the peripheral blood of relapsing patients with condyloma acuminatum(CA) and healthy controls.Methods: Flow cytometry (permeabization and staining procedure with conjugated antibodies) was used.Results: We observed that the expression of Fas protein on CD4+ T lymphocyte subset of CA patients was significantly higher than that of healthy controls( P<0.01 ).Conclusions: Increased expression of Fas proteinon CD4+ T lymphocyte subset may be a cause of de-creased percentage of CD4+ T lymphocyte subset. This induces the increased ratio of CD4+/CD8+.

  20. Methods for Analyzing the Role of DNA Methylation and Chromatin Structure in Regulating T Lymphocyte Gene Expression

    Directory of Open Access Journals (Sweden)

    Lu Qianjin

    2004-01-01

    Full Text Available Chromatin structure, determined in part by DNA methylation, is established during differentiation and prevents expression of genes unnecessary for the function of a given cell type. We reported that DNA methylation and chromatin structure contributes to lymphoid-specific ITGAL (CD11a and PRF1 (perforin expression. We used bisulfite sequencing to compare methylation patterns in the ITGAL promoter and 5' flanking region of T cells and fibroblasts, and in the PRF1 promoter and upstream enhancer of CD4+ and CD8+ T cells with fibroblasts. The effects of methylation on promoter function were tested using regional methylation of reporter constructs, and confirmed by DNA methyltransferase inhibition. The relationship between DNA methylation and chromatin structure was analyzed by DNaseI hypersensitivity. Herein we described the methods and results in greater detail.

  1. Expression of membrane receptor for tumour necrosis factor on human blood lymphocytes.

    Science.gov (United States)

    Zola, H; Flego, L; Weedon, H

    1993-08-01

    Using a monoclonal antibody against the human p75 tumour necrosis factor receptor (TNFR-I) combined with a high-sensitivity immunofluorescence flow cytometric procedure, a proportion of peripheral blood lymphocytes can be shown to express TNFR-I constitutively. Approximately 50% of peripheral blood lymphocytes consisting mostly of CD4 cells and including most CD45R0-positive cells, express TNFR-I. Receptor expression is increased by a variety of activation signals. Only a minority (up to 30%) of tonsil B cells express measurable levels of TNFR-I. The tonsil B cells which express TNFR-I include both cells with a germinal centre cell phenotype and cells with the phenotype of the follicular mantle zone. Activation of B cells with anti-immunoglobulin, alone or in combination with interleukin-4 or interleukin-2, increases receptor expression, particularly in cells with the phenotype of mantle zone cells. The functional significance of constitutive expression of TNFR by blood and tissue lymphocytes is discussed.

  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

    2005-02-01

    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. Expression of the Chemokine Receptors CCR4, CCR5, and CXCR3 by Human Tissue-Infiltrating Lymphocytes

    OpenAIRE

    2002-01-01

    Differential expression of adhesion molecules and chemokine receptors has been useful for identification of peripheral blood memory lymphocyte subsets with distinct tissue and microenvironmental tropisms. Expression of CCR4 by circulating memory CD4+ lymphocytes is associated with cutaneous and other systemic populations while expression of CCR9 is associated with a small intestine-homing subset. CCR5 and CXCR3 are also expressed by discrete memory CD4+ populations in blood, as well as by tis...

  4. Independent expression of the two paralogous CCL4 genes in monocytes and B lymphocytes.

    Science.gov (United States)

    Lu, Jun; Honczarenko, Marek; Sloan, Steven R

    2004-01-01

    The CCL4 chemokine is secreted by a variety of cells following stimulation. CCL4 affects several different types of cells that are important for acute inflammatory responses and are critical for the development of specific immune responses to foreign antigens. The human genome contains two genes for the CCL4 chemokine. Although highly homologous, the two genes encode slightly different proteins. We analyzed the mRNA expressed in monocytes and B lymphocytes and found that while monocytes express predominantly one CCL4 gene, known as ACT-2, peripheral blood B lymphocytes express a mixture of ACT-2 and the second CCL4 gene, lymphocyte activating gene-1 ( LAG-1). Although peripheral blood B cells, CD27(-) B cells, and CD27(+) B cells all express a mixture of LAG-1 and ACT-2, the B-cell lines that were studied regulate the two genes independently. RL, SU-DHL-6, and REH cells predominantly express LAG-1. These studies demonstrate that monocytes and B cells utilize different mechanisms to regulate expression of the two CCL4 genes and suggest that the two genes may not have identical activities.

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

    Energy Technology Data Exchange (ETDEWEB)

    Morzadec, Claudie; Macoch, Mélinda; Robineau, Marc; Sparfel, Lydie [UMR INSERM U1085, Institut de Recherche sur la Santé, l' Environnement et le Travail (IRSET), Université de Rennes 1, 2 avenue du Professeur Léon Bernard, 35043 Rennes (France); Fardel, Olivier [UMR INSERM U1085, Institut de Recherche sur la Santé, l' Environnement et le Travail (IRSET), Université de Rennes 1, 2 avenue du Professeur Léon Bernard, 35043 Rennes (France); Pôle Biologie, Centre Hospitalier Universitaire (CHU) Rennes, 2 rue Henri Le Guilloux, 35033 Rennes (France); Vernhet, Laurent, E-mail: laurent.vernhet@univ-rennes1.fr [UMR INSERM U1085, Institut de Recherche sur la Santé, l' Environnement et le Travail (IRSET), Université de Rennes 1, 2 avenue du Professeur Léon Bernard, 35043 Rennes (France)

    2012-08-01

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

  6. NFAT1 transcription factor regulates cell cycle progression and cyclin E expression in B lymphocytes.

    Science.gov (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

    2016-09-01

    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.

  7. CD40 Ligand Expression on Stimulated T-Helper Lymphocytes in Patients with Common Variable Immunodeficiency

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    Masoud Ravanbakhsh

    2007-09-01

    Full Text Available Common variable immunodeficiency (CVID is the most common symptomatic primary antibody deficiency, characterized by reduced serum immunoglobulins levels and increased susceptibility to recurrent pyogenic infections. In this study, we evaluated CD40 ligand expression on stimulated versus unstimulated T-helper lymphocytes of nine Common variable immunodeficient patients in comparison with fifteen normal controls. Phorbol myristate acetate (PMA and Ionomycin were used to stimulate cells in vitro. After six hours stimulation, the cells were subjected to surface staining with three-color staining procedure. Events were analyzed by flow cytometer, using FloMax software. Results were reported as the percentage of lymphocytes expressing CD markers. We did not find any significant statistical difference in CD40 ligand expression between patients and controls (p>0.05, despite having stimulation documented by CD69 expression as activation marker in each run. The results of this study are in agreement with some other studies, indicating that CD40 ligand expression on stimulated T-helper lymphocytes of Common variable immunodeficiency patients is similar to normal controls.  

  8. Expression of T-Lymphocyte Markers in Human Epidermal Growth Factor Receptor 2-Positive Breast Cancer

    Science.gov (United States)

    Lee, Changro; Kim, Joo Heung; Lim, Sung Mook; Park, Hyung Seok; Kim, Seung Il; Park, Byeong-Woo

    2016-01-01

    Purpose The present study aimed to examine the clinical implications of CD4, CD8, and FOXP3 expression on the prognosis of human epidermal growth factor receptor 2 (HER2)-positive breast cancer using a web-based database, and to compare the immunohistochemical expression of T-lymphocyte markers using primary and metastatic HER2-positive tumor tissues before and after HER2-targeted therapy. Methods Using the cBioPortal for Cancer Genomics and Kaplan-Meier plotter, the mRNA expression, association between T-lymphocyte markers, and survival in HER2-positive cancers were investigated according to various cutoff levels. Immunohistochemistry analysis was performed using paired primary and metastatic tissues of 29 HER2-positive tumors treated with systemic chemotherapy and HER2-directed therapy. Results HER2 mRNA was mutually exclusive of T-lymphocyte markers, and a significant correlation between T-cell markers was observed in the cBioPortal for Cancer Genomics. According to analysis of the Kaplan-Meier plotter, the impact of T-lymphocyte marker expression on survival was statistically insignificant in clinical HER2-positive tumors, irrespective of the cutoff levels. However, in the intrinsic HER2-positive subtype, the individual analyses of T-cell markers except for FOXP3 and combined analysis showed significantly favorable survival irrespective of cutoff points. Although the small clinical sample size made it difficult to show the statistical relevance of immunohistochemistry findings, good responses to neoadjuvant treatments might be associated with positive expression of combined T-lymphocyte markers, and approximately half of the samples showed discordance of combined markers between baseline and resistant tumors. Conclusion T-lymphocyte markers could be favorable prognostic factors in HER2-positive breast cancers; however, a consensus on patient section criteria, detection methods, and cutoff value could not be reached. The resistance to HER2-directed therapy might

  9. Elevated D8/17 expression on B lymphocytes, a marker of rheumatic fever, measured with flow cytometry in tic disorder patients

    NARCIS (Netherlands)

    Hoekstra, PJ; Bijzet, J; Limburg, PC; Steenhuis, MP; Troost, PW; Oosterhoff, MD; Korf, J; Kallenberg, CGM; Minderaa, RB

    2001-01-01

    Objective: Elevated D8/17 expression on B lymphocytes is a known susceptibility marker of rheumatic fever. Previous studies have reported higher than usual D8/ 17 expression on B lymphocytes of patients with tic disorders. The purpose of this study was to assess D8/17 expression on B lymphocytes of

  10. Kinetic analysis of interleukin 2 (IL-2) production and expression of IL-2 receptors by uraemic and normal lymphocytes

    DEFF Research Database (Denmark)

    Langhoff, E; Hofmann, B; Ladefoged, J

    1987-01-01

    The in vitro immune response of lymphocytes from uraemic patients was studied by comparing the in vitro kinetics of interleukin 2 (IL-2) production, the mitogen-induced proliferative response, and the expression of IL-2 receptors by T lymphocytes. The IL-2 production in 26 uraemic cell cultures...

  11. Association of Bax Expression and Bcl2/Bax Ratio with Clinical and Molecular Prognostic Markers in Chronic Lymphocytic Leukemia

    Directory of Open Access Journals (Sweden)

    Vucicevic Ksenija

    2016-04-01

    Full Text Available Background: In chronic lymphocytic leukemia (CLL, in vivo apoptotic resistance of malignant B lymphocytes results, in part, from the intrinsic defects of their apoptotic machinery. These include genetic alterations and aberrant expression of many apoptosis regulators, among which the Bcl2 family members play a central role.

  12. Expression of IL-4 receptor on human T and B lymphocytes.

    Science.gov (United States)

    Zola, H; Flego, L; Weedon, H

    1993-08-01

    The expression of the interleukin-4 receptor on human blood and tonsil lymphocytes has been studied using a monoclonal antibody and high-sensitivity immunofluorescence flow cytometry. While no receptor expression could be detected on circulating or tonsil T cells, a subset of B cells was shown to express the receptor. The IL-4R-positive B cells in tonsil had a phenotype suggesting that they included both germinal centre B cells and B cells outside the germinal centre. The subset of B cells in the blood that expressed the receptor included CD23-positive B cells. Activation of tonsil B cells using anti-IgM, IL-4, IL-2, or combinations of these reagents led to increases in IL-4R expression, but these changes were small compared to changes in the expression of IL-2R p55 (CD25), a known marker of activation. Similarly, activation of T cells led to low-level expression of IL-4R, with IL-4 itself up-regulating IL-4R, especially in CD4 cells. The majority of chronic lymphocytic leukaemia samples were positive for IL-4R expression, whilst most other leukemic samples were negative.

  13. Gene expression of lymphocyte prolactin receptor was suppressed in lactating mothers.

    Science.gov (United States)

    Maeda, Hironobu; Izumi, Shun-ichiro; Kato, Yukio; Cai, Li-yi; Kato, Takako; Suzuki, Takahiro; Nakamura, Eri; Sugiyama, Taro; Fuda, Takayo; Takahashi, Kazumi; Kondo, Akane; Matsumoto, Tadashi; Ishimoto, Hitoshi

    2010-07-20

    Prolactin (PRL) receptor (PRL-R) was proven to be ubiquitously expressed by cells in the immune system, while the physiological role of PRL was established in milk production in mammary glands. We analyzed the mRNA content of PRL-R in human lymphocytes in normo- and hyperprolactinemic conditions to document the presence of functioning PRL-R of human lymphocytes. Blood samples were obtained prior to treatment, and with written informed consent, from outpatients with ovarian dysfunction and hyperprolactinemia (n = 8; 19 ~ 41 y/o), from breast-feeding mothers after normal delivery (n = 12; 27 ~ 36 y/o), and from healthy volunteers: men (n = 9; 33 ~ 40 y/o) and women (n = 9; 26 ~ 36 y/o). Subsequently, total RNA was prepared from the lymphocytes separated. The quantity of PRL-R mRNA was examined by reverse transcription and polymerase chain reaction and normalized with a simultaneously measured amount of b actin. The resultant mRNA level of PRL-R was analyzed for its correlation with serum concentration of PRL measured by immunoassay. PRL-R mRNA levels of lymphocytes were significantly suppressed in lactating mothers, while there was a statistically significant negative correlation between PRL-R mRNA and serum PRL levels. However, there was no significant difference of PRL-R mRNA in the pathological condition of outpatients with ovarian dysfunction and/or hyperprolactinemia. While a few investigators reported the extra-mammary regulation on PRL-R by PRL, our data suggest that the PRL-R levels of circulating lymphocytes could be down-regulated by the elevated serum levels of PRL and that pituitary PRL may participate in regulating the expression of PRL-R genes on cells of the human immune system, especially in physiological circumstances such as in the postpartum period.

  14. Systemic and Brain Pharmacokinetics of Perforin Inhibitor Prodrugs

    DEFF Research Database (Denmark)

    Gynther, Mikko; Pickering, Darryl S; Spicer, Julie;

    2016-01-01

    We have recently reported that by converting a perforin inhibitor into an l-type amino acid transporter 1 (LAT1)-utilizing prodrug its cellular uptake can be greatly increased. The aim of the present study was to determine the in vivo and brain pharmacokinetics of two perforin inhibitors and thei......We have recently reported that by converting a perforin inhibitor into an l-type amino acid transporter 1 (LAT1)-utilizing prodrug its cellular uptake can be greatly increased. The aim of the present study was to determine the in vivo and brain pharmacokinetics of two perforin inhibitors...... and their LAT1-utilizing prodrugs 1 and 2. In addition, the brain uptake mechanism and entry into primary mouse cortical neurons and astrocytes were evaluated. After 23 μmol/kg i.p. bolus injection, the prodrugs’ unbound area under the concentration curve in brain was 0.3 nmol/g × min, whereas the parent drugs...... transporting polypeptides, probenecid, decreased the brain concentrations to 352.4 ± 44.5 and 70.9 ± 7.0 pmol/g, respectively. In addition, in vitro uptake studies showed that at 100 μM prodrug 1 had 3.4-fold and 4.5-fold higher uptake rate into neurons and astrocytes, respectively, compared to its parent drug...

  15. Expression of lymphocyte coding genes in peripheral blood and lymphocyte infiltration in cardiac tissues influenced by cyclosporin A in heterotopic heart transplantation model in rats.

    Science.gov (United States)

    Xu, Xiu-fang; Xin, Yi; Zhang, Ying; Huang, Yi-min; Li, Wen-bin; Li, Na; Lin, Zheng; Zhou, Yu-jie; Zhang, Zhao-guang

    2013-12-01

    To systematically compare the expression of coding genes with pathological changes of transplanted cardiac tissue and peripheral blood lymphocytes in an allo-heterotopic rat cardiac transplant model. Using SD rats as donors and Wistar rats as recipients, animals were divided into two groups, control and cyclosporine A intervention plus heart transplant groups. After transplant at 1, 3, 7, 10 and 12d, we assessed the ability of lymphocytes to infiltrate into cardiac tissues and levels of leukocyte coding genes in peripheral blood. Histopathological changes were monitored in cardiac tissue to determine the level of transplant rejection. (1) 24h after transplant peripheral blood lymphocytes' transcription and expression were temporarily reduced. (2) CD4(+) and CD8(+) lymphocytes infiltrate into cardiac tissue and Grade 1R pathological changes were observed 3d-7d after heart transplant. (3)Cyclosporine A was not able to completely block heart transplant rejection.(4) Although cyclosporine A was not able to effectively suppress CD4(+) T cell gene expression, it did suppress CD8(+) T cell gene transcription. (5) Cyclosporine A did not effectively reduce the rapid infiltration of CD4(+) or CD8(+) infiltration in 3d, but significantly reduced the degree of CD4(+) T cell infiltration in cardiac tissues between 3 and 7d. (6) Differential display (DD-PCR): Graft control group: there were differences in 2,3-bisphosphoglycerate, ribosomal protein S25, 12S ribosomal, gig18, MHC-III and ATPase H(+), which occurred 24h before CD4/CD8 surface protein expression. Cyclosporine A group: there were differences in thrombospondin-1, TCR, 2,3-bisphosphoglycerate, sodium channel beta-1, gig18 and TCR. In the cyclosporine A group 2,3-bisphosphoglycerate positive expression was observed 24h after the control group, which indicates that cyclosporine A slowed down the 2,3-bisphosphoglycerate transcription rate in peripheral lymphocytes and delayed its expression time. Cyclosporine A also

  16. Human yeast-specific CD8 T lymphocytes show a nonclassical effector molecule profile.

    Science.gov (United States)

    Breinig, Tanja; Scheller, Nicoletta; Glombitza, Birgit; Breinig, Frank; Meyerhans, Andreas

    2012-05-01

    Pathogenic yeast and fungi represent a major group of human pathogens. The consequences of infections are diverse and range from local, clinically uncomplicated mycosis of the skin to systemic, life-threatening sepsis. Despite extensive MHC class I-restricted frequencies of yeast-specific CD8 T lymphocytes in healthy individuals and the essential role of the cell-mediated immunity in controlling infections, the characteristics and defense mechanisms of antifungal effector cells are still unclear. Here, we describe the direct analysis of yeast-specific CD8 T lymphocytes in whole blood from healthy individuals. They show a unique, nonclassical phenotype expressing granulysin and granzyme K in lytic granules instead of the major effector molecules perforin and granzyme B. After stimulation in whole blood, yeast-specific CD8 T cells degranulated and, upon cultivation in the presence of IL-2, their granula were refilled with granulysin rather than with perforin and granzyme B. Moreover, yeast-specific stimulation through dendritic cells but not by yeast cells alone led to degranulation of the effector cells. As granulysin is the only effector molecule in lytic granules known to have antifungal properties, our data suggest yeast-specific CD8 T cells to be a nonclassical effector population whose antimicrobial effector machinery seems to be tailor-made for the efficient elimination of fungi as pathogens.

  17. Expression of adhesion and activation molecules on lymphocytes during open-heart surgery with cardiopulmonary bypass

    DEFF Research Database (Denmark)

    Toft, P; Tønnesen, Else Kirstine; Zülow, I

    1997-01-01

    Open-heart surgery with cardiopulmonary bypass (CPB) and abdominal surgery are associated with lymphocytopenia. We measured a panel of adhesion and activation molecules on lymphocytes to clarify possible association of CPB with increased expression of these molecules. Eight patients undergoing open......-heart surgery and eight with abdominal surgery were studied. The adhesion molecules CD11a/CD18 (LFA-1_, CD11c/CD18 and CD44 and the activation molecules CD25, CD69, CD71 and MHCII were measured, using monoclonal antibodies and flow cytometry. Lymphocytopenia was observed during CPB and for some hours after both...... open-heart and abdominal surgery. The proportion of CD11a/CD18-positive lymphocytes rose from 67.6 +/- 8% to 86.4 +/- 3% after aortic declamping (p

  18. The role of perforin-mediated cell apoptosis in murine models of infusion-induced bone marrow failure

    Science.gov (United States)

    Sarcon, Annahita K.; Desierto, Marie J.; Zhou, Wenjun; Visconte, Valeria; Gibellini, Federica; Chen, Jichun; Young, Neal S.

    2009-01-01

    Objective To investigate the role of perforin-mediated cell apoptosis in murine models of immune-mediated bone marrow (BM) failure. Methods We compared C57BL/6J (B6) mice carrying a perforin gene deletion (Prf−/−) with wild type (WT) controls for cellular composition in lymphohematopoietic tissues. Lymph node (LN) cells from Prf−/− mice were co-incubated with BM cells from B10-H2b/LilMcdJ (C.B10) mice in an apoptosis assay in vitro. We then infused Prf−/− and WT B6 LN cells into sublethally-irradiated C.B10 and CByB6F1 recipients with mismatches at the minor- and major-histocompatibility loci, respectively, in order to induce BM failure. Cellular composition was analyzed by flow cytometry. Results Prf−/− mice showed normal lymphoid cell composition but Prf−/− LN cells had reduced ability to induce C.B10 BM cell apoptosis in vitro. Infusion of 5–10 × 106 Prf−/− LN cells produced obvious BM failure in C.B10 and CByB6F1 recipients; pancytopenia and BM hypocellularity were only slightly less severe than those caused by infusion of 5 × 106 WT B6 LN cells. Infused Prf−/− LN cells showed less T cell expansion, normal T cell activation, and higher proportions of T cells expressing gamma-interferon, tissue necrosis factor alpha and Fas ligand CD178, in comparison to infused WT B6 LN cells. Fas expression was equally high in residual BM cells in recipient of both Prf−/− and B6 LN cells. Conclusion Perforin deficiency alters T cell expansion but up-regulates T cell Fas ligand expression. Perforin-mediated cell death appears to play a minor role in mouse models of immune-mediated BM failure. PMID:19216020

  19. Role of extracellular signal-regulated kinase in regulating expression of interleukin 13 in lymphocytes from an asthmatic rat model

    Institute of Scientific and Technical Information of China (English)

    LI Yuan-yuan; LIU Xian-sheng; LIU Chang; XU Yong-jian; XIONG Wei-xing

    2010-01-01

    Background The extracellular signal-regulated kinase (ERK) is widely expressed in mammal cells and involved in airway proliferation and remodeling in asthma. In this study, we intend to explore the role of ERK in the expression of the Th2 cytokine, interleukin 13 (IL-13) in lymphocytes in asthma.Methods Forty Sprague-Dawley rats were randomly divided into two groups: normal control and asthmatic groups. Peripheral blood lymphocytes were isolated and purified from the blood of each rat and divided into five groups: control, asthmatic lymphocytes, asthmatic cells stimulated with ERK activator epidermal growth factor (EGF), or with ERK inhibitor PD98059, or with EGF and PD98059 together. The expression of phosphorylated-ERK (p-ERK) was observed by immunocvtochemical staining, the expression of ERK mRNA was determined by reverse transcriptase-PCR, IL-13 protein in supernatants was measured by ELISA.Results (1) The ERK mRNA level and the percentage of cells with p-ERK in lymphocytes from asthmatic rats were significantly higher than those in normal controls, and were significantly increased by EGF administration. This effect of EGF was significantly inhibited by PD98059 pretreatment. (2) IL-13 protein in supematants of asthmatic lymphocytes was higher than that produced by normal control lymphocytes, and was significantly increased by EGF treatment. This EGF effect was partly blocked by PD98059 pretreatment. (3) There was a significant positive correlation between the percentage of cells with p-ERK in peripheral blood lymphocytes and IL-13 protein in supematants of lymphocytes from asthmatic rats.Conclusions In asthma the ERK expression and activation levels were increased, as was the protein level of IL-13. The ERK signaling pathway may be involved in the increased expression of the Th2 cytokine IL-13 in asthma.

  20. Study of p53 protein expression levels from irradiated peripheral blood lymphocytes for biodosimetry

    Energy Technology Data Exchange (ETDEWEB)

    Cavalcanti, M.B.; Fernandes, T.S. [Universidade Federal de Pernambuco (UFPE), Recife, PE (Brazil). Dept. de Energia Nuclear; Amaral, A. [Universite Paris XII (UPXII) (France); Melo, J.A. [Centro de Radioterapia de Pernambuco (CERAPE), PE (Brazil); Neves, M.A.B.; Machado, C.G.F, E-mail: maribrayner@yahoo.com.br [Fundacao de Hematologia e Hemoterapia de Pernambuco, PE (Brazil)

    2005-07-01

    Biodosimetry can be defined as the investigation of radioinduced biological effects in order to correlate them with the absorbed dose. Scoring of unstable chromosomal aberrations and micronuclei, from in vitro irradiated peripheral blood lymphocytes, is commonly used for biodosimetry based on cytogenetic analysis. However, this method of analysis is time-consuming, which may represent a pitfall when fast investigation of a possible exposure to ionizing radiation (IR) is needed. The interaction of IR with the living cell can cause injuries in the DNA molecules. However, normal cells possess mechanisms of repair that are capable to correct those damages. During the repair process of the DNA various proteins are expressed. Among these proteins, p53 plays an important role. This protein is a transcription factor that helps in the maintenance of the genomic integrity. p53 protein is found into the cytoplasm in reduced concentrations and has a short average life. However, expression of p53 protein can be induced by DNA harmful radioinduced, which increases the concentration and the average life of this protein, making possible its detection. Thus, the correlation between the increasing of p53 expression and the irradiation may constitute a fast and reliable method of individual monitoring in cases of accidental or suspected exposures to IR. In this context, the objective of this research was to evaluate the p53 protein expression levels from lymphocytes of the human peripheral blood after in vitro irradiation. For this, samples of peripheral blood from healthy individuals were irradiated with known doses. Lymphocytes were separated on ficoll gradient by centrifugation and re-suspended at 1x 10{sub 6}/mL in RPMI medium enriched with fetal calf serum. Hence, lymphocytes were incubated in 5% CO{sub 2} at 37 deg C prior to the methodology of flow cytometry, using intranuclear antigens for the quantification of p53. In this report, the methodology performed and the results

  1. Ganoderma lucidum stimulates NK cell cytotoxicity by inducing NKG2D/NCR activation and secretion of perforin and granulysin.

    Science.gov (United States)

    Chang, Chih-Jung; Chen, Yi-Yuan M; Lu, Chia-Chen; Lin, Chuan-Sheng; Martel, Jan; Tsai, Sheng-Hui; Ko, Yun-Fei; Huang, Tsung-Teng; Ojcius, David M; Young, John D; Lai, Hsin-Chih

    2014-04-01

    Ganoderma lucidum (G. lucidum) is a medicinal mushroom long used in Asia as a folk remedy to promote health and longevity. Recent studies indicate that G. lucidum activates NK cells, but the molecular mechanism underlying this effect has not been studied so far. To address this question, we prepared a water extract of G. lucidum and examined its effect on NK cells. We observed that G. lucidum treatment increases NK cell cytotoxicity by stimulating secretion of perforin and granulysin. The mechanism of activation involves an increased expression of NKG2D and natural cytotoxicity receptors (NCRs), as well as increased phosphorylation of intracellular MAPKs. Our results indicate that G. lucidum induces NK cell cytotoxicity against various cancer cell lines by activating NKG2D/NCR receptors and MAPK signaling pathways, which together culminate in exocytosis of perforin and granulysin. These observations provide a cellular and molecular mechanism to account for the reported anticancer effects of G. lucidum extracts in humans.

  2. Increasing a Robust Antigen-Specific Cytotoxic T Lymphocyte Response by FMDV DNA Vaccination with IL-9 Expressing Construct

    Directory of Open Access Journals (Sweden)

    Qiang Zou

    2010-01-01

    Full Text Available Various chemokines and cytokines as adjuvants can be used to improve efficacy of DNA vaccination. In this study, we sought to investigate if a DNA construct expressing IL-9 (designed as proV-IL9 as a molecular adjuvant enhance antigen specific immune responses elicited by the pcD-VP1 DNA vaccination. Mice immunized with pcD-VP1 combined with proV-IL9 developed a strong humoral response. In addition, the coinoculation induced significant higher level of antigen-specific cell proliferation and cytotoxic response. This agreed well with higher expression level of IFN-γ and perforin in CD8+ T cells, but not with IL-17 in these T cells. The results indicate that IL-9 induces the development of IFN-γ-producing CD8+ T cells (Tc1, but not the IL-17-producing CD8+ T cells (Tc17. Up-regulated expressions of BCL-2 and BCL-XL were exhibited in these Tc1 cells, suggesting that IL-9 may trigger antiapoptosis mechanism in these cells. Together, these results demonstrated that IL-9 used as molecular adjuvant could enhance the immunogenicity of DNA vaccination, in augmenting humoral and cellular responses and particularly promoting Tc1 activations. Thus, the IL-9 may be utilized as a potent Tc1 adjuvant for DNA vaccines.

  3. Metallothionein 1 Isoform Gene Expression Induced by Cadmium in Human Peripheral Blood Lymphocytes

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    To study the gene expression of metallothionein 1 (MT-1) isoforms in human peripheral blood lymphocytes (HPBLs). Methods The expression of mRNA representing the seven active MT-1 genes was determined in HPBLs by quantitative RT-PCR before and after exposure to cadmium. Results Basal expressions of MT-1X, and MT-1A in HPBLs were similar to expression of housekeeping gene. In contrast, the basal gene expressions of MT-1H, 1F, 1E, and 1G were a little transcripts in human HPBLs. No signal was detected for MT-1B. There was a sex difference (P<0.05). in basal gene expression of MT-1E. The levels of gene expression of MT-1A, 1E, 1F, 1G, 1H, and 1X increased, but the level of MT-1B did not increase after exposure to cadmium. Conclusions Gene expressions of MT-1G, MT-1H, MT-1F, and MT-1X in HPBLs can be used as a potential biomarker of cadmium exposure.

  4. The Prognostic Value of CD49d Expression in Turkish Patients with Chronic Lymphocytic Leukemia

    Directory of Open Access Journals (Sweden)

    Ant Uzay

    2012-12-01

    Full Text Available OBJECTIVE: The aim of this study was to assess the prognostic value of CD49d expression in Turkish chronic lymphocytic leukemia patients. METHODS: Data for 118 patients from a single center were evaluated. In all, the study included 73 patients for whom complete clinical follow-up data, and flow cytometry test results for CD5/19, CD23/43, CD38, Zap- 70, Kappa, and Lambda light chains, and CD49d were available. The effect of the level of CD49d expression on overall survival (OS and time to treatment (TTT was investigated retrospectively. RESULTS: Patients with high CD49d expression (≥30% had more advanced disease at the time of diagnosis (median Rai stage 3 vs. Rai stage 1, P = 0.03. Patients resistant to treatment had higher CD49d expression than patients that responded to treatment (mean CD49d expression of 58% vs. 46%, P = 0.08. The level of CD49d expression was not associated with OS or TTT. CONCLUSION: The study’s findings show that the patients with high CD49d expression at the time of diagnosis had more advanced disease and poorer response to therapy; however, their overall survival did not differ from that of the patients with advanced disease stage, but lower levels of CD49d expression.

  5. Changes in the Expression of Transcription Factors Involved in Modulating the Expression of EPO-R in Activated Human CD4-Positive Lymphocytes.

    Science.gov (United States)

    Lisowska, Katarzyna A; Frackowiak, Joanna E; Mikosik, Anna; Witkowski, Jacek M

    2013-01-01

    We have recently described the presence of the erythropoietin receptor (EPO-R) on CD4(+) lymphocytes and demonstrated that its expression increases during their activation, reaching a level reported to be typical for erythroid progenitors. This observation suggests that EPO-R expression is modulated during lymphocyte activation, which may be important for the cells' function. Here we investigated whether the expression of GATA1, GATA3 and Sp1 transcription factors is correlated with the expression of EPO-R in human CD4(+) lymphocytes stimulated with monoclonal anti-CD3 antibody. The expression of GATA1, GATA3 and Sp1 transcription factors in CD4(+) cells was estimated before and after stimulation with anti-CD3 antibody by Western Blot and flow cytometry. The expression of EPO-R was measured using real-time PCR and flow cytometry. There was no change in the expression of GATA1 and GATA3 in CD4(+) lymphocytes after stimulation with anti-CD3 antibody. However, stimulation resulted in the significantly increased expression of the Sp1 factor. CD4(+) lymphocytes stimulated with anti-CD3 antibody exhibited an increase in both the expression level of EPOR gene and the number of EPO-R molecules on the cells' surface, the latter being significantly correlated with the increased expression of Sp1. Sp1 is noted to be the single transcription factor among the ones studied whose level changes as a result of CD4(+) lymphocyte stimulation. It seems that Sp1 may significantly affect the number of EPO-R molecules present on the surface of activated CD4(+) lymphocytes.

  6. 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.

  7. Inhibiting the expression of CD28 costimulatory molecule on human lymphocytes by special siRNA

    Institute of Scientific and Technical Information of China (English)

    XU Kai-lin; ZHANG Ying; PAN Xiu-ying; LU Qun-xian

    2005-01-01

    Background The B7/CD28 pathway provides critical costimulatory signals for complete T cell activation, and members of this pathway have served as useful targets for immunotherapeutic strategies. In this study, we investigated the RNA interference (RNAi) effect induced by small interfering RNA (siRNA) targeting CD28 mRNA on human lymphocytes and its specificity.Methods According to CD28 gene sequence, we designed and synthysized three different siRNAs (siRNA-1, siRNA-2, siRNA-3) containing 21 bases using SilencerTM siRNA construction kit. These siRNAs were transfected into freshly isolated human lymphocytes with Lipofectamine 2000 reagent. At 24-hour, 48-hour and 72-hour post transfection, these cells were collected and analyzed. The changes of surface expression of CD28 gene were detected by flow cytometry, and the changes of CD28 mRNA levels were determined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The cell viability of transfected lymphocytes was determined by methyl thiazolyl tetrazolium (MTT) assay and trypan blue dye exclusion assay. Results Three siRNAs (siRNA-1, siRNA-2, siRNA-3) specifically targeting CD28 mRNA were successfully designed and constructed. Flow cytometry analysis showed that a decrease in CD28 expression was detectable at 24-hour post transfection. Different siRNA showed different inhibition effects on CD28 expression. At 48-hour post transfection, the degrees of reduction with siRNA-1, siRNA-2 and siRNA-3 were 22.10%±1.63%, 73.50%±1.02% and 42.90%±0.89% respectively compared with the control (P0.05). Moreover, lymphocytes treated with siRNA-co showed no marked reduction in CD28 expression (5.07%±0.96%) (P>0.05). The results of semi-quantitative RT-PCR assay indicated CD28 mRNA level was inhibited after transfection of specific siRNAs. At least 4-fold of reduction in siRNA-2 group occurred at 48-hour post transfection compared with the control (P0.05). Conclusions Three different siRNAs were

  8. Abnormal adenosine and dopamine receptor expression in lymphocytes of Lesch-Nyhan patients.

    Science.gov (United States)

    García, M G; Puig, J G; Torres, R J

    2009-11-01

    Self-injurious behavior is the most outstanding feature of Lesch-Nyhan syndrome and has recently been ascribed to an obsessive-compulsive behavior. Lesch-Nyhan syndrome results from the complete enzyme deficiency of hypoxanthine-guanine phosphoribosyl transferase (HPRT) but the link between abnormal purine metabolism and its neurological and behavioral manifestations remains largely unknown. Previous studies led us to hypothesize that adenosine and dopamine receptor expression could be altered in HPRT-deficient cells. To test this hypothesis, we examined mRNA expressions of adenosine (ADORA2A and ADORA2B) and dopamine receptors (DRD1 and DRD2 like), and dopamine transporter (DAT1) in peripheral blood lymphocytes (PBLs) from Lesch-Nyhan patients. We also examined the influence of hypoxanthine in these expressions. As compared to normal PBLs, both ADORA2A and DRD5 expression were abnormal in PBLs from Lesch-Nyhan patients. In contrast, DAT1 expression was similar to control values in HPRT deficient PBLs. These results indicate an abnormal adenosine and dopamine receptor expression in HPRT-deficient cells and suggest disrupted adenosine and dopamine neurotransmission may have a significant role in the pathogenesis of the neurological manifestations of Lesch-Nyhan syndrome.

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

    Science.gov (United States)

    Bausinger, Julia; Speit, Günter

    2015-11-01

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

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

    Science.gov (United States)

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

    2000-01-01

    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

  11. Deregulated expression of circadian clock and clock-controlled cell cycle genes in chronic lymphocytic leukemia.

    Science.gov (United States)

    Rana, Sobia; Munawar, Mustafa; Shahid, Adeela; Malik, Meera; Ullah, Hafeez; Fatima, Warda; Mohsin, Shahida; Mahmood, Saqib

    2014-01-01

    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.

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

    Science.gov (United States)

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

    2008-01-01

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

  13. Association between lymphocyte expression of the apoptotic receptor Fas and pain in critically ill patients

    Science.gov (United States)

    Papathanassoglou, Elizabeth DE; Mpouzika, Meropi DA; Giannakopoulou, Margarita; Bozas, Evangelos; Middleton, Nicos; Tsiaousis, George; Karabinis, Andreas

    2017-01-01

    Objective Lymphocyte apoptosis in critical illness is associated with immunosuppression. We explored for the first time the associations between pain ratings and expression of the apoptotic receptor Fas on B and T cells in critically ill patients and the potential mediating effects of adrenocorticotropic hormone (ACTH), cortisol, and substance P (SP). Design This is an exploratory correlational study with repeated measurements (14 days followup) and cross-sectional comparisons. Setting This study was conducted in a state hospital in the metropolitan area of Athens, Greece. Participants The participants were 36 consecutive critically ill patients and 36 matched controls. Outcome measures Pain measured by the self-reported numeric rating scale [NRS], the behavioral pain scale, and the pain assessment scale was the primary outcome measure. Flow cytometry (Fas), electrochemiluminescence (ACTH and cortisol) and enzyme-linked immunosorbent assay (SP) were used. Mixed linear models for repeated measurements and bivariable associations at discrete time points were employed. Results Significant pain at rest was noted. Pain ratings associated with Fas expression on cytotoxic T cells (P=0.041) and B cells (P=0.005), even after adjustment for a number of clinical treatment factors (P=0.006 and P=0.052, respectively). On the day that more patients were able to communicate, Fas on B cells (r=0.897, P=0.029) and cytotoxic T cells (r=0.832; P=0.037) associated with NRS ratings. Associations between pain ratings and ACTH serum levels were noted (P<0.05). When stress neuropeptide levels were added to the model, the statistical significance of the associations between pain ratings and Fas expression was attenuated (P=0.052–0.063), suggesting that stress neuropeptides may partially mediate the association. Conclusion Preliminary evidence for the association between pain and lymphocyte apoptotic susceptibility is provided. The role of pain management in maintaining immunocompetence

  14. Formin like 1 expression is increased on CD4+ T lymphocytes in spontaneous autoimmune uveitis.

    Science.gov (United States)

    Degroote, Roxane L; Uhl, Patrizia B; Amann, Barbara; Krackhardt, Angela M; Ueffing, Marius; Hauck, Stefanie M; Deeg, Cornelia A

    2017-02-10

    The membrane protein expression repertoire of cells changes in course of activation. In equine recurrent uveitis (ERU), a spontaneous autoimmune disease in horses with relapsing and ultimately blinding inner eye inflammation, CD4+ T lymphocytes are the crucial pathogenic cells activated in the periphery directly prior to an inflammatory episode. In order to find relevant changes in the membrane proteome associated to disease, we sorted CD4+ lymphocytes and compared protein abundance from the generated proteome datasets of both healthy horses and ERU cases. We detected formin like 1, a key player in actin dependent cellular processes such as phagocytosis, cell adhesion and cell migration, with significantly higher abundance in the CD4+ cell membrane proteome of horses with ERU. In transmigration experiments, we demonstrated higher migration rate of cells originating from diseased animals connecting formin like 1 to the migratory ability of cells. These findings are the first description of formin like 1 in association to processes involved in migration of inflammatory CD4+ T cells across the blood-retinal barrier in a spontaneous ocular autoimmune disease and suggest formin like 1 to play a role in the molecular mechanisms of ERU disease pathogenesis. Data are available via ProteomeXchange with identifier PXD005384.

  15. Association between lymphocyte expression of the apoptotic receptor Fas and pain in critically ill patients

    Directory of Open Access Journals (Sweden)

    Papathanassoglou EDE

    2017-01-01

    Full Text Available Elizabeth DE Papathanassoglou,1,* Meropi DA Mpouzika,2,* Margarita Giannakopoulou,3 Evangelos Bozas,3 Nicos Middleton,2 George Tsiaousis,2 Andreas Karabinis4,5 1Faculty of Nursing, University of Alberta, Edmonton, AB, Canada; 2Department of Nursing, Cyprus University of Technology, Limassol, Cyprus; 3Department of Nursing, School of Health Sciences, National and Kapodistrian University of Athens, 4Surgical Care Unit, The Onassis Cardiac Surgery Center, Kallithea, 5School of Medicine, National and Kapodistrian University of Athens, Athens, Greece *These authors contributed equally to this work Objective: Lymphocyte apoptosis in critical illness is associated with immunosuppression. We explored for the first time the associations between pain ratings and expression of the apoptotic receptor Fas on B and T cells in critically ill patients and the potential mediating effects of adrenocorticotropic hormone (ACTH, cortisol, and substance P (SP.Design: This is an exploratory correlational study with repeated measurements (14 days follow-up and cross-sectional comparisons.Setting: This study was conducted in a state hospital in the metropolitan area of Athens, Greece.Participants: The participants were 36 consecutive critically ill patients and 36 matched controls.Outcome measures: Pain measured by the self-reported numeric rating scale [NRS], the behavioral pain scale, and the pain assessment scale was the primary outcome measure. Flow cytometry (Fas, electrochemiluminescence (ACTH and cortisol and enzyme-linked immunosorbent assay (SP were used. Mixed linear models for repeated measurements and bivariable associations at discrete time points were employed.Results: Significant pain at rest was noted. Pain ratings associated with Fas expression on cytotoxic T cells (P=0.041 and B cells (P=0.005, even after adjustment for a number of clinical treatment factors (P=0.006 and P=0.052, respectively. On the day that more patients were able to communicate, Fas

  16. B-cell differentiation in the chicken: expression of immunoglobulin genes in the bursal and peripheral lymphocytes.

    Science.gov (United States)

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

    1989-03-01

    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.

  17. Cytokine and adhesion molecule expression evolves between the neutrophilic and lymphocytic phases of viral meningitis.

    Science.gov (United States)

    Makis, Alexandros; Shipway, David; Hatzimichael, Eleftheria; Galanakis, Emmanouil; Pshezhetskiy, Dmitry; Chaliasos, Nikolaos; Stebbing, Justin; Siamopoulou, Antigone

    2010-09-01

    Viral meningitis is characterized by cerebrospinal fluid (CSF) lymphocyte pleocytosis, although neutrophils may predominate in the early phase. The T helper 1 (Th1)/Th2 cytokine balance and expression of adhesion molecules seem to be involved in the CSF chemotaxis. We aimed to determine expression of cytokines and adhesion molecules in enteroviral meningitis. We investigated the serum and CSF levels of adhesion molecules (E-selectin, L-selectin, vascular cell adhesion molecule-1 [VCAM-1], and intracellular adhesion molecule-1 [ICAM-1]) and cytokines (interleukin-12 [IL-12] and IL-4) in 105 children during an outbreak of enteroviral meningitis. Diagnosis was confirmed with positive polymerase chain reaction (PCR) and/or serology for echovirus or Coxsackie virus, and matched with control subjects for clinical features but with negative PCR and/or serology. Apart from VCAM-1, the CSF levels of all investigated inflammatory molecules were significantly increased. In serum, sL-selectin and ICAM-1 levels were significantly higher than control subjects. Serum and CSF L-selectin, serum VCAM-1, and CSF IL-12 were all observed to be expressed in significantly higher levels in the neutrophil-dominant subgroup (72% had duration of symptoms 24 h). Serum and CSF ICAM-1 was found at significantly higher levels in the latter group. Evolving expression of adhesion molecules and cytokines indicates a shift from Th1 to Th2 immune responses as infection progresses.

  18. mRNA expression of dopamine receptors in peripheral blood lymphocytes of computer game addicts.

    Science.gov (United States)

    Vousooghi, Nasim; Zarei, Seyed Zeinolabedin; Sadat-Shirazi, Mitra-Sadat; Eghbali, Fatemeh; Zarrindast, Mohammad Reza

    2015-10-01

    Excessive playing of computer games like some other behaviors could lead to addiction. Addictive behaviors may induce their reinforcing effects through stimulation of the brain dopaminergic mesolimbic pathway. The status of dopamine receptors in the brain may be parallel to their homologous receptors in peripheral blood lymphocytes (PBLs). Here, we have investigated the mRNA expression of dopamine D3, D4 and D5 receptors in PBLs of computer game addicts (n = 20) in comparison to normal subjects (n = 20), using a real-time PCR method. The results showed that the expression level of D3 and D4 dopamine receptors in computer game addicts were not statistically different from the control group. However, the expression of the mRNA of D5 dopamine receptor was significantly down-regulated in PBLs of computer game addicts and reached 0.42 the amount of the control group. It is concluded that unlike with drug addiction, the expression levels of the D3 and D4 dopamine receptors in computer game addicts are not altered compared to the control group. However, reduced level of the D5 dopamine receptor in computer game addicts may serve as a peripheral marker in studies where the confounding effects of abused drugs are unwanted.

  19. CD 13/APN expression in peripheral blood lymphocytes and skin lesions in patients with advanced psoriasis vulgaris

    Institute of Scientific and Technical Information of China (English)

    Liu Taihua; Liu Defang; Chen Yihua; Hu Zonghai; Chen Lu; Luo Chen; Xu Zhejuan

    2009-01-01

    Objective: To observe the expression of CDI3/APN in peripheral blood lymphocytes and skin lesions of patients with advanced psoriasis vulgaris, and discuss its effect on the pathogenesis of psoriasis. Methods: CDI 3 expression in peripheral blood lymphocytes and skin lesions was detected by flow cytometry and immunohistochemical technique, respectively. Results were compared with those of healthy controls. Results: CD13 expression was significantly higher in peripheral blood lymphocytes of patients with advanced psoriasis vulgaris than in that of healthy controls, and in skin lesions than in healthy skin tissues. The expression was mainly in the suprabasal layers of skin lesions, andpositively correlated to PASI (R=0.78029). Conclusion: The significantly higher expression of CDI3 in peripheralblood lymphocytes and skin lesions of the patients with advanced psoriasis vulgaris probably is related to immunological abnormality, blood vessel abnormality and proliferation of keratinocyte in the pathogenic course of psoriasis. It may be a novel and effective way to treat psoriasis with specific CD13 inhibitors.

  20. Accumulation of CD5(+)CD19(+) B lymphocytes expressing PD-1 and PD-1L in hypertrophied pharyngeal tonsils.

    Science.gov (United States)

    Wlasiuk, Paulina; Niedzielski, Artur; Skorka, Katarzyna; Karczmarczyk, Agnieszka; Zaleska, Joanna; Zajac, Malgorzata; Putowski, Maciej; Pac-Kozuchowska, Elzbieta; Giannopoulos, Krzysztof

    2016-11-01

    Programmed death-1 (PD-1) is one of the most important inhibitory co-receptors expressed predominantly on activated T and B lymphocytes whose expression could be sustained by permanent antigenic stimulation accompanying chronic or recurrent tonsillitis. The expression of PD-1 and PD-1L was analyzed using flow cytometry on hypertrophied tonsils collected from 57 children. We observed high expression of PD-1 and PD-1L on certain lymphocytes subpopulations of hypertrophied tonsils; among T cells, the expression of PD-1 on protein level was higher on CD4(+) cells (70.3 %) than on CD8(+) cells (35 %). Interestingly, a limited expression of PD-1 was observed on CD19(+) B lymphocytes (6.5 %), while CD5(+)CD19(+) B cells overexpressed PD-1 (52.5 %). Moreover, the expression of PD-1L was also higher on CD5(+)CD19(+) B cells (16.5 %) than on CD19(+) B cells (3.5 %) and on CD4(+) T cells (20 %) than on CD8(+) T cells (10 %). PD-1 and PD-1L expressions correlated only on CD5(+)CD19(+) cells. In conclusion, high expression of PD-1 and PD-1L on T and B cells could represent hallmark of immune system adaptation to chronic antigenic exposition in patients with tonsillitis.

  1. Expression of the chemokine receptor CXCR4 on lymphocytes of leprosy patients

    Directory of Open Access Journals (Sweden)

    V.A. Mendonça

    2011-12-01

    Full Text Available Leprosy is caused by Mycobacterium leprae, which induces chronic granulomatous infection of the skin and peripheral nerves. The disease ranges from the tuberculoid to the lepromatous forms, depending on the cellular immune response of the host. Chemokines are thought to be involved in the immunopathogenesis of leprosy, but few studies have investigated the expression of chemokine receptors on leukocytes of leprosy patients. In the present study, we evaluated 21 leprosy patients (M/F: 16/5 with a new diagnosis from the Dermatology Outpatient Clinic of the University Hospital, Federal University of Minas Gerais. The control group was composed of 20 healthy members (M/F: 15/5 of the community recruited by means of announcements. The expression of CCR2, CCR3, CCR5, and CXCR4 was investigated by flow cytometry on the surface of peripheral blood lymphocytes. There was a decrease in percentage of CD3+CXCR4+ and CD4+CXCR4+ lymphocytes in the peripheral blood of leprosy patients (median [range], 17.6 [2.7-41.9] and 65.3 [3.9-91.9], respectively compared to the control group (median [range], 43.0 [3.7-61.3] and 77.2 [43.6-93.5], respectively. The percentage of CD4+CXCR4+ was significantly lower in patients with the tuberculoid form (median [range], 45.7 [0.0-83.1] of the disease, but not in lepromatous patients (median [range], 81.5 [44.9-91.9]. The CXCR4 chemokine receptor may play a role in leprosy immunopathogenesis, probably directing cell migration to tissue lesions in tuberculoid leprosy patients.

  2. NPM-ALK and the JunB transcription factor regulate the expression of cytotoxic molecules in ALK-positive, anaplastic large cell lymphoma.

    Science.gov (United States)

    Pearson, Joel D; Lee, Jason K H; Bacani, Julinor T C; Lai, Raymond; Ingham, Robert J

    2011-01-30

    Anaplastic lymphoma kinase-positive, anaplastic large cell lymphoma (ALK+ ALCL) is an aggressive non-Hodgkin lymphoma of T/null immunophenotype that is most prevalent in children and young adults. The normal cellular counterpart of this malignancy is presumed to be the cytotoxic T lymphocyte (CTL), and this presumption is partly based on the observation that these tumour cells often express cytotoxic granules containing Granzyme B (GzB) and Perforin. Chromosomal translocations involving the gene encoding for the ALK tyrosine kinase are also characteristic of ALK+ ALCL, and the resulting fusion proteins (e.g. NPM-ALK) initiate signalling events important in ALK+ ALCL pathogenesis. These events include the elevated expression of JunB; an AP-1 family transcription factor that promotes ALK+ ALCL proliferation. In this report we demonstrate that JunB is a direct transcriptional activator of GzB and that GzB transcription is also promoted by NPM-ALK. We found that Perforin expression was not regulated by JunB, but was promoted by NPM-ALK in some cell lines and inhibited by it in others. In conclusion, our study makes the novel observation that signalling through NPM-ALK and JunB affect the expression of cytotoxic molecules in ALK+ ALCL. Moreover, these findings demonstrate the expression of GzB and Perforin in this lymphoma is not solely due its presumed CTL origin, but that oncogenic signalling is actively influencing the expression of these proteins.

  3. T cell receptor transgenic lymphocytes infiltrating murine tumors are not induced to express foxp3

    Directory of Open Access Journals (Sweden)

    Quatromoni Jon G

    2011-11-01

    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.

  4. Reduced frequency of T lymphocytes expressing CTLA-4 in frontotemporal dementia compared to Alzheimer's disease.

    Science.gov (United States)

    Santos, Rodrigo Ribeiro; Torres, Karen C; Lima, Giselle S; Fiamoncini, Carolina M; Mapa, Filipe C; Pereira, Patricia A; Rezende, Vitor B; Martins, Luiza C; Bicalho, Maria A; Moraes, Edgar N; Reis, Helton J; Teixeira, Antonio L; Romano-Silva, Marco A

    2014-01-03

    Studies suggest that inflammation is involved in the neurodegenerative cascade of dementias. Immunological mechanisms may be part of the pathophysiological process in frontotemporal dementia (FTD), but up till now only vague evidence of such mechanisms has been presented. The B7- CD28/CTLA-4 pathway is an important immunological signaling pathway involved in modulation of T cell activation. The aim of this study was to compare the expression of molecules associated with co-stimulatory signaling in peripheral blood mononuclear cells (PBMC) of FTD to Alzheimer disease (AD) and control groups. Our results confirm the previous demonstrated increased expression of CD80 in CD14+ Alzheimer patients T cells but show, for the first time, a reduction in the expression of CTLA-4 in CD4+ FTD cells. As CTLA-4 is the most potent negative regulators of T-cell activation we speculated that peripheral T lymphocytes in FTD are more activated and this could be involved in the neurodegeneration observed in this dementia.

  5. Cytokine Contents in Chronic Lymphocytic Leukemia: Association with ZAP70 Expression

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    Nilgün Işıksaçan

    2016-08-01

    Full Text Available Objective: Chronic lymphocytic leukemia (CLL is a disease that shows varying clinical progression, and expression of the protein tyrosine kinase ZAP70 has been described as a very valuable prognostic factor. Patients with ZAP70 positivity are characterized by worse clinical course and significantly shorter progression-free and overall survival. In this study, intracytoplasmic interferon gamma (IFN-γ and interleukin-4 (IL-4 content of T, B, and CLL cells in CLL patients and their correlations with Rai staging and ZAP70 positivity were investigated. Materials and Methods: CLL patients newly diagnosed or in followup at the İstanbul University İstanbul Medical Faculty Hematology Department were included in this study. These patients were classified according to Rai staging and ZAP70 expression. IL-4, IFN-γ, and ZAP70 expressions in peripheral blood T, B, and CLL cells were measured by four-color flow cytometry. Results: There was a statistically significant correlation between advanced disease and ZAP70 positivity. IL-4-secreting T cells were significantly increased; however, IFN-γ secretion was significantly decreased in CLL patients compared to healthy individuals, whereas IL-4-secreting B cells were significantly diminished in contrast to T cells. Conclusion: These findings suggest damage in the cellular immunity and that IL-4 might lead to many complications and may be important in disease progression.

  6. Calpains Released by T Lymphocytes Cleave TLR2 To Control IL-17 Expression.

    Science.gov (United States)

    Perez, Joëlle; Dansou, Boris; Hervé, Roxane; Levi, Charlène; Tamouza, Houda; Vandermeersch, Sophie; Demey-Thomas, Emmanuelle; Haymann, Jean-Philippe; Zafrani, Lara; Klatzmann, David; Boissier, Marie-Christophe; Letavernier, Emmanuel; Baud, Laurent

    2016-01-01

    Calpains are intracellular proteases that play a key role in inflammation/immunity. Rare studies show that they are partially externalized. However, the mechanism of this secretion and the functions of exteriorized calpains remain poorly understood. In this study, we found that mouse and human lymphocytes secreted calpains through an ABCA1-driven process. In turn, extracellular calpains inhibited IL-17A expression. We were able to attribute this function to a cleavage of the TLR2 extracellular domain, which prevented TLR2-induced transcription of molecules essential for IL-17A induction. Calpain exteriorization and TLR2 cleavage were critical for the control of IL-17A expression by low doses of IL-2. By using newly developed transgenic mice in which extracellular calpains are specifically inactivated, we provide evidence for the relevance of calpain externalization in vivo in regulating IL-17A expression and function in experimental sterile peritonitis and autoimmune arthritis, respectively. Thus, this study identifies calpain exteriorization as a potential target for immune modulation.

  7. Aberrant microRNA expression in Chinese patients with chronic lymphocytic leukemia.

    Science.gov (United States)

    Zhu, Dan-Xia; Miao, Kou-Rong; Fang, Cheng; Fan, Lei; Zhu, Wei; Zhu, Hua-Yuan; Zhuang, Yun; Hong, Ming; Liu, Peng; Xu, Wei; Li, Jian-Yong

    2011-06-01

    MicroRNAs (miRNAs) are a class of small endogenous RNAs that play important regulatory roles by targeting mRNAs for cleavage or translational repression. Many reports have indicated that miRNAs play a critical role in malignancies, and regulations in the progression of leukemia. However, the miRNAs expression level in Chinese patients with chronic lymphocytic leukemia (CLL), and its prognostic value remain elusive. We identified various degrees of down-regulation of miR-15a, miR-16-1, miR-29b, miR-181a and miR-181b in CLL mononuclear cells. Moreover, we have identified miR-29b and miR-181a/b expression significantly correlated with IGHV mutational status. Transcript levels of predicted target genes BCL-2 and TCL-1 were also determined, and the expression levels were significantly upregulated in CLL patients compared with normal controls (PmiR-181b) and BCL-2 level; furthermore, an inverse correlation was also observed between miRNAs (miR-16-1, miR-181a, miR-181b) and TCL-1, which suggest that these miRNAs may implicate in negatively regulating target mRNA at transcriptional level. These different miRNAs may play an important role in the pathogenesis of CLL and might be applied for the assessment of prognosis in patients with CLL.

  8. Corticosteroids decrease the expression of beta 2-microglobulin and histocompatibility antigens on human peripheral blood lymphocytes in vitro

    DEFF Research Database (Denmark)

    Hokland, M; Larsen, B; Heron, I

    1982-01-01

    . Both antigens were found to be decreased, dexamethasone typically in a concentration of 10-6 mol/l causing a decrease in surface beta 2-microglobulin of 15% after an incubation period of 24 hr. The expression of two other lymphocyte surface antigens, Igm and Thy antigens, measured in parallel with beta...

  9. EXPRESSION OF SURFACE MARKERS ON CD4+T-LYMPHOCYTES IN NORMAL PREGNANCY AND PRE-ECLAMPSIA

    Directory of Open Access Journals (Sweden)

    V. A. Mikhailova

    2012-01-01

    Full Text Available Abstract. In course of physiological pregnancy, peripheral blood CD4+T-lymphocytes normally migrate to the uterine decidual tissue. Hence, this study aimed to compare expression profiles of adhesion molecules and chemokine receptors on the surface of CD4+T-lymphocytes from peripheral blood in healthy pregnancy and in pre-eclampsia. The amounts of CD4+T-lymphocytes expressing CD184, CD119, CD192, CD197, β7 integrin, CD29, CD49d, CD11b were elevated in healthy pregnant women, as compared with non-pregnant women. The amounts of CD4+T-lymphocytes expressing CD49d, CD44, CD47, as well as intensity of CD47, CD29, CD49d, CD44, Integrin β7, CD54 expression proved to be decreased in cases of preeclampsia when compared with healthy pregnancy. This work was supported by grants ГК N 02.740.11.0711 from Russian Ministry of Education and Science, and Presidential grants НШ-3594.2010.7, МД-150.2011.7.

  10. Cooling of heat-stressed cows during the dry period alters lymphocyte but not mammary gland gene expression

    Science.gov (United States)

    Heat stress (HT) during the dry period compromises mammary gland development, decreases future milk production, and impairs immune status of dairy cows. Our objective was to evaluate the effects of cooling heat-stressed cows during the dry period on gene expression of the mammary gland and lymphocyt...

  11. Neoplastic transformation of T lymphocytes through transgenic expression of a virus host modification protein.

    Directory of Open Access Journals (Sweden)

    Sílvia Cristina Paiva Almeida

    Full Text Available Virus host evasion genes are ready-made tools for gene manipulation and therapy. In this work we have assessed the impact in vivo of the evasion gene A238L of the African Swine Fever Virus, a gene which inhibits transcription mediated by both NF-κB and NFAT. The A238L gene has been selectively expressed in mouse T lymphocytes using tissue specific promoter, enhancer and locus control region sequences for CD2. The resulting two independently derived transgenic mice expressed the transgene and developed a metastasic, angiogenic and transplantable CD4(+CD8(+CD69(- lymphoma. The CD4(+CD8(+CD69(- cells also grew vigorously in vitro. The absence of CD69 from the tumour cells suggests that they were derived from T cells at a stage prior to positive selection. In contrast, transgenic mice similarly expressing a mutant A238L, solely inhibiting transcription mediated by NF-κB, were indistinguishable from wild type mice. Expression of Rag1, Rag2, TCRβ-V8.2, CD25, FoxP3, Bcl3, Bcl2 l14, Myc, IL-2, NFAT1 and Itk, by purified CD4(+CD8(+CD69(- thymocytes from A238L transgenic mice was consistent with the phenotype. Similarly evaluated expression profiles of CD4(+CD8(+ CD69(- thymocytes from the mutant A238L transgenic mice were comparable to those of wild type mice. These features, together with the demonstration of (mono-oligoclonality, suggest a transgene-NFAT-dependent transformation yielding a lymphoma with a phenotype reminiscent of some acute lymphoblastic lymphomas.

  12. Variations of the perforin gene in patients with chronic inflammatory demyelinating polyradiculoneuropathy.

    Science.gov (United States)

    Buttini, S; Cappellano, G; Ripellino, P; Briani, C; Cocito, D; Osio, M; Cantello, R; Dianzani, U; Comi, C

    2015-01-01

    Perforin (PRF) has a key role in the function of cytotoxic T and natural killer cells. Rare variations of PRF1 predispose to autoimmunity. Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is an autoimmune disease of the peripheral nervous system, involving defective lymphocyte apoptosis. The aim of this study was to investigate the role of PRF1 in CIDP. The entire coding region of PRF1 was sequenced in 94 patients and 158 controls. We found three missense variations leading to amino acid substitutions and one nonsense variation resulting in a premature stop codon. All variations would decrease PRF activity. Their overall frequency was significantly higher in patients than in controls (odds ratio (OR)=4.47). The most frequent variation was p.Ala91Val (OR=3.92) previously associated with other autoimmune diseases. Clinical analysis showed that PRF1 variations were more frequent in relapsing patients and in patients displaying axonal damage. These data suggest that PRF1 variations may influence CIDP development and course.

  13. Primary Tr1 cells from metastatic melanoma eliminate tumor-promoting macrophages through granzyme B- and perforin-dependent mechanisms.

    Science.gov (United States)

    Yan, Hongxia; Zhang, Ping; Kong, Xue; Hou, Xianglian; Zhao, Li; Li, Tianhang; Yuan, Xiaozhou; Fu, Hongjun

    2017-04-01

    In malignant melanoma, tumor-associated macrophages play multiple roles in promoting tumor growth, such as inducing the transformation of melanocytes under ultraviolet irradiation, increasing angiogenesis in melanomas, and suppressing antitumor immunity. Because granzyme B- and perforin-expressing Tr1 cells could specifically eliminate antigen-presenting cells of myeloid origin, we examined whether Tr1 cells in melanoma could eliminate tumor-promoting macrophages and how the interaction between Tr1 cells and macrophages could affect the growth of melanoma cells. Tr1 cells were characterized by high interleukin 10 secretion and low Foxp3 expression and were enriched in the CD4(+)CD49b(+)LAG-3(+) T-cell fraction. Macrophages derived from peripheral blood monocytes in the presence of modified melanoma-conditioned media demonstrated tumor-promoting capacity, exemplified by improving the proliferation of cocultured A375 malignant melanoma cells. But when primary Tr1 cells were present in the macrophage-A375 coculture, the growth of A375 cells was abrogated. The conventional CD25(+) Treg cells, however, were unable to inhibit macrophage-mediated increase in tumor cell growth. Further analyses showed that Tr1 cells did not directly eliminate A375 cells, but mediated the killing of tumor-promoting macrophages through the secretion of granzyme B and perforin. The tumor-infiltrating interleukin 10(+)Foxp3(-)CD4(+) T cells expressed very low levels of granzyme B and perforin, possibly suggested the downregulation of Tr1 cytotoxic capacity in melanoma tumors. Together, these data demonstrated an antitumor function of Tr1 cells through the elimination of tumor-promoting macrophages, which was not shared by conventional Tregs.

  14. Protection Against Lung Cancer Patient Plasma-Induced Lymphocyte Suppression by Ganoderma Lucidum Polysaccharides

    Directory of Open Access Journals (Sweden)

    Li-Xin Sun

    2014-01-01

    Full Text Available Background/Aims: This study was conducted to determine the potential of Ganoderma lucidum polysaccharides (Gl-PS in protection against lung cancer patient plasma-induced suppression of lymphocytes. Lung cancer is a major cause of disease and loss of life in the United States and worldwide. Cancer cells release immunosuppressive mediators, such as PGE2, TGF-β, IL-10, and VEGF, to inhibit the immune response to escape from immune surveillance. Gl-PS has been shown to counteract this immune inhibition in an animal cell culture model, and thus to facilitate tumor control. The present study explored whether or not such an effect could also be demonstrated in human lung cancer patients. Methods: Immunofluorescence, flow cytometry, MTT, immunocytochemistry, and western blot analysis were used to assess lymphocyte activation with PHA. Results: The plasma of lung cancer patients suppressed proliferation, CD69 expression, and perforin and granzyme B production in lymphocytes upon activation by PHA, effects that were partially of fully reversed by Gl-PS. Conclusion: Lung cancer patient plasma-induced suppression of lymphocyte activation by phytohemagglutinin may be antagonized fully or partially by Gl-PS, an observation suggesting the potential of Gl-PS in cancer therapy.

  15. Flow cytometric analyses of the viability, surface marker expression and function of lymphocytes from children following cryopreservation.

    Science.gov (United States)

    Chen, Xi; Zhang, Hui; Mou, Wenjun; Qi, Zhan; Ren, Xiaoya; Wang, Guoliang; Jiao, Hong; Kong, Xiaohui; Gui, Jingang

    2016-11-01

    Flow cytometric analysis is important for the investigation and clinical preparation of lymphocytes from children. However, the strict requirement of cell freshness and inter‑assay variability limits the application of this methodology for pediatric investigations. Therefore, it is necessary to identify a reliable cryopreservative method capable of maintaining high cell viability and proper cell function in lymphocytes from children. In the present study, eight commonly‑used cell cyropreservative methods were used, and their effects on cell viability, surface marker expression and cell function were examined. In addition, how these methods affect the distribution of T‑cell receptor Vβ subfamilies were also determined. The results of the present study provided valuable experimental evidence, based on which the optimal method for the cryopreservation of lymphocytes from children in pediatric investigations and clinical applications can be selected.

  16. 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

    2004-01-01

    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....

  17. Distinct patterns of mucosal apoptosis in H pylori-associated gastric ulcer are associated with altered FasL and perforin cytotoxic pathways

    Institute of Scientific and Technical Information of China (English)

    Heitor SP Souza; Marcelo S Neves; Celeste CS Elia; Claudio JA Tortori; Ilana Dines; Cesonia A Martinusso; Kalil Madi; Leonardo Andrade; Morgana TL Castelo-Branco

    2006-01-01

    AIM: To analyze the level of apoptosis in different mucosal compartments and the differential expression of Fas/Fas-ligand and perforin in H pylori-associated gastric ulcer.METHODS: Antral specimens from patients withH pylori-related active gastric ulcer (GU),H pylori-related gastritis, and non-infected controls were analysed for densities and distribution of apoptotic cells determined by the TdT-mediated dUDP-biotin nick-end-labelling method. GU patients were submitted to eradication therapy with follow-up biopsy after 60 d. Fas, FasL, and perforin-expressing cells were assessed by immunoperoxidase, and with anti-CD3, anti-CD20 and anti-CD68 by double immunofluorescence and confocal microscopy.Quantitative analysis was performed using a computerassisted image analyser.RESULTS: H pylori-infected antrum showed greater surface epithelial apoptosis which decreased after eradication therapy. In the lamina propria, higher rates of mononuclear cell apoptosis were observed in H pylorigastritis. Co-expression of Fas with T-cell and macrophage markers was reduced in GU. FasL- and perforinexpressing cells were increased in H pylori-infection and correlated with epithelial apoptosis. Perforin-expressing cells were also increased in GU compared with H pylorigastritis.CONCLUSION: Epithelial apoptosis is increased in H pylori-infection and correlates to FasL- and perforinexpression by T cells. Expression of perforin is correlated with the tissue damage, and may represent the enhancement of a distinct cytotoxic pathway in GU. Increased expression of FasL not paralleled by Fas on T-cells and macrophages may indicate a reduced susceptibility to the Fas/FasL-mediated apoptosis of lymphoid cells in H pylori-infection.

  18. Activation and coreceptor expression of T lymphocytes induced by highly active antiretroviral therapy in Chinese HIV/AIDS patients

    Institute of Scientific and Technical Information of China (English)

    ZHANG Zi-ning; SHANG Hong; JIANG Yong-jun; LIU Jing; DAI Di; DIAO Ying-ying; GENG Wen-qing; JIN Xin; WANG Ya-nan

    2006-01-01

    Background At the end of 2005, 650 000 people lived with human immunodeficiency virus type-1 (HIV-1) in (HAART) supported by the "China CARES" program but the immune responses of HAART were seldom reported. This study investigated the effect of HAART on the activation and coreceptor expression of T lymphocytes in Chinese HIV/AIDS patients and evaluated its effect on immune reconstitution.Methods Seventeen HIV/AIDS patients were enrolled and three-color-flow cytometry was used to detect the activation of HLA-DR CD38 and the coreceptor CCR5, CXCR4 expression on T lymphocytes in whole blood samples taken from the patients before and after 3- or 6-month HAART.Results The activation percents of CD4+, CD8+ T lymphocytes were significantly higher before therapy than the normal controls (HLA-DR/CD4: 40.47± 18.85 vs 11.54±4.10; CD38/CD4: 81.34± 10.86 vs 53.34± 11.44;HLA-DR/CD8:63.94±12.71 vs 25.67±9.18; CD38/CD8:86.56±11.41 vs 58.84±6.16, all P<0.01). After 6-month combined antiretroviral treatment, the activation of T lymphocytes in HIV/AIDS patients was significantly decreased (HLA-DR/CD4:28.31 ± 13.48; CD38/CD4:69.88 ± 12.64; HLA-DR/CD8: 46.56±18.64;CD38/CD8: 70.17± 14.54, all P<0.01 compared with the pre-treatment values). Before the treatment, CCR5 expression on CD8+ T lymphocytes was up-regulated while CXCR4 expression on CD8+ T lymphocytes downregulated in HIV/AIDS patients compared with the normal controls (CD8/CCR5:70.9 1± 10.03 vs 52.70 ±7.68; CD8/CXCR4: 24.14± 11.08 vs 50.05± 11.68, all P<0.01). After 6-month HAART, CCR5 expression on CD8+ T lymphocytes significantly decreased (56.35±12.96, P<0.01), while CXCR4 expression on CD8+ T lymphocytes increased (36.95±9.96, P<0.05) compared with the pre-treatment and the normal controls. A significant statistical relationship was observed between the expression of activation markers, CCR5 and the CD4+ T lymphocyte counts after HAART (P<0.05).Conclusions Reduced activation of T lymphocytes

  19. Heterogeneity of p53-pathway Protein Expression in Chemosensitive Chronic Lymphocytic Leukemia: A Pilot Study

    Directory of Open Access Journals (Sweden)

    Michael J Groves, Stephanie F MacCallum, Michael T Boylan, Sally Haydock, Joan Cunningham, Keith Gelly, Duncan Gowans, Ron Kerr, Philip J Coates, Sudhir Tauro

    2012-01-01

    Full Text Available The presence of p53-pathway dysfunction in chronic lymphocytic leukemia (CLL can be used to identify patients with chemotherapy-refractory disease. Therapeutic responses are known to vary between patients with chemosensitive CLL and may relate to differences in p53-pathway activity. We hypothesized that the magnitude or type of p53-pathway protein expression is heterogeneous in patients with chemosensitive disease and could associate with white cell responses. In this pilot study, changes in p53 and its transcriptional targets, p21/waf1 and MDM2 were analyzed by immunoblotting and densitometry in CLL cells from 10 patients immediately prior to the start of chemotherapy, and after culture for 24 hours (h with fludarabine (n=7 or chlorambucil (n=3. The in vitro response was also compared to that in vivo in circulating cells pre-treatment, and at 24h and 96h of chemotherapy. Disease responses were evident in all patients after the first treatment-cycle. Significant p53 induction was observed in CLL cells treated in vitro and in vivo. Greater heterogeneity in the expression-intensity was observed in vivo (σ2=45.15 than in vitro (σ2=1.33 and the results failed to correlate (r2=0.18, p=0.22. p21/waf1 and MDM2 expression-profiles were also dissimilar in vitro and in vivo. Higher in vivo (but not in vitro responses associated with changes in white cell count (p=0.026. Thus, heterogeneity of p53-pathway activity exists in chemosensitive CLL; in unselected patients, in vivo changes do not correlate with those in vitro, but may associate with post-treatment white cell responses.

  20. AKRs expression in peripheral blood lymphocytes from smokers: the role of body mass index.

    Science.gov (United States)

    Barrón-Vivanco, B S; Rothenberg, S J; Medina-Díaz, I M; Robledo-Marenco, L; Rojas-García, A E; Hernández-Cadena, L; Poblete-Naredo, I; Elizondo, G; Albores, A

    2013-04-01

    Aldo-keto reductases (AKRs) metabolize a wide range of substrates, including polycyclic aromatic hydrocarbons (PAHs), generating metabolites (o-quinones) and reactive oxygen species (ROS), which are capable of initiating and promoting carcinogenesis. Exposure to PAHs, their metabolites, and ROS further increase AKRs isoform expression that may amplify oxidative damage. Human AKR enzymes are highly polymorphic, and allelic variants may contribute to different AKRs expression in individuals. Despite the importance of AKRs in PAHs metabolism, there are no studies that evaluate, in general human populations, the effect of PAHs on AKRs expression in peripheral blood lymphocytes (PBLs). The aim of this study was to determine the effect of tobacco smoke exposure, and AKR1A1*2 and AKR1C3*2 polymorphisms, on AKR1A1 and AKR1C1-AKR1C3 messenger RNA (mRNA) levels in PBLs from smokers. In the smoker group, there is a statistically significant positive association between AKR1A1, AKR1C1, and AKR1C3 mRNA induction and urine cotinine levels in individuals with a body mass index (BMI) less than 25. However, AKR1A1*2 and AKR1C3*2 alleles did not influence AKR1A1 and AKR1C1-AKR1C3 mRNA levels. These results suggest that AKRs induction by PAHs in smokers' PBLs is associated with BMI; therefore, the role of adipose tissue accumulation in PAHs' effects needs further investigation.

  1. Unraveling the equine lymphocyte proteome: differential septin 7 expression associates with immune cells in equine recurrent uveitis.

    Science.gov (United States)

    Degroote, Roxane L; Hauck, Stefanie M; Amann, Barbara; Hirmer, Sieglinde; Ueffing, Marius; Deeg, Cornelia A

    2014-01-01

    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.

  2. 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.

  3. 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.

  4. Lymphocyte differentiation in sea bass thymus: CD4 and CD8-alpha gene expression studies.

    Science.gov (United States)

    Picchietti, Simona; Guerra, Laura; Buonocore, Francesco; Randelli, Elisa; Fausto, Anna Maria; Abelli, Luigi

    2009-07-01

    Different developmental stages (from eggs to 1-year-old juveniles) of the teleost fish Dicentrarchus labrax (L.) were assayed for CD4 gene expression. RT-PCR revealed the appearance of CD4 transcripts in post-larvae from 51 days post-hatching (dph). This finding overlaps the first detection of CD8-alpha mRNA. Real-time PCR with specific primers quantified CD4, CD8-alpha and TCR-beta transcripts in larvae and post-larvae (25, 51, 75 and 92 dph) and 1-year-old thymus. At 92 dph, TcR-beta and CD8-alpha transcripts were significantly higher (P overlap, except in the medulla, where CD4(+) thymocytes were isolated, while CD8-alpha(+) ones mainly arranged in cords. These results provide new information about the thymic compartmentalization and lymphocyte differentiation pathways in a teleost, almost demonstrating that double negative thymocytes fill the cortex giving rise to further selection in the medulla.

  5. Germline allele-specific expression of DAPK1 in chronic lymphocytic leukemia.

    Directory of Open Access Journals (Sweden)

    Quan-Xiang Wei

    Full Text Available We previously reported a rare germline variant (c.1-6531 that resulted in allele-specific expression (ASE of death-associated protein kinase 1 (DAPK1 and predisposition to chronic lymphocytic leukemia (CLL. We investigated a cohort of CLL patients lacking this mutation for the presence of ASE of DAPK1. We developed a novel strategy that combines single-nucleotide primer extension (SNuPE with MALDI-TOF mass spectrometry, and detected germline DAPK1 ASE in 17 out of 120 (14.2% CLL patients associated with a trend towards younger age at diagnosis. ASE was absent in 63 healthy controls. Germline cells of CLL patients with ASE showed increased levels of DNA methylation in the promoter region, however, neither genetic nor further epigenetic aberrations could be identified in the DAPK1 5' upstream regulatory region, within distinct exons or in the 3'-UTR. We identified B-lymphoid malignancy related cell line models harboring allelic imbalance and found that allele-specific methylation in DAPK1 is associated with ASE. Our data indicate that ASE at the DAPK1 gene locus is a recurrent event, mediated by epigenetic mechanisms and potentially predisposing to CLL.

  6. Cell-type-specific expression of STAT transcription factors in tissue samples from patients with lymphocytic thyroiditis.

    Science.gov (United States)

    Staab, Julia; Barth, Peter J; Meyer, Thomas

    2012-09-01

    Expression of cytokine-regulated signal transducer and activator of transcription (STAT) proteins was histochemically assessed in patients diagnosed as having Hashimoto's disease or focal lymphocytic thyroiditis (n = 10). All surgical specimens showed histological features of lymphocytic thyroiditis, including a diffuse infiltration with mononuclear cells and an incomplete loss of thyroid follicles, resulting in the destruction of glandular tissue architecture. Immunohistochemical analysis demonstrated differential expression patterns of the various members of the STAT transcription factors examined, indicating that each member of this conserved protein family has its distinct functions in the development of the disease. Using an antibody that specifically recognized the phosphorylated tyrosine residue in position 701, we detected activated STAT1 dimers in numerous germinal macrophages and infiltrating lymphocytes as well as in oncocytes. In contrast, STAT3 expression was restricted to epithelial cells and showed a clear colocalization with the antiapoptotic protein Bcl-2. Moreover, expression of phospho-STAT3 was associated with low levels of stromal fibrosis, suggesting that STAT3 serves as a protective factor in the remodeling of the inflamed thyroid gland. Phospho-STAT5 immunoreactivity was detected in numerous infiltrating cells of hematopoietic origin and, additionally, in hyperplastic follicular epithelia. This tissue distribution demonstrated that activated STAT5 molecules participate in both lymphocytopoiesis and possibly also in the buildup of regenerating thyroid follicles. Taken together, the cell-type-specific expression patterns of STAT proteins in human lymphocytic thyroiditis reflect their distinct and partially antagonistic roles in orchestrating the balance between degenerating and regenerating processes within a changing cytokine environment.

  7. Abnormal lipid rafts related ganglioside expression and signaling in T lymphocytes in immune thrombocytopenia patients.

    Science.gov (United States)

    Zhang, Xian; Zhang, Donglei; Liu, Wenjie; Li, Huiyuan; Fu, Rongfeng; Liu, Xiaofan; Xue, Feng; Yang, Renchi

    2016-01-01

    Aberrant T lymphocytes signaling is considered to play a crucial role in the abnormal immune state of primary immune thrombocytopenia (ITP). Lipid raft has been verified to engage in the T cell receptor (TCR)-mediated T lymphocytes signal transduction. Whether lipid raft-associated T cells signal transduction has impact on the pathogenesis of ITP is still unconfirmed. In this study, we aimed to reveal the abnormality in structure and function of lipid rafts (LRs) in CD4(+) and CD8(+) T lymphocytes of patients with ITP. Our results showed that there was an increased lipid raft aggregation in ITP patients, while this kind of increase would not be influenced by platelet counts or therapeutic regimes. Stimulation by anti-CD3/CD28 monoclonal antibodies promoted enhanced lipid raft clustering in T lymphocytes of ITP patients compared with negative controls. Methyl-β-cyclodextrin (MβCD) could block the abnormal lipid raft aggregation and disrupt the TCR-mediated T cells proliferation and cytokines secretion, including both proinflammatory cytokines and anti-inflammatory cytokines. The spontaneous activation of T lymphocytes from ITP patients might be due to the elevated co-localization of protein tyrosine phosphatase (PTP) CD45 and lipid rafts in patients' CD4(+) and CD8(+) T lymphocytes. These findings suggest that the autoactivation of T lymphocytes from ITP patients may lead to the abnormality in lipid raft structure and raft-anchored proteins, and the changes conversely promote the TCR-mediated T cells activation of ITP patients.

  8. 生理状态下外周血白细胞穿孔素基因表达的荧光定量PCR检测%Quantitative Real- time Detection of Spontaneous Perforin(PFR) Gene Expression in Peripheral Blood Mononuclear Cell (PBMC) by Polymerase Chain Reaction

    Institute of Scientific and Technical Information of China (English)

    王茹; 陈佩杰; 董强刚

    2006-01-01

    目的:建立穿孔素(perforin,PFR)基因表达的绝对定量实时PCR检测技术.方法:应用实时定量PCR方法检测25名健康运动员生理状态下处周血PFR mRNA的表达.主要结果和结论:应用实时定量PCR方法能够对健康受试者外周血白细胞自发性PFR mRNA表达进行定量分析,相关系数r=1.

  9. Regulation of Mcl-1 Expression in Context to Bone Marrow Stromal Microenvironment in Chronic Lymphocytic Leukemia

    Directory of Open Access Journals (Sweden)

    Kumudha Balakrishnan, PhD

    2014-12-01

    Full Text Available A growing body of evidence suggests that the resistance of CLL cells to apoptosis is partly mediated through the interactions between leukemia cells and adjacent stromal cells residing in the lymphatic tissue or bone marrow microenvironment. Mcl-1, an anti-apoptotic protein that is associated with failure to treatment is up-regulated in CLL lymphocytes after interaction with microenvironment. However, the regulation of its expression in context to microenvironment is unclear. We evaluated and compared changes in Mcl-1 in CLL B-cells in suspension culture and when co-cultured on stromal cells. The blockade of apoptosis in co-cultured CLL cells is associated with diminution in caspase-3 and PARP cleavage and is not dependent on cytogenetic profile or prognostic factors of the disease. Stroma-derived resistance to apoptosis is associated with a cascade of transcriptional events such as increase in levels of total RNA Pol II and its phosphorylation at Ser2 and Ser5, increase in the rate of global RNA synthesis, and amplification of Mcl-1 transcript levels. The latter is associated with increase in Mcl-1 protein level without an impact on the levels of Bcl-2 and Bcl-xL. Post-translational modifications of protein kinases show increased phosphorylation of Akt at Ser473, Erk at Thr202/Tyr204 and Gsk-3β at Ser9 and augmentation of total Mcl-1 accumulation along with phosphorylation at Ser159/Thr163 sites. Collectively, stroma-induced apoptosis resistance is mediated through signaling proteins that regulate transcriptional and translational expression and post-translational modification of Mcl-1 in CLL cells in context to bone marrow stromal microenvironment.

  10. Regulation of Mcl-1 expression in context to bone marrow stromal microenvironment in chronic lymphocytic leukemia.

    Science.gov (United States)

    Balakrishnan, Kumudha; Burger, Jan A; Fu, Min; Doifode, Tejaswini; Wierda, William G; Gandhi, Varsha

    2014-12-01

    A growing body of evidence suggests that the resistance of CLL cells to apoptosis is partly mediated through the interactions between leukemia cells and adjacent stromal cells residing in the lymphatic tissue or bone marrow microenvironment. Mcl-1, an anti-apoptotic protein that is associated with failure to treatment is up-regulated in CLL lymphocytes after interaction with microenvironment. However, the regulation of its expression in context to microenvironment is unclear. We evaluated and compared changes in Mcl-1 in CLL B-cells in suspension culture and when co-cultured on stromal cells. The blockade of apoptosis in co-cultured CLL cells is associated with diminution in caspase-3 and PARP cleavage and is not dependent on cytogenetic profile or prognostic factors of the disease. Stroma-derived resistance to apoptosis is associated with a cascade of transcriptional events such as increase in levels of total RNA Pol II and its phosphorylation at Ser2 and Ser5, increase in the rate of global RNA synthesis, and amplification of Mcl-1 transcript levels. The latter is associated with increase in Mcl-1 protein level without an impact on the levels of Bcl-2 and Bcl-xL. Post-translational modifications of protein kinases show increased phosphorylation of Akt at Ser473, Erk at Thr202/Tyr204 and Gsk-3β at Ser9 and augmentation of total Mcl-1 accumulation along with phosphorylation at Ser159/Thr163 sites. Collectively, stroma-induced apoptosis resistance is mediated through signaling proteins that regulate transcriptional and translational expression and post-translational modification of Mcl-1 in CLL cells in context to bone marrow stromal microenvironment. Copyright © 2014 Neoplasia Press, Inc. Published by Elsevier Inc. All rights reserved.

  11. CCR5 Expression Levels Influence NFAT Translocation, IL-2 Production, and Subsequent Signaling Events during T Lymphocyte Activation1

    OpenAIRE

    2009-01-01

    Ligands of CCR5, the major coreceptor of HIV-1, costimulate T lymphocyte activation. However, the full impact of CCR5 expression on T cell responses remains unknown. Here, we show that compared with CCR5+/+, T cells from CCR5−/− mice secrete lower amounts of IL-2, and a similar phenotype is observed in humans who lack CCR5 expression (CCR5-Δ32/Δ32 homozygotes) as well as after Ab-mediated blockade of CCR5 in human T cells genetically intact for CCR5 expression. Conversely, overexpression of C...

  12. CD56(bright)perforin(low) noncytotoxic human NK cells are abundant in both healthy and neoplastic solid tissues and recirculate to secondary lymphoid organs via afferent lymph.

    Science.gov (United States)

    Carrega, Paolo; Bonaccorsi, Irene; Di Carlo, Emma; Morandi, Barbara; Paul, Petra; Rizzello, Valeria; Cipollone, Giuseppe; Navarra, Giuseppe; Mingari, Maria Cristina; Moretta, Lorenzo; Ferlazzo, Guido

    2014-04-15

    As limited information is available regarding the distribution and trafficking of NK cells among solid organs, we have analyzed a wide array of tissues derived from different human compartments. NK cells were widely distributed in most solid tissues, although their amount varied significantly depending on the tissue/organ analyzed. Interestingly, the distribution appeared to be subset specific, as some tissues were preferentially populated by CD56(bright)perforin(low) NK cells, with others by the CD56(dim)perforin(high) cytotoxic counterpart. Nevertheless, most tissues were highly enriched in CD56(bright)perforin(low) cells, and the distribution of NK subsets appeared in accordance with tissue gene expression of chemotactic factors, for which receptors are differently represented in the two subsets. Remarkably, chemokine expression pattern of tissues was modified after neoplastic transformation. As a result, although the total amount of NK cells infiltrating the tissues did not significantly change upon malignant transformation, the relative proportion of NK subsets infiltrating the tissues was different, with a trend toward a tumor-infiltrating NK population enriched in noncytotoxic cells. Besides solid tissues, CD56(bright)perforin(low) NK cells were also detected in seroma fluids, which represents an accrual of human afferent lymph, indicating that they may leave peripheral solid tissues and recirculate to secondary lymphoid organs via lymphatic vessels. Our results provide a comprehensive mapping of NK cells in human tissues, demonstrating that discrete NK subsets populate and recirculate through most human tissues and that organ-specific chemokine expression patterns might affect their distribution. In this context, chemokine switch upon neoplastic transformation might represent a novel mechanism of tumor immune escape.

  13. Consequences of cytotoxic T lymphocyte interaction with major histocompatibility complex class I-expressing neurons in vivo

    OpenAIRE

    1995-01-01

    Neurons have evolved strategies to evade immune surveillance that include an inability to synthesize the heavy chain of the class I major histocompatibility complex (MHC), proteins that are necessary for cytotoxic T lymphocyte (CTL) recognition of target cells. Multiple viruses have taken advantage of the lack of CTL-mediated recognition and killing of neurons by establishing persistent neuronal infections and thereby escaping attack by antiviral CTL. We have expressed a class I MHC molecule ...

  14. Expressions of programmed death-1 and programmed death ligand on the surface of peripheral blood lymphocytes in patients with tuberculosis

    Institute of Scientific and Technical Information of China (English)

    胥萍

    2014-01-01

    Objective To describe the expressions of programmed death-1(PD-1)and its ligand PD-L1 on the surface of peripheral blood lymphocytes in patients with tuberculosis.Methods A total of 77 cases of pulmonary tuberculosis were recruited,of which 27 were single infection,41 were coincident with bacterial or fungal infection and 9 patients with diabetes millitus.Twenty-nine

  15. ER-mitochondria contacts control surface glycan expression and sensitivity to killer lymphocytes in glioma stem-like cells.

    Science.gov (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

    2017-06-01

    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.

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

    Directory of Open Access Journals (Sweden)

    Xiuying Li

    2016-01-01

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

  17. Association of Bax Expression and Bcl2/Bax Ratio with Clinical and Molecular Prognostic Markers in Chronic Lymphocytic Leukemia.

    Science.gov (United States)

    Vucicevic, Ksenija; Jakovljevic, Vladimir; Colovic, Natasa; Tosic, Natasa; Kostic, Tatjana; Glumac, Irena; Pavlovic, Sonja; Karan-Djurasevic, Teodora; Colovic, Milica

    2016-04-01

    In chronic lymphocytic leukemia (CLL), in vivo apoptotic resistance of malignant B lymphocytes results, in part, from the intrinsic defects of their apoptotic machinery. These include genetic alterations and aberrant expression of many apoptosis regulators, among which the Bcl2 family members play a central role. The aim of this study was to investigate the association of pro-apoptotic Bax gene expression and Bcl2/Bax ratio with the clinical features of CLL patients as well as with molecular prognostic markers, namely the mutational status of rearranged immunoglobulin heavy variable (IGHV) genes and lipoprotein lipase (LPL) gene expression. We analyzed the expression of Bax mRNA and Bcl2/Bax mRNA ratio in the peripheral blood mononuclear cells of 58 unselected CLL patients and 10 healthy controls by the quantitative reverse-transcriptase polymerase chain reaction. We detected significant Bax gene overexpression in CLL samples compared to non-leukemic samples (p=0.003), as well as an elevated Bcl2/Bax ratio (p=Bax ratio showed a negative correlation to lymphocyte doubling time (r=-0.307; p=0.0451), while high-level Bax expression was associated with LPL-positive status (p=0.035). Both the expression of Bax and Bcl2/Bax ratio were higher in patients with unmutated vs. mutated IGHV rearrangements, but this difference did not reach statistical significance. Our results suggest that dysregulated expression of Bcl2 and Bax, which leads to a high Bcl2/Bax ratio in leukemic cells, contributes to the pathogenesis and clinical course of CLL.

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

    DEFF Research Database (Denmark)

    Hofmann, B; Moller, J; Langhoff, E

    1989-01-01

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

  19. B lymphocyte-specific c-Myc expression stimulates early and functional expansion of the vasculature and lymphatics during lymphomagenesis.

    Science.gov (United States)

    Ruddell, Alanna; Mezquita, Pau; Brandvold, Kimberly A; Farr, Andrew; Iritani, Brian M

    2003-12-01

    Expression of the c-myc proto-oncogene is deregulated in many human cancers. We examined the role of c-Myc in stimulating angiogenesis and lymphangiogenesis in a highly metastatic murine model of Burkitt's lymphoma (E micro -c-myc), where c-Myc is expressed exclusively in B lymphocytes. Immunohistochemical analysis of bone marrow and lymph nodes from young (preneoplastic) E micro -c-myc transgenic mice revealed increased growth of blood vessels, which are functional by dye flow assay. Lymphatic sinuses also increased in size and number within the lymph nodes, as demonstrated by immunostaining for with a lymphatic endothelial marker 10.1.1. The 10.1.1 antibody recognizes VEGFR-2- and VEGFR-3-positive lymphatic sinuses and vessels within lymph nodes, and also recognizes lymphatic vessels in other tissues. Subcutaneously injected dye traveled more efficiently through draining lymph nodes in E micro -c-myc mice, indicating that these hypertrophic lymphatic sinuses increase lymph flow. Purified B lymphocytes and lymphoid tissues from E micro -c-myc mice expressed increased levels of vascular endothelial growth factor (VEGF) by immunohistochemical or immunoblot assays, which could promote blood and lymphatic vessel growth through interaction with VEGFR-2, which is expressed on the endothelium of both vessel types. These results indicate that constitutive c-Myc expression stimulates angiogenesis and lymphangiogenesis, which may promote the rapid growth and metastasis of c-Myc-expressing cancer cells, respectively.

  20. Perforin is detrimental to controlling [corrected] C. muridarum replication in vitro, but not in vivo.

    Directory of Open Access Journals (Sweden)

    Raymond M Johnson

    Full Text Available CD4 T cells are critical for clearing experimental Chlamydia muridarum genital tract infections. Two independent in vitro CD4 T cell mechanisms have been identified for terminating Chlamydia replication in epithelial cells. One mechanism, requiring IFN-γ and T cell-epithelial cell contact, terminates infection by triggering epithelial production of nitric oxide to chlamydiacidal levels; the second is dependent on T cell degranulation. We recently demonstrated that there are two independent in vivo clearance mechanisms singly sufficient for clearing genital tract infections within six weeks; one dependent on iNOS, the other on Plac8. Redundant genital tract clearance mechanisms bring into question negative results in single-gene knockout mice. Two groups have shown that perforin-knockout mice were not compromised in their ability to clear C. muridarum genital tract infections. Because cell lysis would be detrimental to epithelial nitric oxide production we hypothesized that perforin was not critical for iNOS-dependent clearance, but posited that perforin could play a role in Plac8-dependent clearance. We tested whether the Plac8-dependent clearance was perforin-dependent by pharmacologically inhibiting iNOS in perforin-knockout mice. In vitro we found that perforin was detrimental to iNOS-dependent CD4 T cell termination of Chlamydia replication in epithelial cells. In vivo, unexpectedly, clearance in perforin knockout mice was delayed to the end of week 7 regardless of iNOS status. The discordant in vitro/in vivo results suggest that the perforin's contribution to bacterial clearance in vivo is not though enhancing CD4 T cell termination of Chlamydia replication in epithelial cells, but likely via a mechanism independent of T cell-epithelial cell interactions.

  1. Qa-2 expression levels is related with tumor-infiltrating lymphocytes profile during solid Ehrlich tumor development.

    Science.gov (United States)

    da Silva, Istéfani Luciene; Veloso, Emerson Soares; Gonçalves, Ivy Nayra Nascimento; Braga, Ariadne Duarte; Lopes, Miriam Teresa Paz; Cassali, Geovanni Dantas; Quintanilla, Miguel; Simões, Renata Toscano; Ferreira, Enio

    2017-08-01

    The Qa-2 has been described as Human Leucocyte Antigen G (HLA-G) murine homolog. This homology is well accepted to gene and protein structure, in different pathology process and embryos implantation. However, in some neoplasm, this homology is questioned, where Qa-2 has been proposed as an immunogenic molecule, associated to tumor rejection. In this way, the aim of this study was to describe the pattern of Qa-2 expression and its relationship with the profile of tumor-infiltrating lymphocytes in solid Ehrlich tumor. The Ehrlich tumor growth was evaluated in Balb/c female mice in different tumor stages. The inflammatory infiltration features were determined by histopathology and, both lymphocyte type and tissue Qa-2 expression by immunohistochemistry. ELISA kit was used to determine soluble Qa-2 in the serum from the animals. We observed that Qa-2 in neoplastic cells increases in intermediate tumor development stages, while, serum Qa-2 increases in the late stage. Qa-2 increasing is correlated with CD3+ increase. Our results suggest that Qa-2 has a role opposite to HLA-G in Ehrlich solid carcinoma, and may be modulating the immune response by attracting the inflammatory infiltrate, especially T CD8+ Lymphocytes. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  2. [Dexamethasone affect on the expression of bcl-2 and mTOR genes in T-lymphocytes from healthy donors].

    Science.gov (United States)

    Fatkhullina, A R; Abramov, S N; Skibo, Iu V; Abramova, Z I

    2014-01-01

    Synthetic glucocorticoids are able to activate apoptosis in the cells by regulating the transcription of the respective genes. Effect of dexamethasone on apoptosis is an established fact. However, its influence on another program of cell death autophagy, is currently unproven. Therefore, in this paper we have analyzed the influence of dexamethasone on the expression of bcl-2 and mTOR genes in T-lymphocytes from healthy donors. The results showed that dexamethasone reduced the expression of bcl-2 and mTOR genes. However, the nature of the effect of dexamethasone on mTOR and bcl-2 expression was different: the expression of bcl-2 gene in the long-term cultivation was maintained at the same reduced level, while the expression of mTOR was first reduced and then increased.

  3. Chimeric approach for narrowing a membrane-inserting region within human perforin.

    Science.gov (United States)

    Neely, Amy E; Mandigo, Kimberly A; Robinson, Rebekah L; Ness, Traci L; Weiland, Mitch H

    2017-02-01

    Perforin is a pore-forming, immune protein that functions to deliver an apoptotic cocktail of proteins into a target pathogen. Recent studies of the bacterial cholesterol-dependent cytolysins (CDCs) have provided a model for perforin's pore-forming mechanism. Both perforin and CDC family members share a conserved β-sheet flanked by two clusters of α-helices. Within the CDCs, these helices refold into two transmembrane β-hairpins, TMH1 and TMH2. Based upon structural conservation and electron microscopy imaging, the analogous helices within perforin are predicted to also be membrane inserting; however, these regions are approximately twice the length of the CDC TMHs. To test the membrane-insertion potential of one of these regions, chimeras were created using a well-characterized CDC, perfringolysin-O (PFO), as the backbone of these constructs. PFO's TMH2 region was replaced with perforin's corresponding helical region. Although hemolytic activity was observed, the chimera was poorly soluble. A second chimera contained the same region truncated to match the length of the PFO TMH2 region. The truncated chimera demonstrated improved solubility, significant hemolytic activity and the ability to form pores characteristic of those created by PFO. These results provide the first evidence that perforin's helices function as TMHs and more importantly narrows the residues responsible for membrane insertion. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  4. Differences in APOBEC3G expression in CD4+ T helper lymphocyte subtypes modulate HIV-1 infectivity.

    Directory of Open Access Journals (Sweden)

    Michael L Vetter

    2009-02-01

    Full Text Available The cytidine deaminases APOBEC3G and APOBEC3F exert anti-HIV-1 activity that is countered by the HIV-1 vif protein. Based on potential transcription factor binding sites in their putative promoters, we hypothesized that expression of APOBEC3G and APOBEC3F would vary with T helper lymphocyte differentiation. Naive CD4+ T lymphocytes were differentiated to T helper type 1 (Th1 and 2 (Th2 effector cells by expression of transcription factors Tbet and GATA3, respectively, as well as by cytokine polarization. APOBEC3G and APOBEC3F RNA levels, and APOBEC3G protein levels, were higher in Th1 than in Th2 cells. T cell receptor stimulation further increased APOBEC3G and APOBEC3F expression in Tbet- and control-transduced, but not in GATA3-transduced, cells. Neutralizing anti-interferon-gamma antibodies reduced both basal and T cell receptor-stimulated APOBEC3G and APOBEC3F expression in Tbet- and control-transduced cells. HIV-1 produced from Th1 cells had more virion APOBEC3G, and decreased infectivity, compared to virions produced from Th2 cells. These differences between Th1- and Th2-produced virions were greater for viruses lacking functional vif, but also seen with vif-positive viruses. Over-expression of APOBEC3G in Th2 cells decreased the infectivity of virions produced from Th2 cells, and reduction of APOBEC3G in Th1 cells increased infectivity of virions produced from Th1 cells, consistent with a causal role for APOBEC3G in the infectivity difference. These results indicate that APOBEC3G and APOBEC3F levels vary physiologically during CD4+ T lymphocyte differentiation, that interferon-gamma contributes to this modulation, and that this physiological regulation can cause changes in infectivity of progeny virions, even in the presence of HIV-1 vif.

  5. Deep immune profiling by mass cytometry links human T and NK cell differentiation and cytotoxic molecule expression patterns.

    Science.gov (United States)

    Bengsch, Bertram; Ohtani, Takuya; Herati, Ramin Sedaghat; Bovenschen, Niels; Chang, Kyong-Mi; Wherry, E John

    2017-03-19

    The elimination of infected or tumor cells by direct lysis is a key T and NK cell effector function. T and NK cells can kill target cells by coordinated secretion of cytotoxic granules containing one or both pore-forming proteins, perforin and granulysin and combinations of granzyme (Gzm) family effector proteases (in humans: Gzm A, B, K, M and H). Understanding the pattern of expression of cytotoxic molecules and the relationship to different states of T and NK cells may have direct relevance for immune responses in autoimmunity, infectious disease and cancer. Approaches capable of simultaneously evaluating expression of multiple cytotoxic molecules with detailed information on T and NK differentiation state, however, remain limited. Here, we established a high dimensional mass cytometry approach to comprehensively interrogate single cell proteomic expression of cytotoxic programs and lymphocyte differentiation. This assay identified a coordinated expression pattern of cytotoxic molecules linked to CD8 T cell differentiation stages. Coordinated high expression of perforin, granulysin, Gzm A, Gzm B and Gzm M was associated with markers of late effector memory differentiation and expression of chemokine receptor CX3CR1. However, classical gating and dimensionality reduction approaches also identified other discordant patterns of cytotoxic molecule expression in CD8 T cells, including reduced perforin, but high Gzm A, Gzm K and Gzm M expression. When applied to non-CD8 T cells, this assay identified different patterns of cytotoxic molecule co-expression by CD56(hi) versus CD56(dim) defined NK cell developmental stages; in CD4 T cells, low expression of cytotoxic molecules was found mainly in TH1 phenotype cells, but not in Tregs or T follicular helper cells (TFH). Thus, this comprehensive, single cell, proteomic assessment of cytotoxic protein co-expression patterns demonstrates specialized cytotoxic programs in T cells and NK cells linked to their differentiation

  6. CXCR3, CCR5, and CRTH2 Chemokine Receptor Expression in Lymphocytes Infiltrating Thyroid Nodules with Coincident Hashimoto’s Thyroiditis Obtained by Fine Needle Aspiration Biopsy

    Directory of Open Access Journals (Sweden)

    Jan Jiskra

    2016-01-01

    Full Text Available Objective. To determine the expression of chemokine receptors in lymphocytes from thyroid nodules and peripheral blood in patients with and without Hashimoto’s thyroiditis (HT. Patients and Methods. The study included 46 women with thyroid nodules and HT and 60 women with thyroid nodules without HT (controls who underwent a fine needle aspiration biopsy (FNAB. Expression of chemokine receptors CXCR3, CCR5, and CRTH2 was assessed by flow cytometry in lymphocytes from FNAB samples and from peripheral blood. Results. The percentage of CRTH2+ lymphocytes was higher in nodules with HT in comparison with controls, both in FNAB samples (13.95 versus 6.7%, p=0.008 and in peripheral blood (6.7 versus 5.13%, p=0.047, and positively correlated with serum antibodies to thyroid peroxidase (r=0.243; p=0.026 and negatively correlated with thyroid volume (r=-0.346; p=0.008. Lymphocytes from neoplastic nodules showed a higher expression of both CXCR3 and CCR5 than those from hyperplastic ones. Conclusion. Flow cytometry performed in FNAB samples may serve as a good tool in investigation of intrathyroidal expression of immunological parameters. In our study, the CRTH2 expression on thyroid-infiltrating lymphocytes as well as on lymphocytes from peripheral blood was increased in HT as compared to controls.

  7. CXCR3, CCR5, and CRTH2 Chemokine Receptor Expression in Lymphocytes Infiltrating Thyroid Nodules with Coincident Hashimoto's Thyroiditis Obtained by Fine Needle Aspiration Biopsy

    Science.gov (United States)

    Antošová, Marie; Krátký, Jan; Vítková, Hana; Límanová, Zdeňka; Marečková, Helena; Potluková, Eliška

    2016-01-01

    Objective. To determine the expression of chemokine receptors in lymphocytes from thyroid nodules and peripheral blood in patients with and without Hashimoto's thyroiditis (HT). Patients and Methods. The study included 46 women with thyroid nodules and HT and 60 women with thyroid nodules without HT (controls) who underwent a fine needle aspiration biopsy (FNAB). Expression of chemokine receptors CXCR3, CCR5, and CRTH2 was assessed by flow cytometry in lymphocytes from FNAB samples and from peripheral blood. Results. The percentage of CRTH2+ lymphocytes was higher in nodules with HT in comparison with controls, both in FNAB samples (13.95 versus 6.7%, p = 0.008) and in peripheral blood (6.7 versus 5.13%, p = 0.047), and positively correlated with serum antibodies to thyroid peroxidase (r = 0.243; p = 0.026) and negatively correlated with thyroid volume (r = −0.346; p = 0.008). Lymphocytes from neoplastic nodules showed a higher expression of both CXCR3 and CCR5 than those from hyperplastic ones. Conclusion. Flow cytometry performed in FNAB samples may serve as a good tool in investigation of intrathyroidal expression of immunological parameters. In our study, the CRTH2 expression on thyroid-infiltrating lymphocytes as well as on lymphocytes from peripheral blood was increased in HT as compared to controls. PMID:27872865

  8. Expression of Hsp27 and Hsp70 in lymphocytes and plasma in healthy workers and coal miners with lung cancer.

    Science.gov (United States)

    Wang, Haijiao; Xing, Jingcai; Wang, Feng; Han, Wenhui; Ren, Houmao; Wu, Tangchun; Chen, Weihong

    2010-08-01

    In coal mines, main occupational hazard is coal-mine dust, which can cause health problem including coal workers' pneumoconiosis and lung cancer. Some heat shock proteins (Hsps) have been reported as an acute response to a wide variety of stressful stimuli. Whether Hsps protect against chronic environmental coal-mine dust over years is unknown. It is also interesting to know that whether the expression of Hsp27 and Hsp70 proteins as a marker for exposure is associated risk of lung cancer among coal miners. We investigated the association between levels of Hsp27 and Hsp70 expression in lymphocytes and plasma and levels of coal-mine dust exposure in workplace or risk of lung cancer in 42 cancer-free non-coal miners, 99 cancer-free coal miners and 51 coal miners with lung cancer in Taiyuan city in China. The results showed that plasma Hsp27 levels were increased in coal miners compared to non-coal miners (P<0.01). Except high cumulative coal-mine dust exposure (OR=13.62, 95%CI=6.05-30.69) and amount of smoking higher than 24 pack-year (OR=2.72, 95% CI=1.37-5.42), the elevated levels of plasma Hsp70 (OR=13.00, 95% CI=5.14-32.91) and plasma Hsp27 (OR=2.97, 95% CI=1.40-6.32) and decreased expression of Hsp70 in lymphocytes (OR=2.36, 95% CI=1.05-5.31) were associated with increased risk of lung cancer. These findings suggest that plasma Hsp27 may be a potential marker for coal-mine dust exposure. And the expression of Hsp27 and Hsp70 levels in plasma and lymphocytes may be used as biomarkers for lung cancer induced by occupational coal-mine dust exposure.

  9. Abnormally High Expression of BAFF on T Lymphocytes from Lung Cancer-associated Pleural Effusions and Its Potent Anti-tumor Effect

    Institute of Scientific and Technical Information of China (English)

    Haiyan XU; Xiaozhou HE; Yibei ZHU; Tiangzan YAN; Hongbin MA; Xueguang ZHANG

    2007-01-01

    In the present study, the expressions of B cell activating factor belonging to the tumor necrosis factor family (BAFF) and its receptors (BAFF-R and TACI) on T lymphocytes from malignant pleural effusion (MPE) were examined by fluorescence-activated cell sorting (FACS) analysis, and compared with those on the T lymphocytes from non-malignant pleural effusion (NMPE) and healthy controls. It was found that CD3 positive T lymphocytes (including CD4, CD8, and part of CD25 and CD69 positive cells) of MPE in lung cancer highly and consistently expressed the BAFF molecule, while high expressions of BAFF could only be found in phytohemagglutinin (PHA) or interleukin 2 (IL-2) induced T lymphocytes from NMPE or healthy controls. These results were consistent with the results from BAFF mRNA detection by real-time PCR. In addition, T lymphocytes from MPE expressed significantly more BAFF-R than those from NMPE or healthy controls, while the expression of TACI was increased on CD4+ T cells but decreased on CD8+ T cells when compared with controls. The Annexin/PI assay suggested that recombinant human BAFF (rhBAFF) could promote the survival rate of T lymphocytes from MPE, while the decoy receptor TACI-Fc fusion protein could promote the apoptosis rate of T lymphocytes. Cytokines in the supernatant detected by ELISA assay showed that rhBAFF could significantly upregulate the secretion of IFN-γ in vitro,and the IFN-γ level in the TACI-Fc-treated group resembled that of the control groups. All of these results indicated that the abnormally high expression of BAFF on T lymphocytes from MPE may play a role of antitumor effect.

  10. Propionic acid secreted from propionibacteria induces NKG2D ligand expression on human-activated T lymphocytes and cancer cells

    DEFF Research Database (Denmark)

    Andresen, Lars; Hansen, Karen Aagaard; Jensen, Helle

    2009-01-01

    We found that propionic acid secreted from propionibacteria induces expression of the NKG2D ligands MICA/B on activated T lymphocytes and different cancer cells, without affecting MICA/B expression on resting peripheral blood cells. Growth supernatant from propionibacteria or propionate alone could...... activity. Other short-chain fatty acids such as lactate, acetate, and butyrate could also induce MICA/B expression. We observed a striking difference in the molecular signaling pathways that regulate MICA/B. A functional glycolytic pathway was essential for MICA/B expression after exposure to propionate...... that propionate, produced either by bacteria or during cellular metabolism, has significant immunoregulatory function and may be cancer prophylactic....

  11. Ratios of CD64 expressed on neutrophils, monocytes, and lymphocytes may be a novel method for diagnosis of neonatal sepsis.

    Science.gov (United States)

    Fang, Dai-Hua; Fan, Cong-Hai; Li, Juan; An, Qi; Yao, Hong; Ji, Qiang; Niu, Gao

    2015-02-19

    Neutrophil CD64 expression has been demonstrated as an improved diagnostic marker of infection and sepsis. The purpose of this study was to develop a new method to evaluate neutrophil CD64 expression for diagnosis of neonatal sepsis. Eighty neonates with neonatal sepsis (21 culture positive, 59 negative) were enrolled in this prospective study along with 19 neonates with no symptoms or signs of infection as controls. Expressions of CD64 on monocytes, lymphocytes, and neutrophils were evaluated with flow cytometry (FCM). Ratios were calculated with these levels of CD64 expression. Blood culture and other laboratory exams were done at the same time for the diagnosis of neonatal sepsis. Results were compared between the neonatal sepsis and control groups. CD64 ratios showed significant difference between the groups (p neonatal sepsis identification. The novel CD64 evaluation method, CD64 ratio, can be used as a supplementary method for diagnosis of neonatal sepsis.

  12. Expression of lymphocyte-derived growth hormone (GH) and GH-releasing hormone receptors in aging rats.

    Science.gov (United States)

    Weigent, Douglas A

    2013-04-01

    In the present study, we show that higher levels of lymphocyte GH are expressed in spleen cells from aging animals compared to young animals. Further, leukocytes from primary and secondary immune tissues and splenic T and B cells from aging rats all express higher levels of GHRH receptors compared to younger animals. Bone marrow and splenic T cells express the highest levels of GHRH receptor in aging animals. Spleen cells from aging animals showed no significant change in proliferation or GH induction after treatment with GHRH. Taken together, the data for the first time show alterations in GH synthesis and expression of the GHRH receptor on cells of the immune system that may play a role in the immune response in aging.

  13. Biological analysis of chronic lymphocytic leukemia: integration of mRNA and microRNA expression profiles.

    Science.gov (United States)

    Dong, L; Bi, K H; Huang, N; Chen, C Y

    2016-01-08

    Chronic lymphocytic leukemia (CLL) is a disease that involves progressive accumulation of nonfunctioning lymphocytes and has a low cure rate. There is an urgent requirement to determine the molecular mechanism underlying this disease in order to improve the early diagnosis and treatment of CLL. In this study, genes differentially expressed between CLL samples and age-matched controls were identified using microRNA (miRNA) and mRNA expression profiles. Differentially expressed (DE) miRNA targets were predicted by combining five algorithms. Common genes were obtained on overlapping the DE mRNA and DE miRNA targets. Then, network and module analyses were performed. A total of 239 miRNA targets were predicted and 357 DE mRNAs were obtained. On intersecting miRNA targets and DE mRNAs, 33 common genes were obtained. The protein-protein interaction network and module analysis identified several crucial genes and modules that might be associated with the development of CLL. These DE mRNAs were significantly enriched in the hematopoietic cell lineage (P = 2.58E-4), mitogen-activated protein kinase signaling pathway (P = 0.0025), and leukocyte transendothelial migration pathway (P = 0.0026). Thus, we conducted biological analysis on integration of DE mRNAs and DE miRNAs in CLL, determined gene expression patterns, and screened out several important genes that might be related to CLL.

  14. Expression of HLA class Ⅰ and Ⅱ on peripheral blood lymphocytes in HBV infection

    Institute of Scientific and Technical Information of China (English)

    WANG Chuan-xin; WANG Jin-feng; LIU Min; ZOU Xiong; YU Xiu-ping; YANG Xiao-jing; ZHENG Gui-xi

    2006-01-01

    @@ Persistent hepatitis B virus (HBV) infection is the most important reason for chronic hepatitis B,hepatic cirrhosis, and hepatocellular carcinoma.1 T lymphocytes, including CD4+ and CD8+ T cells, are major composition of host cellular immunity.Furthermore, CD8+ cells play a primary role in host immune reaction of anti-tumor and anti-infection.

  15. Expression of nerve growth factor mRNA in splenic lymphocytes of bronchial asthma rats and its influencing actors

    Institute of Scientific and Technical Information of China (English)

    Jihong Dai; Yonghong Wang; Haixia He

    2008-01-01

    BACKGROUND: Previous research has proved that nerve growth factor (NGF) participates in the onset of asthma by the induction of neurogenic inflammation.OBJECTIVE: To investigate the effect of interleukin-13 (IL-13) and interferon-γ(IFN-γ) on the expression of NGF mRNA in the splenic lymphocytes of bronchial asthma rats.DESIGN, TIME AND SETTING: The experiment, a completely randomized study based on cellular immunology, was performed in the Laboratory of Neurology in Chongqing Medical University and the Department of Clinical Pharmacy in College of Clinical Medicine, Chongqing Medical University (Chongqing, China) from January 2006 to April 2007.MATERIALS: Four adult male Wistar rats were used in this study. Rat IL-13, IFN-γprobe and the total RNA extraction kit were produced by Shanghai Sangon Biological Technology & Services Co., Ltd (China). The NGF ELISA kit was a product of Wuhan Boster Bioengineering Co., Ltd (China). A Du-70 automatic UV spectrophotometer was produced by Beckman Company (USA).METHODS: Rats were subjected to 1-mL intraperitoneal injections each containing 100 mg of ovalbumin, and were sensitized by using antigen solution, which was sensitized with 5×109 Bacillus pertussis and 100 mg aluminum hydroxide powder. Four rats were challenged with 1% ovalbumin using an ultrasonic nebulizer for 60 minutes to establish an asthmatic model. After rats were anesthetized, splenic lymphocytes were isolated and cultured in medium, which was supplemented with IL-13 or IFN-γ, for 0, 12, 24 or 48 hours. A parallel study was conducted with cultured splenic lymphocytes, which were divided into a control group, an IL-13 group and an IFN-γ group. Culture medium was added with different concentrations of IL-13 (10, 50, 100 μg/L) and IFN-γ (1, 10, 50 μg/L); 24 hours later, all samples were harvested.MAIN OUTCOME MEASURES: The expression levels of NGF mRNA were detected by reverse transcription-polymerase chain reaction.RESULTS: In the control group, the

  16. Homing receptor expression is deviated on CD56+ blood lymphocytes during pregnancy in Type 1 diabetic women.

    Directory of Open Access Journals (Sweden)

    Suzanne D Burke

    Full Text Available Type 1 Diabetes Mellitus (T1DM is characterized by an augmented pro-inflammatory immune state. This contributes to the increased risk for gestational complications observed in T1DM mothers. In normal pregnancies, critical immunological changes occur, including the massive recruitment of lymphocytes, particularly CD56bright NK cells, into early decidua basalis and a 2nd trimester shift towards Type 2 immunity. Decidual CD56bright NK cells arise at least partly from circulating progenitors expressing adhesion molecules SELL and ITGA4 and the chemokine receptors CXCR3 and CXCR4. In vitro studies show that T1DM reduces interactions between blood CD56+ NK cells and decidual endothelial cells by reducing SELL and ITGA4-based interactions. To address the mechanisms by which specific lymphocyte subsets may be recruited from the circulation during pregnancy and whether these mechanisms are altered in T1DM, flow cytometry was used to examine eight peripheral blood lymphocyte subsets (Type 1 (IL18R1+ and Type 2 (IL1RL1+ CD56bright NK, CD56dim NK, NKT and T cells from control and T1DM women. Blood was collected serially over pregnancy and postpartum, and lymphocytes were compared for expression of homing receptors SELL, ITGA4, CXCR3, and CXCR4. The decline of Type 1/Type 2 immune cells in normal pregnancy was driven by an increase in Type 2 cells that did not occur in T1DM. CD56bright NK cells from control women had the highest expression of all four receptors with greatest expression in 2nd trimester. At this time, these receptors were expressed at very low levels by CD56bright NK cells from TIDM patients. Type 1/Type 2 NKT cell ratios were not influenced by either pregnancy or TIDM. Our results suggest that T1DM alters immunological balances during pregnancy with its greatest impact on CD56bright NK cells. This implicates CD56bright NK cells in diabetic pregnancy complications.

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

    Directory of Open Access Journals (Sweden)

    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.

  18. Determination of quantitative and site-specific DNA methylation of perforin by pyrosequencing

    Directory of Open Access Journals (Sweden)

    Rajeevan Mangalathu S

    2009-06-01

    Full Text Available Abstract Background Differential expression of perforin (PRF1, a gene with a pivotal role in immune surveillance, can be attributed to differential methylation of CpG sites in its promoter region. A reproducible method for quantitative and CpG site-specific determination of perforin methylation is required for molecular epidemiologic studies of chronic diseases with immune dysfunction. Findings We developed a pyrosequencing based method to quantify site-specific methylation levels in 32 out of 34 CpG sites in the PRF1 promoter, and also compared methylation pattern in DNAs extracted from whole blood drawn into PAXgene blood DNA tubes (whole blood DNA or DNA extracted from peripheral blood mononuclear cells (PBMC DNA from the same normal subjects. Sodium bisulfite treatment of DNA and touchdown PCR were highly reproducible (coefficient of variation 1.63 to 2.18% to preserve methylation information. Application of optimized pyrosequencing protocol to whole blood DNA revealed that methylation level varied along the promoter in normal subjects with extremely high methylation (mean 86%; range 82–92% in the distal enhancer region (CpG sites 1–10, a variable methylation (range 49%–83% in the methylation sensitive region (CpG sites 11–17, and a progressively declining methylation level (range 12%–80% in the proximal promoter region (CpG sites 18–32 of PRF1. This pattern of methylation remained the same between whole blood and PBMC DNAs, but the absolute values of methylation in 30 out of 32 CpG sites differed significantly, with higher values for all CpG sites in the whole blood DNA. Conclusion This reproducible, site-specific and quantitative method for methylation determination of PRF1 based on pyrosequencing without cloning is well suited for large-scale molecular epidemiologic studies of diseases with immune dysfunction. PBMC DNA may be better suited than whole blood DNA for examining methylation levels in genes associated with immune

  19. 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.

  20. Receptor Activator of Nuclear Factor κB Ligand (RANKL) Protein Expression by B Lymphocytes Contributes to Ovariectomy-induced Bone Loss*

    Science.gov (United States)

    Onal, Melda; Xiong, Jinhu; Chen, Xinrong; Thostenson, Jeff D.; Almeida, Maria; Manolagas, Stavros C.; O'Brien, Charles A.

    2012-01-01

    Production of the cytokine receptor activator of NFκB ligand (RANKL) by lymphocytes has been proposed as a mechanism by which sex steroid deficiency causes bone loss. However, there have been no studies that functionally link RANKL expression in lymphocytes with bone loss in this condition. Herein, we examined whether RANKL expression in either B or T lymphocytes contributes to ovariectomy-induced bone loss in mice. Mice harboring a conditional RANKL allele were crossed with CD19-Cre or Lck-Cre mice to delete RANKL in B or T lymphocytes, respectively. Deletion of RANKL from either cell type had no impact on bone mass in estrogen-replete mice up to 7 months of age. However, mice lacking RANKL in B lymphocytes were partially protected from the bone loss caused by ovariectomy. This protection occurred in cancellous, but not cortical, bone and was associated with a failure to increase osteoclast numbers in the conditional knock-out mice. Deletion of RANKL from T lymphocytes had no impact on ovariectomy-induced bone loss. These results demonstrate that lymphocyte RANKL is not involved in basal bone remodeling, but B cell RANKL does contribute to the increase in osteoclasts and cancellous bone loss that occurs after loss of estrogen. PMID:22782898

  1. Expression of blood serum proteins and lymphocyte differentiation clusters after chronic occupational exposure to ionizing radiation.

    Science.gov (United States)

    Rybkina, Valentina L; Azizova, Tamara V; Scherthan, Harry; Meineke, Viktor; Doerr, Harald; Adamova, Galina V; Teplyakova, Olga V; Osovets, Sergey V; Bannikova, Maria V; Zurochka, Alexander V

    2014-11-01

    This study aimed to assess effects of chronic occupational exposure on immune status in Mayak workers chronically exposed to ionizing radiation (IR). The study cohort consists of 77 workers occupationally exposed to external gamma-rays at total dose from 0.5 to 3.0 Gy (14 individuals) and workers with combined exposure (external gamma-rays at total dose range 0.7-5.1 Gy and internal alpha-radiation from incorporated plutonium with a body burden of 0.3-16.4 kBq). The control group consists of 43 age- and sex-matched individuals who never were exposed to IR, never involved in any cleanup operations following radiation accidents and never resided at contaminated areas. Enzyme-linked immunoassay and flow cytometry were used to determine the relative concentration of lymphocytes and proteins. The concentrations of T-lymphocytes, interleukin-8 and immunoglobulins G were decreased in external gamma-exposed workers relative to control. Relative concentrations of NKT-lymphocytes, concentrations of transforming growth factor-β, interferon gamma, immunoglobulins A, immunoglobulins M and matrix proteinase-9 were higher in this group as compared with control. Relative concentrations of T-lymphocytes and concentration of interleukin-8 were decreased, while both the relative and absolute concentration of natural killers, concentration of immunoglobulins A and M and matrix proteinase-9 were increased in workers with combined exposure as compared to control. An inverse linear relation was revealed between absolute concentration of T-lymphocytes, relative and absolute concentration of T-helpers cells, concentration of interferon gamma and total absorbed dose from external gamma-rays in exposed workers. For workers with incorporated plutonium, there was an inverse linear relation of absolute concentration of T-helpers as well as direct linear relation of relative concentration of NKT-lymphocytes to total absorbed red bone marrow dose from internal alpha-radiation. In all, chronic

  2. Expression of blood serum proteins and lymphocyte differentiation clusters after chronic occupational exposure to ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Rybkina, Valentina L.; Azizova, Tamara V.; Adamova, Galina V.; Teplyakova, Olga V.; Osovets, Sergey V.; Bannikova, Maria V. [Southern Urals Biophysics Institute, Ozyorsk, Chelyabinsk Region (Russian Federation); Scherthan, Harry; Meineke, Viktor; Doerr, Harald [University of Ulm, Bundeswehr Institute of Radiobiology, Munich (Germany); Zurochka, Alexander V. [Immunology Institute, Yekaterinburg (Russian Federation)

    2014-11-15

    This study aimed to assess effects of chronic occupational exposure on immune status in Mayak workers chronically exposed to ionizing radiation (IR). The study cohort consists of 77 workers occupationally exposed to external gamma-rays at total dose from 0.5 to 3.0 Gy (14 individuals) and workers with combined exposure (external gamma-rays at total dose range 0.7-5.1 Gy and internal alpha-radiation from incorporated plutonium with a body burden of 0.3-16.4 kBq). The control group consists of 43 age- and sex-matched individuals who never were exposed to IR, never involved in any cleanup operations following radiation accidents and never resided at contaminated areas. Enzyme-linked immunoassay and flow cytometry were used to determine the relative concentration of lymphocytes and proteins. The concentrations of T-lymphocytes, interleukin-8 and immunoglobulins G were decreased in external gamma-exposed workers relative to control. Relative concentrations of NKT-lymphocytes, concentrations of transforming growth factor-β, interferon gamma, immunoglobulins A, immunoglobulins M and matrix proteinase-9 were higher in this group as compared with control. Relative concentrations of T-lymphocytes and concentration of interleukin-8 were decreased, while both the relative and absolute concentration of natural killers, concentration of immunoglobulins A and M and matrix proteinase-9 were increased in workers with combined exposure as compared to control. An inverse linear relation was revealed between absolute concentration of T-lymphocytes, relative and absolute concentration of T-helpers cells, concentration of interferon gamma and total absorbed dose from external gamma-rays in exposed workers. For workers with incorporated plutonium, there was an inverse linear relation of absolute concentration of T-helpers as well as direct linear relation of relative concentration of NKT-lymphocytes to total absorbed red bone marrow dose from internal alpha-radiation. In all, chronic

  3. Occupational Toluene Exposure Induces Cytochrome P450 2E1 mRNA Expression in Peripheral Lymphocytes

    OpenAIRE

    Mendoza-Cantú, Ania; Castorena-Torres, Fabiola; León,Mario Bermúdez de; Cisneros, Bulmaro; López-Carrillo, Lizbeth; Rojas-García, Aurora E.; Aguilar-Salinas, Alberto; Manno, Maurizio; Albores, Arnulfo

    2005-01-01

    Print workers are exposed to organic solvents, of which the systemic toxicant toluene is a main component. Toluene induces expression of cytochrome P450 2E1 (CYP2E1), an enzyme involved in its own metabolism and that of other protoxicants, including some procarcinogens. Therefore, we investigated the association between toluene exposure and the CYP2E1 response, as assessed by mRNA content in peripheral lymphocytes or the 6-hydroxychlorzoxazone (6OH-CHZ)/chlorzoxazone (CHZ) quotient (known as ...

  4. Time-dependent changes in the expression of lymphocyte and monocyte cell adhesion molecules after meals of different composition.

    Science.gov (United States)

    Torrecilla, Esther; González-Muñoz, Miguel; Lahoz, Carlos; Mostaza, Jose

    2010-12-01

    The objective of the present study was to compare the acute effect of meals of different composition on the expression of adhesion molecules that play a key role in leucocyte trafficking. A total of twenty apparently healthy subjects randomly consumed three isoenergetic meals 1 week apart: enriched in carbohydrates (CHO), enriched in monounsaturated fat and enriched in saturated fat. Blood samples were obtained before the meals and at 2, 4, 6, 8 and 10 h after meal ingestion. Samples were analysed for LDL resistance to Cu-mediated oxidation and for the surface expression on peripheral blood mononuclear cells (PBMC) of CD62L, CD162, CD11a, CD11b, CD49d and CD54 by flow cytometry. The present results showed that there were no changes in LDL susceptibility to oxidation within and among the meals. After the CHO-enriched meal, there was a time-dependent increased expression of CD162, CD49d, CD11a and CD54 on PBMC that returned to basal values after 8-10 h. These changes were significantly greater than the ones observed after the consumption of the monounsaturated fat- and the saturated fat-enriched meals and were more evident in lymphocytes than in monocytes. In conclusion, acute ingestion of a CHO-enriched meal induces higher increases of lymphocyte activation markers than fat-enriched meals. These results suggest that long-term consumption of CHO-enriched diets may be associated with a sustained pro-inflammatory state.

  5. Isopentenyl pyrophosphate-activated CD56+ {gamma}{delta} T lymphocytes display potent antitumor activity toward human squamous cell carcinoma.

    Science.gov (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

    2008-07-01

    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.

  6. Association between Up-regulation of Fas Ligand Expression and Apoptosis of Tumor-infiltrating Lymphocytes in Human Breast Cancer

    Institute of Scientific and Technical Information of China (English)

    CHENG Bo

    2006-01-01

    In order to study the significance of FasL expression in immune escape of breast cancer,FasL protein expression and the number of tumor-infiltrating lymphocytes (TILs) in 40 specimens of breast cancer were detected by immunohistochemitry. The expression of FasL mRNA was measured by in situ hybridization in the consecutive tissue slices of 40 breast cancers respectively. By using terminal deoxynucleotidyl transferase-mediaed dUTP nick end labeling (TUNEL), apoptotic cells were detected in 40 specimens of breast cancer. The expression of FasL was detected in all 40 specimens to varying degrees. In the consecutive tissue slices, the location of expression of FasL protein corresponded with that of FasL mRNA. In those with FasL extensive expression, the number of TILs was less (P<0.05), the apoptotic index (AI) of TILs was higher and the AI of tumor cells was lower (P<0.01) than those with FasL weak expression respectively. The AI of TILs was correlated with that of tumor cells (r=-0.629, P<0.01). In conclusion, breast cancer cells can induce the apoptosis of TILs through the expression of FasL, which can counterattack the immune system. This may be a mechanism of immune evasion in breast cancer.

  7. Cytotoxic T lymphocyte-dependent tumor growth inhibition by a vascular endothelial growth factor-superantigen conjugate

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Qingwen [Shanghai Chest Hospital, Shanghai 200433 (China); State Key Laboratory of Genetic Engineering, Fudan University, Shanghai 200433 (China); Jiang, Songmin [State Key Laboratory of Genetic Engineering, Fudan University, Shanghai 200433 (China); Han, Baohui [Shanghai Chest Hospital, Shanghai 200433 (China); Sun, Tongwen [Wuhan Junyu Innovation Pharmaceuticals, Inc., Wuhan 430079 (China); Li, Zhengnan; Zhao, Lina; Gao, Qiang [College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457 (China); Sun, Jialin, E-mail: jialin_sun@126.com [Wuhan Junyu Innovation Pharmaceuticals, Inc., Wuhan 430079 (China)

    2012-11-02

    Highlights: Black-Right-Pointing-Pointer We construct and purify a fusion protein VEGF-SEA. Black-Right-Pointing-Pointer VEGF-SEA strongly repressed the growth of murine solid sarcoma 180 (S180) tumors. Black-Right-Pointing-Pointer T cells driven by VEGF-SEA were accumulated around tumor cells bearing VEGFR by mice image model. Black-Right-Pointing-Pointer VEGF-SEA can serve as a tumor targeting agent and sequester CTLs into the tumor site. Black-Right-Pointing-Pointer The induced CTLs could release the cytokines, perforins and granzyme B to kill the tumor cells. -- Abstract: T cells are major lymphocytes in the blood and passengers across the tumor vasculature. If these T cells are retained in the tumor site, a therapeutic potential will be gained by turning them into tumor-reactive cytotoxic T lymphocytes (CTLs). A fusion protein composed of human vascular endothelial growth factor (VEGF) and staphylococcal enterotoxin A (SEA) with a D227A mutation strongly repressed the growth of murine solid sarcoma 180 (S180) tumors (control versus VEGF-SEA treated with 15 {mu}g, mean tumor weight: 1.128 g versus 0.252 g, difference = 0.876 g). CD4{sup +} and CD8{sup +} T cells driven by VEGF-SEA were accumulated around VEGFR expressing tumor cells and the induced CTLs could release the tumoricidal cytokines, such as interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). Meanwhile, intratumoral CTLs secreted cytolytic pore-forming perforin and granzyme B proteins around tumor cells, leading to the death of tumor cells. The labeled fusion proteins were gradually targeted to the tumor site in an imaging mice model. These results show that VEGF-SEA can serve as a tumor targeting agent and sequester active infiltrating CTLs into the tumor site to kill tumor cells, and could therefore be a potential therapeutical drug for a variety of cancers.

  8. Novel function of perforin in negatively regulating CD4+T cell activation by affecting calcium signaling

    Institute of Scientific and Technical Information of China (English)

    Enguang Bi; Kairui Mao; Jia Zou; Yuhan Zheng; Bing Sun; Chunjian Huang; Yu Hu; Xiaodong Wu; Weiwen Deng; Guomei Lin; Zhiduo Liu; Lin Tian; Shuhui Sun

    2009-01-01

    Perforin is a pore-forming protein engaged mainly in mediating target T cell death and is employed by cytotoxic Tlymphocytes (CTLs) and natural killer cells. However, whether it also plays a role in conventional CD4+ T cell func-tion remains unclear. Here we report that in perforin-deficient (PKO) mice, CD4+ T cells are hyperproliferative in response to T cell receptor (TCR) stimulation. This feature of hyperproliferation is accompanied by the enhancement both in cell division and in IL-2 secretion. It seems that the perforin deficiency does not influence T cell development in thymus spleen and lymph node. In vivo, perforin deficiency results in increased antigen-specific T cell prolifera-tion and antibody production. Furthermore, PKO mice are more susceptible to experimental autoimmune uveitis. To address the molecular mechanism, we found that after TCR stimulation, CD44 T cells from PKO mice display an increased intracellular calcium flux and subsequently enhance activation of transcription factor NFATI. Our results indicate that perforin plays a negative role in regulating CD4+ T cell activation and immune response by affecting TCR-dependent Ca2+ signaling.

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

    Directory of Open Access Journals (Sweden)

    Smith Lorraine P

    2010-05-01

    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

  10. LDOC1 mRNA is differentially expressed in chronic lymphocytic leukemia and predicts overall survival in untreated patients

    Science.gov (United States)

    Duzkale, Hatice; Schweighofer, Carmen D.; Coombes, Kevin R.; Barron, Lynn L.; Ferrajoli, Alessandra; O'Brien, Susan; Wierda, William G.; Pfeifer, John; Majewski, Tadeusz; Czerniak, Bogdan A.; Jorgensen, Jeffrey L.; Medeiros, L. Jeffrey; Freireich, Emil J; Keating, Michael J.

    2011-01-01

    We previously identified LDOC1 as one of the most significantly differentially expressed genes in untreated chronic lymphocytic leukemia (CLL) patients with respect to the somatic mutation status of the immunoglobulin heavy-chain variable region genes. However, little is known about the normal function of LDOC1, its contribution to the pathophysiology of CLL, or its prognostic significance. In this study, we have investigated LDOC1 mRNA expression in a large cohort of untreated CLL patients, as well as in normal peripheral blood B-cell (NBC) subsets and primary B-cell lymphoma samples. We have confirmed that LDOC1 is dramatically down-regulated in mutated CLL cases compared with unmutated cases, and have identified a new splice variant, LDOC1S. We show that LDOC1 is expressed in NBC subsets (naive > memory), suggesting that it may play a role in normal B-cell development. It is also expressed in primary B-cell lymphoma samples, in which its expression is associated with somatic mutation status. In CLL, we show that high levels of LDOC1 correlate with biomarkers of poor prognosis, including cytogenetic markers, unmutated somatic mutation status, and ZAP70 expression. Finally, we demonstrate that LDOC1 mRNA expression is an excellent predictor of overall survival in untreated CLL patients. PMID:21310924

  11. Low dose decitabine treatment induces CD80 expression in cancer cells and stimulates tumor specific cytotoxic T lymphocyte responses.

    Science.gov (United States)

    Wang, Li-Xin; Mei, Zhen-Yang; Zhou, Ji-Hao; Yao, Yu-Shi; Li, Yong-Hui; Xu, Yi-Han; Li, Jing-Xin; Gao, Xiao-Ning; Zhou, Min-Hang; Jiang, Meng-Meng; Gao, Li; Ding, Yi; Lu, Xue-Chun; Shi, Jin-Long; Luo, Xu-Feng; Wang, Jia; Wang, Li-Li; Qu, Chunfeng; Bai, Xue-Feng; Yu, Li

    2013-01-01

    Lack of immunogenicity of cancer cells has been considered a major reason for their failure in induction of a tumor specific T cell response. In this paper, we present evidence that decitabine (DAC), a DNA methylation inhibitor that is currently used for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and other malignant neoplasms, is capable of eliciting an anti-tumor cytotoxic T lymphocyte (CTL) response in mouse EL4 tumor model. C57BL/6 mice with established EL4 tumors were treated with DAC (1.0 mg/kg body weight) once daily for 5 days. We found that DAC treatment resulted in infiltration of IFN-γ producing T lymphocytes into tumors and caused tumor rejection. Depletion of CD8(+), but not CD4(+) T cells resumed tumor growth. DAC-induced CTL response appeared to be elicited by the induction of CD80 expression on tumor cells. Epigenetic evidence suggests that DAC induces CD80 expression in EL4 cells via demethylation of CpG dinucleotide sites in the promoter of CD80 gene. In addition, we also showed that a transient, low-dose DAC treatment can induce CD80 gene expression in a variety of human cancer cells. This study provides the first evidence that epigenetic modulation can induce the expression of a major T cell co-stimulatory molecule on cancer cells, which can overcome immune tolerance, and induce an efficient anti-tumor CTL response. The results have important implications in designing DAC-based cancer immunotherapy.

  12. Low dose decitabine treatment induces CD80 expression in cancer cells and stimulates tumor specific cytotoxic T lymphocyte responses.

    Directory of Open Access Journals (Sweden)

    Li-Xin Wang

    Full Text Available Lack of immunogenicity of cancer cells has been considered a major reason for their failure in induction of a tumor specific T cell response. In this paper, we present evidence that decitabine (DAC, a DNA methylation inhibitor that is currently used for the treatment of myelodysplastic syndrome (MDS, acute myeloid leukemia (AML and other malignant neoplasms, is capable of eliciting an anti-tumor cytotoxic T lymphocyte (CTL response in mouse EL4 tumor model. C57BL/6 mice with established EL4 tumors were treated with DAC (1.0 mg/kg body weight once daily for 5 days. We found that DAC treatment resulted in infiltration of IFN-γ producing T lymphocytes into tumors and caused tumor rejection. Depletion of CD8(+, but not CD4(+ T cells resumed tumor growth. DAC-induced CTL response appeared to be elicited by the induction of CD80 expression on tumor cells. Epigenetic evidence suggests that DAC induces CD80 expression in EL4 cells via demethylation of CpG dinucleotide sites in the promoter of CD80 gene. In addition, we also showed that a transient, low-dose DAC treatment can induce CD80 gene expression in a variety of human cancer cells. This study provides the first evidence that epigenetic modulation can induce the expression of a major T cell co-stimulatory molecule on cancer cells, which can overcome immune tolerance, and induce an efficient anti-tumor CTL response. The results have important implications in designing DAC-based cancer immunotherapy.

  13. Occupational Toluene Exposure Induces Cytochrome P450 2E1 mRNA Expression in Peripheral Lymphocytes

    Science.gov (United States)

    Mendoza-Cantú, Ania; Castorena-Torres, Fabiola; de León, Mario Bermúdez; Cisneros, Bulmaro; López-Carrillo, Lizbeth; Rojas-García, Aurora E.; Aguilar-Salinas, Alberto; Manno, Maurizio; Albores, Arnulfo

    2006-01-01

    Print workers are exposed to organic solvents, of which the systemic toxicant toluene is a main component. Toluene induces expression of cytochrome P450 2E1 (CYP2E1), an enzyme involved in its own metabolism and that of other protoxicants, including some procarcinogens. Therefore, we investigated the association between toluene exposure and the CYP2E1 response, as assessed by mRNA content in peripheral lymphocytes or the 6-hydroxychlorzoxazone (6OH-CHZ)/chlorzoxazone (CHZ) quotient (known as CHZ metabolic ratio) in plasma, and the role of genotype (5′-flanking region RsaI/PstI polymorphic sites) in 97 male print workers. The geometric mean (GM) of toluene concentration in the air was 52.80 ppm (10–760 ppm); 54% of the study participants were exposed to toluene concentrations that exceeded the maximum permissible exposure level (MPEL). The GM of urinary hippuric acid at the end of a work shift (0.041 g/g creatinine) was elevated relative to that before the shift (0.027 g/g creatinine; p < 0.05). The GM of the CHZ metabolic ratio was 0.33 (0–9.3), with 40% of the subjects having ratios below the GM. However, the average CYP2E1 mRNA level in peripheral lymphocytes was 1.07 (0.30–3.08), and CYP2E1 mRNA levels within subjects correlated with the toluene exposure ratio (environmental toluene concentration:urinary hippuric acid concentration) (p = 0.014). Genotype did not alter the association between the toluene exposure ratio and mRNA content. In summary, with further validation, CYP2E1 mRNA content in peripheral lymphocytes could be a sensitive and noninvasive biomarker for the continuous monitoring of toluene effects in exposed persons. PMID:16581535

  14. Occupational toluene exposure induces cytochrome P450 2E1 mRNA expression in peripheral lymphocytes.

    Science.gov (United States)

    Mendoza-Cantú, Ania; Castorena-Torres, Fabiola; Bermúdez de León, Mario; Cisneros, Bulmaro; López-Carrillo, Lizbeth; Rojas-García, Aurora E; Aguilar-Salinas, Alberto; Manno, Maurizio; Albores, Arnulfo

    2006-04-01

    Print workers are exposed to organic solvents, of which the systemic toxicant toluene is a main component. Toluene induces expression of cytochrome P450 2E1 (CYP2E1), an enzyme involved in its own metabolism and that of other protoxicants, including some procarcinogens. Therefore, we investigated the association between toluene exposure and the CYP2E1 response, as assessed by mRNA content in peripheral lymphocytes or the 6-hydroxychlorzoxazone (6OH-CHZ)/chlorzoxazone (CHZ) quotient (known as CHZ metabolic ratio) in plasma, and the role of genotype (5 -flanking region RsaI/PstI polymorphic sites) in 97 male print workers. The geometric mean (GM) of toluene concentration in the air was 52.80 ppm (10-760 ppm); 54% of the study participants were exposed to toluene concentrations that exceeded the maximum permissible exposure level (MPEL). The GM of urinary hippuric acid at the end of a work shift (0.041 g/g creatinine) was elevated relative to that before the shift (0.027 g/g creatinine; p < 0.05). The GM of the CHZ metabolic ratio was 0.33 (0-9.3), with 40% of the subjects having ratios below the GM. However, the average CYP2E1 mRNA level in peripheral lymphocytes was 1.07 (0.30-3.08), and CYP2E1 mRNA levels within subjects correlated with the toluene exposure ratio (environmental toluene concentration:urinary hippuric acid concentration) (p = 0.014). Genotype did not alter the association between the toluene exposure ratio and mRNA content. In summary, with further validation, CYP2E1 mRNA content in peripheral lymphocytes could be a sensitive and noninvasive biomarker for the continuous monitoring of toluene effects in exposed persons.

  15. Lack of galectin-3 up-regulates IgA expression by peritoneal B1 lymphocytes during B cell differentiation.

    Science.gov (United States)

    Oliveira, Felipe L; Bernardes, Emerson S; Brand, Camila; dos Santos, Sofia N; Cabanel, Mariana P; Arcanjo, Kátia D; Brito, José M; Borojevic, Radovan; Chammas, Roger; El-Cheikh, Márcia C

    2016-02-01

    Galectin-3 is a β-galactoside-binding protein with an inhibitory role in B cell differentiation into plasma cells in distinct lymphoid tissues. We use a model of chronic schistosomiasis, a well-characterized experimental disease hallmarked by polyclonal B cell activation, in order to investigate the role of galectin-3 in controlling IgA production through peritoneal B1 cells. Chronically infected, galectin-3-deficient mice (Lgals3(-/-)) display peritoneal fluid hypercellularity, increased numbers of atypical peritoneal IgM(+)/IgA(+) B1a and B1b lymphocytes and histological disturbances in plasma cell niches when compared with Lgals3(+/+) mice. Similar to our infection model, peritoneal B1 cells from uninfected Lgals3(-/-) mice show enhanced switching to IgA after in vitro treatment with interleukin-5 plus transforming growth factor-β (IL-5 + TGF-β1). A higher number of IgA(+) B1a lymphocytes was found in the peritoneal cavity of Lgals3(-/-)-uninfected mice at 1 week after i.p. injection of IL-5 + TGF-β1; this correlates with the increased levels of secreted IgA detected in the peritoneal fluid of these mice after cytokine treatment. Interestingly, a higher number of degranulated mast cells is present in the peritoneal cavity of uninfected and Schistosoma mansoni-infected Lgals3(-/-) mice, indicating that, at least in part, mast cells account for the enhanced differentiation of B1 into IgA-producing B cells found in the absence of galectin-3. Thus, a novel role is revealed for galectin-3 in controlling the expression of surface IgA by peritoneal B1 lymphocytes; this might have important implications for manipulating the mucosal immune response.

  16. Prognostic value of the CD4+/CD8+ ratio of tumour infiltrating lymphocytes in colorectal cancer and HLA-DR expression on tumour cells

    DEFF Research Database (Denmark)

    Diederichsen, Axel Cosmus Pyndt; Hjelmborg, J v B; Christensen, Per B

    2003-01-01

    clinical course, with significantly higher 5-year survival, p=0.046, independent of the Dukes stage and age. Our results have implications for tumour immunology; colorectal cancer cells might be a target for cytotoxic T-lymphocytes, however the tumour cells are not able to initiate an immune response......The purpose of this study was to clarify whether HLA-DR expression of colorectal tumour cells or the CD4+/CD8+ ratio of the tumour infiltrating lymphocytes is significantly associated with the prognosis of colorectal cancer. Using flow cytometry, we studied the tumour cell expression of the HLA...... class II in 70 enzymatically dissociated colorectal cancers and the phenotype of tumour infiltrating lymphocytes (TILs) in 41 cases. There was no trend in 5-year survival between three levels (low, medium, high) of HLA-DR expression on the tumour cells. Patients with low CD4+/CD8+ ratios had a better...

  17. Using the geometric mean fluorescence intensity index method to measure ZAP-70 expression in patients with chronic lymphocytic leukemia

    Directory of Open Access Journals (Sweden)

    Wu YJ

    2016-02-01

    Full Text Available Yu-Jie Wu, Hui Wang, Jian-Hua Liang, Yi Miao, Lu Liu, Hai-Rong Qiu, Chun Qiao, Rong Wang, Jian-Yong Li Department of Hematology, First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Nanjing, People’s Republic of China Abstract: Expression of ζ-chain-associated protein kinase 70 kDa (ZAP-70 in chronic lymphocytic leukemia (CLL is associated with more aggressive disease and can help differentiate CLL from cases expressing mutated or unmutated immunoglobulin heavy chain variable region (IgHV genes. However, standardizing ZAP-70 expression by flow cytometric analysis has proved unsatisfactory. The key point is that ZAP-70 is weakly expressed with a continuous expression pattern rather than a clear discrimination between positive and negative CLL cells, which means that the resulting judgment is subjective. Thus, in this study, we aimed at assessing the reliability and repeatability of ZAP-70 expression using the geometric mean fluorescence intensity (geo MFI index method based on flow cytometry with 256-channel resolution in a series of 402 CLL patients and to compare ZAP-70 with other biological and clinical prognosticators. According to IgHV mutational status, we were able to confirm that the optimal cut-off point for the geo MFI index was 3.5 in the test set. In multivariate analyses that included the major clinical and biological prognostic markers for CLL, the prognostic impact of ZAP-70 expression appeared to have stronger discriminatory power when the geo MFI index method was applied. In addition, we found that ZAP-70-positive patients according to the geo MFI index method had shorter time to first treatment or overall survival (P=0.0002, P=0.0491. This is the first report showing that ZAP-70 expression can be evaluated by a new approach, the geo MFI index, which could be a useful prognostic method as it is more reliable, less subjective, and therefore better associated with improvement of CLL prognostication

  18. The role of Ly49E receptor expression on murine intraepithelial lymphocytes in intestinal cancer development and progression.

    Science.gov (United States)

    Van Acker, Aline; Louagie, Els; Filtjens, Jessica; Taveirne, Sylvie; Van Ammel, Els; Kerre, Tessa; Elewaut, Dirk; Taghon, Tom; Vandekerckhove, Bart; Plum, Jean; Leclercq, Georges

    2016-11-01

    Ly49E is a member of the Ly49 family of NK receptors and is distinct from other members of this family on the basis of its structural properties, expression pattern and ligand recognition. Importantly, Ly49E receptor expression is high on small intestinal and colonic intraepithelial lymphocytes (IELs). Intestinal IELs are regulators of the mucosal immune system and contribute to front-line defense at the mucosal barrier, including anti-tumor immune response. Whereas most Ly49 receptors have MHC class-I ligands, we showed that Ly49E is instead triggered by urokinase plasminogen activator (uPA). uPA has been extensively implicated in tumor development, where increased uPA expression correlates with poor prognosis. As such, we investigated the role of Ly49E receptor expression on intestinal IELs in the anti-tumor immune response. For this purpose, we compared Ly49E wild-type mice to Ly49E knockout mice in two established tumor models: Apc(Min/+)-mediated and azoxymethane-induced intestinal cancer. Our results indicate that Ly49E expression on IELs does not influence the development or progression of intestinal cancer.

  19. Cytotoxic T lymphocytes promote cytarabine-induced acute myeloid leukemia cell apoptosis via inhibiting Bcl-2 expression.

    Science.gov (United States)

    Deng, Rui; Fan, Fang-Yi; Yi, Hai; Fu, Li; Zeng, Yan; Wang, Yi; Miao, Xiao-Juan; Shuai, Yan-Rong; He, Guang-Cui; Su, Yi

    2017-08-01

    Acute myeloid leukemia (AML) remains difficult to cure due to its drug tolerance and refractoriness. Immunotherapy is a growing area of cancer research, which has been applied for the treatment of numerous types of cancer, including leukemia. The present study generated AML cell-specific cytotoxic T lymphocytes (CTLs) in vitro and investigated the effect of combining CTL treatment with one of the most commonly used drugs for the treatment of hematological malignancies, cytarabine, on AML cell apoptosis. Firstly, it was observed that monocyte-depleted peripheral blood lymphocytes from healthy donors could be used to generate large numbers of CD3(+)CD8(+) CTLs through immune stimulation. These CD3(+)CD8(+) CTLs could effectively recognize and induce the apoptosis of human Kasumi-3 AML cells. In addition, cytarabine-induced AML cell apoptosis was enhanced by CTL treatment. Western blotting revealed that Bcl-2 expression was downregulated in AML cells following cytarabine and CTL treatment, indicating that the synergistic effect of this treatment on AML cell apoptosis is due to the downregulation of Bcl-2. These results highlight the potential application of CTL immunotherapy for the treatment of AML. Further studies optimizing the specificity and potency of CTLs, and identifying favorable combinations with other chemotherapeutic drug are required.

  20. Construction, Expression and in vitro Biological Effects of Idiotype Ig Fab Fragment of B-Chronic Lymphocytic Leukemia

    Institute of Scientific and Technical Information of China (English)

    Feng WANG; Ping LEI; Ping HU; Lijuan ZHU; Huifeng ZHU; Yue ZHANG; Jing YANG; Guanxin SHEN

    2008-01-01

    Summary: The purpose of this study was to construct expression vectors of idiotype (Id) Smlg in patients with B-chronic lymphocytic leukemia and to express them in E.coli to obtain recombinant Id,and to investigate the effect of the protein on the proliferation and secretion of IL-2 and IFN-γ of stimulated peripheral blood mononuclear cells (PBMC) in vitro. Light chain gene and Fd fragment of heavy chain gene were inserted into fd-tet-DOG2 vector to construct fd-tet-DOG2-Fab. Fab gene was further cloned into expression vector pHEN2 to construct the soluble expression vector pHEN2-Fab. After induction by IPTG, Fab protein was purified by Ni-NTA-chromatography. MTT was used to determine the effects of purified protein on the proliferation of stimulated PBMC in vitro and the concentrations of IL-2 and IFN-γ in the culture supernatants were detected by ELISA. The results showed that recombinant pHEN2-Fab expression vector was constructed successfully. Fab protein was expressed in positive clone after induced by 1PTG and two specific bands at 24-25 kD position were observed by SDS-PAGE electrophoresis. Proliferation of PBMC could be induced by purified Fab and the concentrations of IL-2 and IFN-γ, in culture supernatants were increased significantly after induction. It was suggested that the expression vector of SmIg Fab fragment was constructed successfully, and expressed and secreted from E. Coli. The Fab protein could induce proliferation of PBMC and promote secretion of IL-2 and IFN-γ.

  1. 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

    2009-01-01

    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.

  2. Genotype-Associated Differential NKG2D Expression on CD56+CD3+ Lymphocytes Predicts Response to Pegylated-Interferon/Ribavirin Therapy in Chronic Hepatitis C.

    Directory of Open Access Journals (Sweden)

    Po-sung Chu

    Full Text Available Hepatitis C virus (HCV genotype 1 infections are significantly more difficult to eradicate with PEG-IFN/ribavirin therapy, compared to HCV genotype 2. The aim of this work is to investigate the difference of immunological impairments underlying this phenomenon. Pre-treatment NKG2D expression on peripheral CD56+CD3+ lymphocytes and CD56+CD3- NK cells from cases of chronic hepatitis C were analyzed and assessed by treatment effect. Two strains of HCV were used to co-incubate with immune cells in vitro. NKG2D expression on peripheral CD56+CD3+ lymphocytes, but not NK cells, was significantly impaired in genotype 1 infection, compared to genotype 2. When peripheral blood mononuclear cells from healthy donors were co-incubated with TNS2J1, a genotype 1b/2a chimera strain, or with JFH1, a genotype 2a strain, genotype-specific decrease of NKG2D on CD56+CD3+ lymphocytes, but not NK cells, was observed. Pre-treatment NKG2D expression on peripheral CD56+CD3+ lymphocytes significantly correlated with reduction in serum HCV RNA levels from week 0 to week 4, and predicted treatment response. Ex vivo stimulation of peripheral CD56+CD3+ lymphocytes showed NKG2D expression-correlated IFN-γ production. In conclusion, Decreased NKG2D expression on CD56+CD3+ lymphocytes in chronic HCV genotype 1 infection predicts inferior treatment response to PEG-IFN/ribavirin therapy compared to genotype 2.

  3. Consequences of cytotoxic T lymphocyte interaction with major histocompatibility complex class I-expressing neurons in vivo.

    Science.gov (United States)

    Rall, G F; Mucke, L; Oldstone, M B

    1995-11-01

    Neurons have evolved strategies to evade immune surveillance that include an inability to synthesize the heavy chain of the class I major histocompatibility complex (MHC), proteins that are necessary for cytotoxic T lymphocyte (CTL) recognition of target cells. Multiple viruses have taken advantage of the lack of CTL-mediated recognition and killing of neurons by establishing persistent neuronal infections and thereby escaping attack by antiviral CTL. We have expressed a class I MHC molecule (Db) in neurons of transgenic mice using the neuron-specific enolase (NSE) promoter to determine the pathogenic consequences of CTL recognition of virally infected, MHC-expressing central nervous system (CNS) neurons. The NSE-Db transgene was expressed in H-2b founder mice, and transgene-derived messenger RNA was detected by reverse transcriptase-polymerase chain reaction in transgenic brains from several lines. Purified primary neurons from transgenic but not from nontransgenic mice adhered to coverslips coated with a conformation-dependent monoclonal antibody directed against the Dv molecule and presented viral peptide to CTL in an MHC-restricted manner, indicating that the Db molecule was expressed on transgenic neurons in a functional form. Transgenic mice infected with the neurotropic lymphocytic choriomeningitis virus (LCMV) and given anti-LCMV, MHC-restricted CTL displayed a high morbidity and mortality when compared with controls receiving MHC-mismatched CTL or expressing alternative transgenes. After CTL transfer, transgenic brains showed an increased number of CD8+ cells compared with nontransgenic controls as well as an increased rate of clearance of infectious virus from the CNS. Additionally, an increase in blood-brain barrier permeability was detected during viral clearance in NSE-Db transgenic mice and lasted several months after clearance of virus from neurons. In contrast, LCMV-infected, nontransgenic littermates and mice expressing other gene products from the

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

    Directory of Open Access Journals (Sweden)

    Ochoa-Hernández Alejandra B

    2012-02-01

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

  5. Expression of selectins and P-selectin glycoprotein ligand-1 in dogs with lymphocytic-plasmacytic enteritis.

    Science.gov (United States)

    Okanishi, Hiroki; Kagawa, Yumiko; Watari, Toshihiro

    2014-09-15

    Lymphocytic-plasmacytic enteritis (LPE) is the most common form of inflammatory bowel disease (IBD) affecting the canine small intestine; however, the molecular pathogenesis of this disease remains unclear. Although selectins and their ligands play a critical role as cell adhesion molecules during inflammation, there is very little information about their involvement in canine LPE. The aim of this study was to evaluate transcript expression of selectins (E-, L-, and P- selectin) and P-selectin glycoprotein ligand-1 (PSGL-1) in the duodenal mucosa of 21 dogs with LPE and 10 healthy laboratory beagles. Duodenal expression of E-selectin, L-selectin, P-selectin, and PSGL-1 was quantified by real-time reverse-transcription PCR. Correlations between clinical severity, histopathological grade, selectins, and PSGL-1 were analyzed by Spearman's rank test. Transcript expression of duodenal E- and P-selectins and PSGL-1 was higher in dogs with LPE than in healthy laboratory beagles; however, there was no difference in L-selectin expression. Positive correlations between E- and L-selectin and between L- and P-selectin were observed in the duodenum of LPE dogs. The selectins and ligand may recruit circulating inflammatory cells into the lesion. These findings improve our understanding of the inflammatory cascade of canine LPE. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Increased expression of SLAM receptors SLAMF3 and SLAMF6 in systemic lupus erythematosus T lymphocytes promotes Th17 differentiation.

    Science.gov (United States)

    Chatterjee, Madhumouli; Rauen, Thomas; Kis-Toth, Katalin; Kyttaris, Vasileios C; Hedrich, Christian M; Terhorst, Cox; Tsokos, George C

    2012-02-01

    Altered T cell function in systemic lupus erythematosus (SLE) is determined by various molecular and cellular abnormalities, including increased IL-17 production. Recent evidence suggests a crucial role for signaling lymphocyte activation molecules (SLAMs) in the expression of autoimmunity. In this study, we demonstrate that SLAMF3 and SLAMF6 expression is increased on the surface of SLE T cells compared with normal cells. SLAM coengagement with CD3 under Th17 polarizing conditions results in increased IL-17 production. SLAMF3 and SLAMF6 T cell surface expression and IL-17 levels significantly correlate with disease activity in SLE patients. Both naive and memory CD4(+) T cells produce more IL-17 in response to SLAM costimulation as compared with CD28 costimulation. In naive CD4(+) cells, IL-17 production after CD28 costimulation peaks on day 3, whereas costimulation with anti-SLAMF3 and anti-SLAMF6 Abs results in a prolonged and yet increasing production during 6 d. Unlike costimulation with anti-CD28, SLAM costimulation requires the presence of the adaptor molecule SLAM-associated protein. Thus, engagement of SLAMF3 and SLAMF6 along with Ag-mediated CD3/TCR stimulation represents an important source of IL-17 production, and disruption of this interaction with decoy receptors or blocking Abs should mitigate disease expression in SLE and other autoimmune conditions.

  7. High expression of PI3K core complex genes is associated with poor prognosis in chronic lymphocytic leukemia

    DEFF Research Database (Denmark)

    Kristensen, Louise; Kielsgaard Kristensen, Thomas; Abildgaard, Niels;

    2015-01-01

    Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults in the Western world. Autophagy is a highly conserved process in eukaryotic cells. In CLL autophagy is involved in mediating the effect of chemotherapy but the role of autophagy in CLL pathogenesis remains unknown....... In the present study, we used real-time RT-PCR to analyze expression of the PIK3C3, PIK3R4, and BECN1 genes. These genes encode the components of the PI3K core complex, which is central to initiation of autophagy. A consecutive series of 149 well-characterized CLL cases from Region of Southern Denmark were...... on the role of autophagy in CLL, and they may further represent targets of treatment....

  8. Activated peripheral lymphocytes with increased expression of cell adhesion molecules and cytotoxic markers are associated with dengue fever disease

    Directory of Open Access Journals (Sweden)

    Elzinandes L Azeredo

    2006-06-01

    Full Text Available The immune mechanisms involved in dengue fever and dengue hemorrhagic/dengue shock syndrome are not well understood. The ex vivo activation status of immune cells during the dengue disease in patients was examined. CD4and CD8 T cells were reduced during the acute phase. Interestingly, CD8 T cells co-expressing activation marker HLA-DR, Q, P, and cytolytic granule protein-Tia-1 were significantly higher in dengue patients than in controls. Detection of adhesion molecules indicated that in dengue patients the majority of T cells (CD4 and CD8 express the activation/memory phenotype, characterized as CD44HIGH and lack the expression of the naïve cell marker, CD62L LOW. Also, the levels of T cells co-expressing ICAM-1 (CD54, VLA-4, and LFA-1 (CD11a were significantly increased. CD8 T lymphocytes expressed predominantly low levels of anti-apoptotic molecule Bcl-2 in the acute phase, possibly leading to the exhibition of a phenotype of activated/effector cells. Circulating levels of IL-18, TGF-b1 and sICAM-1 were significantly elevated in dengue patients. Early activation events occur during acute dengue infection which might contribute to viral clearance. Differences in expression of adhesion molecules among CD4 and CD8 T cells might underlie the selective extravasation of these subsets from blood circulation into lymphoid organs and/or tissues. In addition, activated CD8 T cells would be more susceptible to apoptosis as shown by the alteration in Bcl-2 expression. Cytokines such as IL-18, TGF-b1, and sICAM-1 may be contributing by either stimulating or suppressing the adaptative immune response, during dengue infection, thereby perhaps establishing a relationship with disease severity.

  9. Activated peripheral lymphocytes with increased expression of cell adhesion molecules and cytotoxic markers are associated with dengue fever disease.

    Science.gov (United States)

    Azeredo, Elzinandes L; Zagne, Sonia M O; Alvarenga, Allan R; Nogueira, Rita M R; Kubelka, Claire F; de Oliveira-Pinto, Luzia M

    2006-06-01

    The immune mechanisms involved in dengue fever and dengue hemorrhagic/dengue shock syndrome are not well understood. The ex vivo activation status of immune cells during the dengue disease in patients was examined. CD4 and CD8 T cells were reduced during the acute phase. Interestingly, CD8 T cells co-expressing activation marker HLA-DR, Q, P, and cytolytic granule protein-Tia-1 were significantly higher in dengue patients than in controls. Detection of adhesion molecules indicated that in dengue patients the majority of T cells (CD4 and CD8) express the activation/memory phenotype, characterized as CD44HIGH and lack the expression of the naïve cell marker, CD62L LOW. Also, the levels of T cells co-expressing ICAM-1 (CD54), VLA-4, and LFA-1 (CD11a) were significantly increased. CD8 T lymphocytes expressed predominantly low levels of anti-apoptotic molecule Bcl-2 in the acute phase, possibly leading to the exhibition of a phenotype of activated/effector cells. Circulating levels of IL-18, TGF-b1 and sICAM-1 were significantly elevated in dengue patients. Early activation events occur during acute dengue infection which might contribute to viral clearance. Differences in expression of adhesion molecules among CD4 and CD8 T cells might underlie the selective extravasation of these subsets from blood circulation into lymphoid organs and/or tissues. In addition, activated CD8 T cells would be more susceptible to apoptosis as shown by the alteration in Bcl-2 expression. Cytokines such as IL-18, TGF-b1, and sICAM-1 may be contributing by either stimulating or suppressing the adaptative immune response, during dengue infection, thereby perhaps establishing a relationship with disease severity.

  10. Dicer Gene Expression as a Prognostic Factor in Acute Lymphoblastic Leukemia and Chronic Lymphocytic Leukemia in Fars Province

    Directory of Open Access Journals (Sweden)

    Mohamad Reza Farzaneh

    2016-05-01

    Full Text Available Alterations in the expression of microRNAs (miRNAs have been proposed to play a role in the pathogenesis of acute lymphoblastic leukemia (ALL and chronic lymphocytic leukemia (CLL. Dicer is one of the main regulators of miRNA biogenesis, and deregulation of its expression has been indicated as a possible cause of miRNA alterations observed in various cancers. Our aim was to analyze the expression of the Dicer protein and its relationship with ALL and CLL. This cross-sectional study was performed from 2010 to 2012 in Shahid Faghihi Hospital, Shiraz, Iran. In this study, 30 patients with CLL, 21 patients with ALL, 10 child healthy donors, and 19 adult healthy donors were recruited. The patients’ samples were checked via flow cytometry, immunohistochemistry, and immunocytochemistry. The controls’ samples were also examined in the hematology ward. Total RNA was extracted from the bone marrow and peripheral blood samples of the patients and controls. Then, reverse-transcription polymerase chain reaction was used to estimate the level of Dicer miRNA. The outcomes of the expression analysis of Dicer revealed statistically significant differences between the ALL patients/child healthy controls (mean±SD, 0.19±0.28 vs. 0.73±0.12; P<0.001 and the CLL patients/adult healthy controls (mean±SD, 0.24±0.25 vs. 0.41±0.28; P=0.033. This is the first piece of evidence showing that the expression of the Dicer gene greatly decreased in the patients with ALL in comparison to the child controls. The expression of the Dicer gene was also downregulated in the patients with CLL compared to the adult controls. Given the above findings, the expression of Dicer may play an important role in the progression and prognosis of these diseases.

  11. Prognostic value of ZAP-70 expression in chronic lymphocytic leukemia as assessed by quantitative polymerase chain reaction and flow cytometry.

    Science.gov (United States)

    Adams, Rebecca L C; Cheung, Catherine; Banh, Raymond; Saal, Russell; Cross, Donna; Gill, Devinder; Self, Marlene; Klein, Kerenaftali; Mollee, Peter

    2014-03-01

    Chronic lymphocytic leukemia (CLL) is a disorder in which the tempo of disease progression is highly variable, and prognostic markers that can be utilized at diagnosis are regarded as clinically important. Currently, there are several prognostic factors, such as immunoglobulin heavy chain (IgVH) mutational status, and ZAP-70 protein expression in neoplastic B-cells, that have demonstrated significant discriminative power in the prognostication of CLL. They are, however, largely unavailable in the routine diagnostic laboratory setting. In this study, we characterized the IgVH status and ZAP-70 expression by molecular techniques in a cohort of 108 patients with CLL, and correlated these results with three different methods of ZAP-70 expression by flow cytometry. We then assessed the results of these methods in terms of prognostic power as characterized by time to first treatment (TTFT). By comparing three different flow cytometry methods using receiver–operator curve (ROC) analysis, we identified that by utilizing a corrected mean fluorescence intensity (CorrMFI) algorithm for assessing ZAP-70 expression, there was good correlation with both IgVH mutational status, and ZAP-70 expression as assessed by qPCR. We were also able to show that ZAP-70 expression, as assessed by both qPCR and the CorrMFI method, was prognostic of TTFT. While confirmation in a larger patient cohort, with longer follow-up is required, we believe that the CorrMFI represents the most promising method currently available in a routine diagnostic setting for the assessment of ZAP-70 expression in CLL patients. © 2013 International Clinical Cytometry Society.

  12. Evaluation of ZAP-70 expression by flow cytometry in chronic lymphocytic leukemia: A multicentric international harmonization process.

    Science.gov (United States)

    Letestu, Remi; Rawstron, Andy; Ghia, Paolo; Villamor, Neus; Boeckx, Nancy; Leuven, Nancy Boeckx; Boettcher, Sebastian; Buhl, Anne Mette; Duerig, Jan; Ibbotson, Rachel; Kroeber, Alexander; Langerak, Anton; Le Garff-Tavernier, Magali; Mockridge, Ian; Morilla, Alison; Padmore, Ruth; Rassenti, Laura; Ritgen, Matthias; Shehata, Medhat; Smolewski, Piotr; Staib, Peter; Ticchioni, Michel; Walker, Clare; Ajchenbaum-Cymbalista, Florence

    2006-07-15

    The clinical course of patients with chronic lymphocytic leukemia (CLL) is heterogeneous with some patients requiring early therapy whereas others will not be treated for years. The evaluation of an individual CLL patient's prognosis remains a problematic issue. The presence or absence of somatic mutations in the IgVH genes is currently the gold-standard prognostic factor, but this technique is labor intensive and costly. Genomic studies uncovered that 70 kDa zeta-associated protein (ZAP-70) expression was associated with unmutated IgVH genes and ZAP-70 protein expression was proposed as a surrogate for somatic mutational status. Among the available techniques for ZAP-70 detection, flow cytometry is most preferable as it allows the simultaneous quantification of ZAP-70 protein expression levels in CLL cells and residual normal lymphocyte subsets. However, several factors introduce variability in the results reported from different laboratories; these factors include the anti-ZAP-70 antibody clone and conjugate, the staining procedure, the gating strategy, and the method of reporting the results. The need for standardization of the approach led to the organization of an international working group focused on harmonizing all aspects of the technique. During this workshop, a technical consensus was reached on the methods for cell permeabilization and immunophenotyping procedures. An assay was then designed that allowed comparison of two clones of anti-ZAP-70 antibody and the identification of the expression of this molecule in B, T, and NK cells identified in a four multicolor analysis. This procedure was applied to three stabilized blood samples, provided by the UK NEQAS group to all participating members of this study, in order to minimize variability caused by sample storage and shipment. Analysis was performed in 20 laboratories providing interpretable data from 14 centers. Various gating strategies were used and the ZAP-70 levels were expressed as percentage

  13. HLA-DR-expressing cells and T-lymphocytes in sural nerve biopsies

    DEFF Research Database (Denmark)

    Schrøder, H D; Olsson, T; Solders, G;

    1988-01-01

    was confirmed. HLA-DR expression was found in all biopsies and thus was not restricted to any particular type of neuropathy. The HLA-DR expression appeared to correlate with severity and activity of the neuropathy. HLA-DR-expressing macrophages wrapping myelinated fibers were prominent in primary demyelinating......Thirty-five sural nerve biopsies were stained immunohistochemically for HLA-DR antigen. HLA-DR was expressed on nonmyelinating Schwann cells, macrophages, vascular endothelium, and perineurium. By means of double immunofluorescence staining the identity of the HLA-DR presenting structures...

  14. Isopentenyl pyrophosphate activated CD56+ γδ T lymphocytes display potent anti-tumor activity towards human squamous cell carcinoma

    Science.gov (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.

    2008-01-01

    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

  15. Igs Expressed by Chronic Lymphocytic Leukemia B Cells Show Limited Binding-Site Structure Variability

    KAUST Repository

    Marcatili, P.

    2013-05-01

    Ag selection has been suggested to play a role in chronic lymphocytic leukemia (CLL) pathogenesis, but no large-scale analysis has been performed so far on the structure of the Ag-binding sites (ABSs) of leukemic cell Igs. We sequenced both H and L chain V(D)J rearrangements from 366 CLL patients and modeled their three-dimensional structures. The resulting ABS structures were clustered into a small number of discrete sets, each containing ABSs with similar shapes and physicochemical properties. This structural classification correlates well with other known prognostic factors such as Ig mutation status and recurrent (stereotyped) receptors, but it shows a better prognostic value, at least in the case of one structural cluster for which clinical data were available. These findings suggest, for the first time, to our knowledge, on the basis of a structural analysis of the Ab-binding sites, that selection by a finite quota of antigenic structures operates on most CLL cases, whether mutated or unmutated. Copyright © 2013 by The American Association of Immunologists, Inc.

  16. The sphingosine-1-phosphate transporter Spns2 expressed on endothelial cells regulates lymphocyte trafficking in mice.

    Science.gov (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

    2012-04-01

    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.

  17. Maternal T-lymphocytes in equine colostrum express a primarily inflammatory phenotype.

    Science.gov (United States)

    Perkins, Gillian A; Goodman, Laura B; Wimer, Christine; Freer, Heather; Babasyan, Susanna; Wagner, Bettina

    2014-10-15

    The purpose of this study was to characterize maternal immune cells in colostrum of mares. Cell phenotypes and cytokine secretion from mare peripheral blood mononuclear cells (PBMC) and cells from colostrum were analyzed by flow cytometry and by multiplex cytokine analysis. Equine colostral leukocytes were composed of mainly CD8(+) and CD4(+) lymphocytes. CD8(+) cells were significantly enriched in colostrum compared to PBMC (n=35). Colostral T-cells (n=13) responded to stimulation with PMA/ionomycin with a significantly higher magnitude of IL-17 (p=0.037) and similar IFN-γ concentrations (p=0.305), while IL-4 (p=0.0002) and IL-10 (p=0.0002) production was decreased compared to PBMC. CD4(+) and CD8(+) T-cells in colostrum produced IFN-γ (n=4). The findings show that colostrum T-cells can produce all four cytokines investigated here but most cells are polarized toward IL-17 and IFN-γ production and an inflammatory phenotype. Maternal T-cells likely migrate to the colostrum in a selective manner and may have specific roles in neonatal immune development.

  18. Cloning, sequence analysis, and expression of the large subunit of the human lymphocyte activation antigen 4F2

    Energy Technology Data Exchange (ETDEWEB)

    Lumadue, J.A.; Glick, A.B.; Ruddle, F.H.

    1987-12-01

    Among the earliest expressed antigens on the surface of activated human lymphocytes is the surface antigen 4F2. The authors have used DNA-mediated gene transfer and fluorescence-activated cell sorting to obtain cell lines that contain the gene encoding the large subunit of the human 4F2 antigen in a mouse L-cell background. Human DNAs cloned from these cell lines were subsequently used as hybridization probes to isolate a full-length cDNA clone expressing 4F2. Sequence analysis of the coding region has revealed an amino acid sequence of 529 residues. Hydrophobicity plotting has predicted a probable structure for the protein that includes an external carboxyl terminus, an internal leader sequence, a single hydrophobic transmembrane domain, and two possible membrane-associated domains. The 4F2 cDNA detects a single 1.8-kilobase mRNA in T-cell and B-cell lines. RNA gel blot analysis of RNA derived from quiescent and serum-stimulated Swiss 3T3 fibroblasts reveals a cell-cycle modulation of 4F2 gene expression: the mRNA is present in quiescent fibroblasts but increases 8-fold 24-36 hr after stimulation, at the time of maximal DNA synthesis.

  19. Induced gene expression of the hypusine-containing protein eukaryotic initiation factor 5A in activated human T lymphocytes.

    Science.gov (United States)

    Bevec, D; Klier, H; Holter, W; Tschachler, E; Valent, P; Lottspeich, F; Baumruker, T; Hauber, J

    1994-11-08

    The hypusine-containing protein eukaryotic initiation factor 5A (eIF-5A) is a cellular cofactor critically required for the function of the Rev transactivator protein of human immunodeficiency virus type 1 (HIV-1). eIF-5A localizes in the nuclear and cytoplasmic compartments of mammalian cells, suggesting possible activities on the level of regulated mRNA transport and/or protein translation. In this report we show that eIF-5A gene expression is constitutively low but inducible with T-lymphocyte-specific stimuli in human peripheral blood mononuclear cells (PBMCs) of healthy individuals. In contrast, eIF-5A is constitutively expressed at high levels in human cell lines as well as in various human organs. Comparison of eIF-5A levels in the PBMCs of uninfected and HIV-1-infected donors shows a significant upregulation of eIF-5A gene expression in the PBMCs of HIV-1 patients, compatible with a possible role of eIF-5A in HIV-1 replication during T-cell activation.

  20. Global Gene Expression Profiling in Interleukin-12-Induced Activation of CD8+ Cytotoxic T Lymphocytes against Mouse Mammary Carcinoma

    Institute of Scientific and Technical Information of China (English)

    Shanjin Cao; Zhaoying Xiang; Xiaojing Ma

    2004-01-01

    Interleukin-12 (IL-12) is a critical cytokine representing the link between the cellular and humoral branches of host immune defense apparatus. IL-12-induced cytotoxic lymphocyte (CTL) development is a central mechanism in immune responses against intracellular infectious agents as well as malignant growth. However,the molecular basis of tumor-specific CTL responses mediated by IL-12 remains poorly defined. In this study,we addressed this issue in a comprehensive manner to probe into IL-12-induced anti-tumor responses by global gene expression profiling of mRNA expression in CD8+T cells in a transplantable syngeneic mouse mammary carcinoma model treated or not with recombinant IL-12. A strong tumor regression was induced by the IL-12 treatment. An introspection of differential gene expression at an early stage of the IL-12-initiated CTL activation reveals interesting genes and molecular pathways that may account for the marked tumor regression,and is likely to provide a rich source of potential targets for further research and development of effective therapeutic modalities. Cellular & Molecular Immunology. 2004;1(5):357-366.

  1. Characterization of CRTAM gene promoter: AP-1 transcription factor control its expression in human T CD8 lymphocytes.

    Science.gov (United States)

    Valle-Rios, Ricardo; Patiño-Lopez, Genaro; Medina-Contreras, Oscar; Canche-Pool, Elsy; Recillas-Targa, Felix; Lopez-Bayghen, Esther; Zlotnik, Albert; Ortiz-Navarrete, Vianney

    2009-10-01

    Class-I MHC-restricted T-cell associated molecule (CRTAM) is a member of the Nectin-like adhesion molecule family. It is rapidly induced in NK, NKT and CD8(+) T cells. Interaction with its ligand Nectin-like 2 results in increased secretion of IFN-gamma by activated CD8(+) T lymphocytes. Through sequential bioinformatic analyses of the upstream region of the human CRTAM gene, we detected cis-elements potentially important for CRTAM gene transcription. Analyzing 2kb upstream from the ATG translation codon by mutation analysis in conjunction with luciferase reporter assays, electrophoretic mobility shify assay (EMSA) and supershift assays, we identified an AP-1 binding site, located at 1.4kb from the ATG translation codon of CRTAM gene as an essential element for CRTAM expression in activated but not resting human CD8(+) T cells. CRTAM expression was reduced in activated CD8(+) T cells treated with the JNK inhibitor SP600125, indicating that CRTAM expression is driven by the JNK-AP-1 signaling pathway. This study represents the first CRTAM gene promoter analysis in human T cells and indicates that AP-1 is a positive transcriptional regulator of this gene, a likely important finding because CRTAM has recently been shown to play a role in IFN-gamma and IL-17 production and T cell proliferation.

  2. Differences of CD4+ T lymphocyte miRNA gene expression in acute coronary artery syndrome (ACS patients and the effects of rosuvastatin on its expressions

    Directory of Open Access Journals (Sweden)

    Hong TAN

    2014-03-01

    Full Text Available Objective To investigate the effects of rosuvastatin on the expression profile of peripheral blood CD4+ T lymphocytes miRNA gene in the patients with acute coronary syndrome (ACS and screen the differentially expressed miRNAs before and after treatment, and elucidate the mechanisms of rosuvastatin calcium in the treatment of patients with ACS. Methods Nine cases were selected from the patients with ACS treated in the General Hospital of Jinan Military Command from Mar. to Jul. of 2012, with other 9 cases selected as controls, whose degree of coronary artery stenosis was less than 50% as confirmed by 320CT. Peripheral blood mononuclear cells were isolated by density gradient centrifugation, and CD4+ T lymphocytes were isolated by immunomagnetic beads method. miRNAs of CD4+ T lymphocytes were detected by miRNA gene chip technology. The differentially expressed miRNAs between ACS patients and normal control, and those in ACS patients before and after treatment were screened. Three of the maximum difference miRNAs were selected and followed by verification by real-time polymerase chain reaction (RT-PCR. Results More than 1900 genes were detected by gene microarray, of which more than 300 genes showed significant differences in expression. Comparing the ACS patients and normal controls, 126 genes were significantly up-regulated, including miRNA-21, miRNA-142, and miRNA-20a; and 202 genes were significantly down-regulated, including miRNA-4734, miRNA-1182, and miRNA-1273f. A total of 157 genes were significantly up-regulated after treatment with rosuvastatin calcium (20mg/d×10d, such as miRNA-4734, miRNA-1182, and miRNA-663b; and 137 genes were significantly down-regulated, such as miRNA-4789, miRNA-5692c, and miRNA-26a. The results were validated by RT-PCR and they were same as miRNA microarray. Conclusion Rosuvastain may play a role in the treatment of patients with ACS by regulating a series of miRNAs such as miRNA-4734, miRNA-1182, and mi

  3. 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

    2013-01-01

    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...

  4. Expressions of HSP 70 and NF-κB in the peripheral blood lymphocyte of chronic gastritis patients of different syndrome patterns

    Institute of Scientific and Technical Information of China (English)

    胡玲

    2012-01-01

    Objective To study the expressions of heat shock protein 70 (HSP 70) and nuclear factor-kappa B(NF-κB) in the peripheral blood lymphocyte of chronic gastritis (CG) patients of Pi-Wei hygropyrexia syndrome (PWHS) and Pi-qi deficiency syndrome(PQDS),and to explore their correlation with Helicobacter pylori(Hp) infection. Methods

  5. In vivo diabetogenic action of CD4+ T lymphocytes requires Fas expression and is independent of IL-1 and IL-18.

    Science.gov (United States)

    Wen, Li; Green, Elizabeth A; Stratmann, Thomas; Panosa, Anaïs; Gomis, Ramon; Eynon, Elizabeth E; Flavell, Richard A; Mezquita, Jovita A; Mora, Conchi

    2011-05-01

    CD4(+) T lymphocytes are required to induce spontaneous autoimmune diabetes in the NOD mouse. Since pancreatic β cells upregulate Fas expression upon exposure to pro-inflammatory cytokines, we studied whether the diabetogenic action of CD4(+) T lymphocytes depends on Fas expression on target cells. We assayed the diabetogenic capacity of NOD spleen CD4(+) T lymphocytes when adoptively transferred into a NOD mouse model combining: (i) Fas-deficiency, (ii) FasL-deficiency, and (iii) SCID mutation. We found that CD4(+) T lymphocytes require Fas expression in the recipients' target cells to induce diabetes. IL-1β has been described as a key cytokine involved in Fas upregulation on mouse β cells. We addressed whether CD4(+) T cells require IL-1β to induce diabetes. We also studied spontaneous diabetes onset in NOD/IL-1 converting enzyme-deficient mice, in NOD/IL-1β-deficient mice, and CD4(+) T-cell adoptively transferred diabetes into NOD/SCID IL-1β-deficient mice. Neither IL-1β nor IL-18 are required for either spontaneous or CD4(+) T-cell adoptively transferred diabetes. We conclude that CD4(+) T-cell-mediated β-cell damage in autoimmune diabetes depends on Fas expression, but not on IL-1β unveiling the existing redundancy regarding the cytokines involved in Fas upregulation on NOD β cells in vivo. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Dynamic T-lymphocyte chemokine receptor expression induced by interferon-beta therapy in multiple sclerosis

    DEFF Research Database (Denmark)

    Krakauer, M; Sorensen, P S; Khademi, M;

    2006-01-01

    as these influence central nervous system (CNS) transmigration and inflammation. At 'steady state' (>/=1 day after the most recent IFN-beta injection), IFN-beta treatment increased CD4(+) T-cell surface expression of CC chemokine receptor (CCR)4, CCR5 and CCR7 after 3 months of treatment, whereas that of CXC...... chemokine receptor (CXCR)3 was unaltered. Conversely, at 9-12 h after the most recent IFN-beta injection, CCR4, CCR5 and CCR7 expressions were unaltered, while CXCR3 expression was reduced. CD4(+) T-cell surface expression of CCR4 was significantly lower in untreated MS patients compared with healthy...... volunteers. Of the plasma chemokines, only CXCL10 was increased by IFN-beta treatment; CCL3, CCL4, CCL5 and CXCL9 were unaltered. CCR5 mRNA expression in blood mononuclear cells correlated with the expression of T-helper type 1 (Th1)-associated genes whereas CCR4 and CCR7 mRNA expression correlated with Th2...

  7. CCR7 is mainly expressed in teleost gills, where it defines an IgD+IgM- B lymphocyte subset.

    Science.gov (United States)

    Castro, Rosario; Bromage, Erin; Abós, Beatriz; Pignatelli, Jaime; González Granja, Aitor; Luque, Alfonso; Tafalla, Carolina

    2014-02-01

    Chemokine receptor CCR7, the receptor for both CCL19 and CCL21 chemokines, regulates the recruitment and clustering of circulating leukocytes to secondary lymphoid tissues, such as lymph nodes and Peyer's patches. Even though teleost fish do not have either of these secondary lymphoid structures, we have recently reported a homolog to CCR7 in rainbow trout (Oncorhynchus mykiss). In the present work, we have studied the distribution of leukocytes bearing extracellular CCR7 in naive adult tissues by flow cytometry, observing that among the different leukocyte populations, the highest numbers of cells with membrane (mem)CCR7 were recorded in the gill (7.5 ± 2% CCR7(+) cells). In comparison, head kidney, spleen, thymus, intestine, and peripheral blood possessed CCR7(+) cells. When CCR7 was studied at early developmental stages, we detected a progressive increase in gene expression and protein CCR7 levels in the gills throughout development. Surprisingly, the majority of the CCR7(+) cells in the gills were not myeloid cells and did not express membrane CD8, IgM, nor IgT, but expressed IgD on the cell surface. In fact, most IgD(+) cells in the gills expressed CCR7. Intriguingly, the IgD(+)CCR7(+) population did not coexpress memIgM. Finally, when trout were bath challenged with viral hemorrhagic septicemia virus, the number of CCR7(+) cells significantly decreased in the gills while significantly increased in head kidney. These results provide evidence of the presence of a novel memIgD(+)memIgM(-) B lymphocyte subset in trout that expresses memCCR7 and responds to viral infections. Similarities with IgD(+)IgM(-) subsets in mammals are discussed.

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

    Science.gov (United States)

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

    2017-07-01

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

  9. Curcumin blocks interleukin (IL)-2 signaling in T-lymphocytes by inhibiting IL-2 synthesis, CD25 expression, and IL-2 receptor signaling

    Energy Technology Data Exchange (ETDEWEB)

    Forward, Nicholas A.; Conrad, David M. [Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia (Canada); Power Coombs, Melanie R.; Doucette, Carolyn D. [Department of Pathology, Dalhousie University, Halifax, Nova Scotia (Canada); Furlong, Suzanne J. [Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia (Canada); Lin, Tong-Jun [Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia (Canada); Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia (Canada); Hoskin, David W., E-mail: d.w.hoskin@dal.ca [Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia (Canada); Department of Pathology, Dalhousie University, Halifax, Nova Scotia (Canada); Department of Surgery, Dalhousie University, Halifax, Nova Scotia (Canada)

    2011-04-22

    Highlights: {yields} Curcumin inhibits CD4{sup +} T-lymphocyte proliferation. {yields} Curcumin inhibits interleukin-2 (IL-2) synthesis and CD25 expression by CD4{sup +} T-lymphocytes. {yields} Curcumin interferes with IL-2 receptor signaling by inhibiting JAK3 and STAT5 phosphorylation. {yields} IL-2-dependent regulatory T-lymphocyte function and Foxp3 expression is downregulated by curcumin. -- Abstract: Curcumin (diferulomethane) is the principal curcuminoid in the spice tumeric and a potent inhibitor of activation-induced T-lymphocyte proliferation; however, the molecular basis of this immunosuppressive effect has not been well studied. Here we show that micromolar concentrations of curcumin inhibited DNA synthesis by mouse CD4{sup +} T-lymphocytes, as well as interleukin-2 (IL-2) and CD25 ({alpha} chain of the high affinity IL-2 receptor) expression in response to antibody-mediated cross-linking of CD3 and CD28. Curcumin acted downstream of protein kinase C activation and intracellular Ca{sup 2+} release to inhibit I{kappa}B phosphorylation, which is required for nuclear translocation of the transcription factor NF{kappa}B. In addition, IL-2-dependent DNA synthesis by mouse CTLL-2 cells, but not constitutive CD25 expression, was impaired in the presence of curcumin, which demonstrated an inhibitory effect on IL-2 receptor (IL-2R) signaling. IL-2-induced phosphorylation of STAT5A and JAK3, but not JAK1, was diminished in the presence of curcumin, indicating inhibition of critical proximal events in IL-2R signaling. In line with the inhibitory action of curcumin on IL-2R signaling, pretreatment of CD4{sup +}CD25{sup +} regulatory T-cells with curcumin downregulated suppressor function, as well as forkhead box p3 (Foxp3) expression. We conclude that curcumin inhibits IL-2 signaling by reducing available IL-2 and high affinity IL-2R, as well as interfering with IL-2R signaling.

  10. Expression of TLR-7, MyD88, NF-kB and INF-α in B lymphocytes of Mayan Women with Systemic Lupus Erythematosus, in Mexico

    Directory of Open Access Journals (Sweden)

    Guillermo eValencia Pacheco

    2016-02-01

    Full Text Available Background: Systemic Lupus Erythematosus (SLE is a chronic inflammatory autoimmune disease involving multiple organs. It is currently accepted that several genetic, environmental, and hormonal factors contributing to its development. Innate immunity may have a great influence in autoimmunity through Toll-like receptors (TLR. TLR-7 recognizing single-strand RNA has been involved in SLE. Its activation induces intracellular signal with attraction of MyD88 and NF-kB, leading to IFN-α synthesis which correlate with disease activity. Objective: To assess the expression of TLR-7, MyD88 and NF-kB in mononuclear cells of Mayan women with SLE. Methods: One hundred patients with SLE and one hundred healthy controls, all them Mayan women, were included. TLR-7 was analyzed on B and T lymphocytes, and MyD88 and NF-kB only in B lymphocytes. Serum INF-α level was evaluated by ELISA. Results: Significant expression (p < 0.0001 of TLR-7 in B and T lymphocytes and serum IFN-α increased (p = 0.034 was observed in patients. MyD88 and NF-kB were also increased in B lymphocytes of patients. TLR-7 and NF-kB expression correlated, but no correlation with INF-α and disease activity was detected. Conclusion: Data support the role of TLR-7 and signal proteins in the pathogenesis of SLE in the Mayan population of Yucatán.

  11. The mRNA Expression Status of Dopamine Receptor D2, Dopamine Receptor D3 and DARPP-32 in T Lymphocytes of Patients with Early Psychosis

    Directory of Open Access Journals (Sweden)

    Yin Cui

    2015-11-01

    Full Text Available Peripheral blood lymphocytes are an attractive tool because there is accumulating evidence indicating that lymphocytes may be utilized as a biomarker in the field of psychiatric study as they could reveal the condition of cells distributed in the brain. Here, we measured the mRNA expression status of dopamine receptor D2 (DRD2, DRD3, and dopamine and cyclic adenosine 3′,5′-monophosphate regulated phosphoprotein-32 (DARPP-32 in T lymphocytes of patients with early psychosis by quantitative real-time polymerase chain reaction (q-PCR and explored the relationships between their mRNA levels and the psychopathological status of patients. The present study demonstrated that the mRNA expression levels of DRD3 in T lymphocytes were significantly different among controls, and in patients with psychotic disorder not otherwise specified (NOS and schizophrenia/schizophreniform disorder. However, no significant differences in mRNA expression levels of DRD2 and DARPP-32 were found among the three groups. We found a significant positive correlation between the DRD2 mRNA level and the score of the excited factor of the Positive and Negative Syndrome Scale (PANSS in patients with schizophrenia/schizophreniform disorder. These findings suggest that DRD3 mRNA levels may serve as a potential diagnostic biomarker differentiating patients with early psychosis from controls.

  12. Effects of Fluoride on the Expression of p38MAPK Signaling Pathway-Related Genes and Proteins in Spleen Lymphocytes of Mice.

    Science.gov (United States)

    Shi, Zeyu; Zhan, Yaqi; Zhao, Junxing; Wang, Jinming; Ma, Haili

    2016-10-01

    This study investigated the effects of sodium fluoride on the expression of p38MAPK signaling pathway-related genes and proteins in the spleen lymphocytes of mice, revealing the mechanism of the toxicity of fluoride to the immune system. The spleen lymphocytes, isolated from mice consuming different NaF doses (0, 50, 100, and 150 mg/L) for 60 days, were cultured in medium with bacteria lipopolysaccharide, and the cells' proliferation ability was analyzed through MTT; real-time PCR detected the change of MLK3/MKK6/p38MAPK/MSK1/ATF1 on mRNA, and the difference of protein expression of MKK6/p38MAPK were detected through the Western blotting. The results suggested that the proliferation ability of spleen lymphocytes isolated from mice consuming different NaF doses is lower, and the expression of genes and proteins of MKK6/p38MAPK showed a decreasing trend. These results demonstrate that fluoride can suppress the activation of p38MAPK pathway in mice spleen lymphocytes and further influences the function of the immune system.

  13. Blockade of CTLA-4 promotes the development of effector CD8+ T lymphocytes and the therapeutic effect of vaccination with an attenuated protozoan expressing NY-ESO-1.

    Science.gov (United States)

    Dos Santos, Luara Isabela; Galvão-Filho, Bruno; de Faria, Paula Cristina; Junqueira, Caroline; Dutra, Miriam Santos; Teixeira, Santuza Maria Ribeiro; Rodrigues, Maurício Martins; Ritter, Gerd; Bannard, Oliver; Fearon, Douglas Thomas; Antonelli, Lis Ribeiro; Gazzinelli, Ricardo Tostes

    2015-03-01

    The development of cancer immunotherapy has long been a challenge. Here, we report that prophylactic vaccination with a highly attenuated Trypanosoma cruzi strain expressing NY-ESO-1 (CL-14-NY-ESO-1) induces both effector memory and effector CD8(+) T lymphocytes that efficiently prevent tumor development. However, the therapeutic effect of such a vaccine is limited. We also demonstrate that blockade of Cytotoxic T Lymphocyte Antigen 4 (CTLA-4) during vaccination enhances the frequency of NY-ESO-1-specific effector CD8(+) T cells producing IFN-γ and promotes lymphocyte migration to the tumor infiltrate. As a result, therapy with CL-14-NY-ESO-1 together with anti-CTLA-4 is highly effective in controlling the development of an established melanoma.

  14. Epstein–Barr Virus MicroRNAs are Expressed in Patients with Chronic Lymphocytic Leukemia and Correlate with Overall Survival

    Directory of Open Access Journals (Sweden)

    Alessandra Ferrajoli

    2015-06-01

    Full Text Available Although numerous studies highlighted the role of Epstein–Barr Virus (EBV in B-cell transformation, the involvement of EBV proteins or genome in the development of the most frequent adult leukemia, chronic lymphocytic leukemia (CLL, has not yet been defined. We hypothesized that EBV microRNAs contribute to progression of CLL and demonstrated the presence of EBV miRNAs in B-cells, in paraffin-embedded bone marrow biopsies and in the plasma of patients with CLL by using three different methods (small RNA-sequencing, quantitative reverse transcription PCR [q-RT-PCR] and miRNAs in situ hybridization [miRNA-ISH]. We found that EBV miRNA BHRF1-1 expression levels were significantly higher in the plasma of patients with CLL compared with healthy individuals (p < 0 · 0001. Notably, BHRF1-1 as well as BART4 expression were detected in the plasma of either seronegative or seropositive (anti-EBNA-1 IgG and EBV DNA tested patients; similarly, miRNA-ISH stained positive in bone marrow specimens while LMP1 and EBER immunohistochemistry failed to detect viral proteins and RNA. We also found that BHRF1-1 plasma expression levels were positively associated with elevated beta-2-microglobulin levels and advanced Rai stages and observed a correlation between higher BHRF1-1 expression levels and shorter survival in two independent patients' cohorts. Furthermore, in the majority of CLL cases where BHRF1-1 was exogenously induced in primary malignant B cells the levels of TP53 were reduced. Our findings suggest that EBV may have a role in the process of disease progression in CLL and that miRNA RT-PCR and miRNAs ISH could represent additional methods to detect EBV miRNAs in patients with CLL.

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

    Institute of Scientific and Technical Information of China (English)

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

    2006-01-01

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

  16. Epstein–Barr Virus MicroRNAs are Expressed in Patients with Chronic Lymphocytic Leukemia and Correlate with Overall Survival☆

    Science.gov (United States)

    Ferrajoli, Alessandra; Ivan, Cristina; Ciccone, Maria; Shimizu, Masayoshi; Kita, Yoshiaki; Ohtsuka, Masahisha; D'Abundo, Lucilla; Qiang, Jun; Lerner, Susan; Nouraee, Nazila; Rabe, Kari G.; Rassenti, Laura Z.; Van Roosbroeck, Katrien; Manning, John T.; Yuan, Yuan; Zhang, Xinna; Shanafelt, Tait D.; Wierda, William G.; Sabbioni, Silvia; Tarrand, Jeffrey J.; Estrov, Zeev; Radovich, Milan; Liang, Han; Negrini, Massimo; Kipps, Thomas J.; Kay, Neil E.; Keating, Michael; Calin, George A.

    2015-01-01

    Although numerous studies highlighted the role of Epstein–Barr Virus (EBV) in B-cell transformation, the involvement of EBV proteins or genome in the development of the most frequent adult leukemia, chronic lymphocytic leukemia (CLL), has not yet been defined. We hypothesized that EBV microRNAs contribute to progression of CLL and demonstrated the presence of EBV miRNAs in B-cells, in paraffin-embedded bone marrow biopsies and in the plasma of patients with CLL by using three different methods (small RNA-sequencing, quantitative reverse transcription PCR [q-RT-PCR] and miRNAs in situ hybridization [miRNA-ISH]). We found that EBV miRNA BHRF1-1 expression levels were significantly higher in the plasma of patients with CLL compared with healthy individuals (p < 0 · 0001). Notably, BHRF1-1 as well as BART4 expression were detected in the plasma of either seronegative or seropositive (anti-EBNA-1 IgG and EBV DNA tested) patients; similarly, miRNA-ISH stained positive in bone marrow specimens while LMP1 and EBER immunohistochemistry failed to detect viral proteins and RNA. We also found that BHRF1-1 plasma expression levels were positively associated with elevated beta-2-microglobulin levels and advanced Rai stages and observed a correlation between higher BHRF1-1 expression levels and shorter survival in two independent patients' cohorts. Furthermore, in the majority of CLL cases where BHRF1-1 was exogenously induced in primary malignant B cells the levels of TP53 were reduced. Our findings suggest that EBV may have a role in the process of disease progression in CLL and that miRNA RT-PCR and miRNAs ISH could represent additional methods to detect EBV miRNAs in patients with CLL. PMID:26288818

  17. Epstein-Barr Virus MicroRNAs are Expressed in Patients with Chronic Lymphocytic Leukemia and Correlate with Overall Survival.

    Science.gov (United States)

    Ferrajoli, Alessandra; Ivan, Cristina; Ciccone, Maria; Shimizu, Masayoshi; Kita, Yoshiaki; Ohtsuka, Masahisha; D'Abundo, Lucilla; Qiang, Jun; Lerner, Susan; Nouraee, Nazila; Rabe, Kari G; Rassenti, Laura Z; Van Roosbroeck, Katrien; Manning, John T; Yuan, Yuan; Zhang, Xinna; Shanafelt, Tait D; Wierda, William G; Sabbioni, Silvia; Tarrand, Jeffrey J; Estrov, Zeev; Radovich, Milan; Liang, Han; Negrini, Massimo; Kipps, Thomas J; Kay, Neil E; Keating, Michael; Calin, George A

    2015-06-01

    Although numerous studies highlighted the role of Epstein-Barr Virus (EBV) in B-cell transformation, the involvement of EBV proteins or genome in the development of the most frequent adult leukemia, chronic lymphocytic leukemia (CLL), has not yet been defined. We hypothesized that EBV microRNAs contribute to progression of CLL and demonstrated the presence of EBV miRNAs in B-cells, in paraffin-embedded bone marrow biopsies and in the plasma of patients with CLL by using three different methods (small RNA-sequencing, quantitative reverse transcription PCR [q-RT-PCR] and miRNAs in situ hybridization [miRNA-ISH]). We found that EBV miRNA BHRF1-1 expression levels were significantly higher in the plasma of patients with CLL compared with healthy individuals (p < 0 · 0001). Notably, BHRF1-1 as well as BART4 expression were detected in the plasma of either seronegative or seropositive (anti-EBNA-1 IgG and EBV DNA tested) patients; similarly, miRNA-ISH stained positive in bone marrow specimens while LMP1 and EBER immunohistochemistry failed to detect viral proteins and RNA. We also found that BHRF1-1 plasma expression levels were positively associated with elevated beta-2-microglobulin levels and advanced Rai stages and observed a correlation between higher BHRF1-1 expression levels and shorter survival in two independent patients' cohorts. Furthermore, in the majority of CLL cases where BHRF1-1 was exogenously induced in primary malignant B cells the levels of TP53 were reduced. Our findings suggest that EBV may have a role in the process of disease progression in CLL and that miRNA RT-PCR and miRNAs ISH could represent additional methods to detect EBV miRNAs in patients with CLL.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-01-16

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

  19. Expression of granzyme B,granulysin and perforin decreased significantly in peripheral blood mononuclear cells(PBMC)from patients with Cryptococcal Meningitis%隐球菌性脑炎患者颗粒酶B、颗粒溶素、穿孔素的表达及临床意义

    Institute of Scientific and Technical Information of China (English)

    周晔; 邓安梅; 谷明莉; 陈燕; 陈波; 陈孙孝; 仲人前

    2010-01-01

    目的 研究颗粒酶B(granzyme B,GrB)、颗粒溶素(granulysin,GNLY)及穿孔素(perforin,又称pore-forming protein,PFP)三种免疫效应分子在隐球菌性脑膜炎(cryptococcal meningitis)外周血单个核细胞中的表达及其临床意义.方法 首先用密度梯度离心法分离得到25例隐球菌性脑膜炎患者和30例健康人外周血单个核细胞(peripheral blood mononuclear cells,PBMC),其次用免疫印迹(western blot)的方法检测其中的GrB、GNLY、及PFP的蛋白含量,用实时荧光定量(RFQ)-PCR方法测定其中GrB、GNLY、及PFP的mRNA含量.并分析隐球菌性脑膜炎患者蛋白含量与一些免疫指标的相关性.结果 与健康对照组比较,隐球菌性脑膜炎患者PBMC中GNLY、PFP蛋白含量和mRNA均明显降低,而GrB无明显变化.隐球菌性脑膜炎患者中GNLY蛋白表达水平与血清IgG、IFN-γ水平成正相关(r=0.477,P<0.05;r=0.534,P<0.05),与IL-10水平成负相关(r=-0.505,P<0.05).结论 GNLY、PFP可能参与隐球菌性脑膜炎的疾病进程,这为探讨隐球菌性脑膜炎的病情监控和有效治疗提供了新的线索.

  20. Expression profiling of clonal lymphocyte cell cultures from Rett syndrome patients

    Science.gov (United States)

    More than 85% of Rett syndrome (RTT) patients have heterozygous mutations in the X-linked MECP2 gene which encodes methyl-CpG-binding protein 2, a transcriptional repressor that binds methylated CpG sites. Because MECP2 is subject to X chromosome inactivation (XCI), girls with RTT express either the...

  1. Malignant transformation of CD4+ T lymphocytes mediated by oncogenic kinase NPM/ALK recapitulates IL-2-induced cell signaling and gene expression reprogramming

    DEFF Research Database (Denmark)

    Marzec, Michal; Halasa, Krzysztof; Liu, Xiaobin

    2013-01-01

    by the STAT5 and STAT3 transcription factors, whereas transcription of Egr-1 and Fosl-1 was induced by the MEK-ERK pathway. Finally, we found that Egr-1, a protein not associated previously with either IL-2 or ALK, contributes to the cell proliferation. These findings indicate that NPM/ALK transforms......Anaplastic lymphoma kinase (ALK), physiologically expressed only by nervous system cells, displays a remarkable capacity to transform CD4(+) T lymphocytes and other types of nonneural cells. In this study, we report that activity of nucleophosmin (NPM)/ALK chimeric protein, the dominant form of ALK...... expressed in T cell lymphomas (TCLs), closely resembles cell activation induced by IL-2, the key cytokine supporting growth and survival of normal CD4(+) T lymphocytes. Direct comparison of gene expression by ALK(+) TCL cells treated with an ALK inhibitor and IL-2-dependent ALK(-) TCL cells stimulated...

  2. Failure of lymphocyte-membrane HLA-A and -B expression in two siblings with combined immunodeficiency

    NARCIS (Netherlands)

    Schuurman, R.K.B.; Rood, J.J. van; Vossen, J.M.; Schellekens, P.Th.A.; Feltkamp-Vroom, Th.M.; Doyer, E.; Gmelig Meyling, F.H.J.; Visser, H.K.A.

    1979-01-01

    A diagnosis of partial combined immunodeficiency was made in two Turkish siblings with a history of multiple pyogenic infections and persistent candidiasis. They demonstrated severe hypo-γ-globulinemia, with B-lymphocytes, but deficient plasma cell differentiation. T-Lymphocytes were decreased in nu

  3. Novel primary thymic defect with T lymphocytes expressing gamma delta T cell receptor

    DEFF Research Database (Denmark)

    Geisler, C; Pallesen, G; Platz, P

    1989-01-01

    Flow cytometric analysis of the peripheral blood mononuclear cells in a six year old girl with a primary cellular immune deficiency showed a normal fraction of CD3 positive T cells. Most (70%) of the CD3 positive cells, however, expressed the gamma delta and not the alpha beta T cell receptor....... Immunoprecipitation and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that most of the gamma delta T cell receptors existed as disulphide-linked heterodimers. Proliferative responses to mitogens were severely reduced, but specific antibody responses after vaccination could be detected...... deficiency associated with a high proportion of T cells expressing the gamma delta T cell receptor has been described in nude mice, and it is suggested that the immune deficiency of this patient may represent a human analogue....

  4. A new 500 kb haplotype associated with high CD8+ T-lymphocyte numbers predicts a less severe expression of hereditary hemochromatosis

    Directory of Open Access Journals (Sweden)

    Mascarenhas Cláudia

    2008-11-01

    Full Text Available Abstract Background Hereditary Hemochromatosis(HH is a common genetic disorder of iron overload where the large majority of patients are homozygous for one ancestral mutation in the HFE gene. In spite of this remarkable genetic homogeneity, the condition is clinically heterogeneous, varying from a severe disease to an asymptomatic phenotype with only abnormal biochemical parameters. The recent recognition of the variable penetrance of the HH mutation in different large population studies demands the need to search for new modifiers of its phenotypic expression. The present study follows previous observations that MHC class-I linked genetic markers, associated with the setting of CD8+ T-lymphocyte numbers, could be clinically relevant modifiers of the phenotypic expression in HH, and aimed to find new markers that could be used as more reliable prognostic variables. Methods Haplotype analysis, including seven genetic markers within a 1 Mb region around the microsatellite D6S105 was performed in a group of 56 previously characterized C282Y homozygous Portuguese patients. Parameters analyzed in this study were total body iron stores, clinical manifestations related with HH and immunological parameters (total lymphocyte numbers, CD4+ and CD8+ T-lymphocyte numbers. An independent group of 10 C282Y homozygous patients from Vancouver, Canada, were also included in this study and analyzed for the same parameters. Results A highly conserved ancestral haplotype defined by the SNP markers PGBD1-A, ZNF193-A, ZNF165-T (designated as A-A-T was found associated with both abnormally low CD8+ T-lymphocyte numbers and the development of a severe clinical expression of HH. In a small proportion of patients, another conserved haplotype defined by the SNP markers PGBD1-G, ZNF193-G, ZNF165-G (designated as G-G-G was found associated with high CD8+ T-lymphocyte numbers and a milder clinical expression. Remarkably, the two conserved haplotypes defined in Portuguese

  5. Rapid gene expression changes in peripheral blood lymphocytes upon practice of a comprehensive yoga program.

    Directory of Open Access Journals (Sweden)

    Su Qu

    Full Text Available One of the most common integrative medicine (IM modalities is yoga and related practices. Previous work has shown that yoga may improve wellness in healthy people and have benefits for patients. However, the mechanisms of how yoga may positively affect the mind-body system are largely unknown. Here we have assessed possible rapid changes in global gene expression profiles in the peripheral blood mononuclear cells (PBMCs in healthy people that practiced either a comprehensive yoga program or a control regimen. The experimental sessions included gentle yoga postures, breathing exercises, and meditation (Sudarshan Kriya and Related Practices--SK&P compared with a control regimen of a nature walk and listening to relaxing music. We show that the SK&P program has a rapid and significantly greater effect on gene expression in PBMCs compared with the control regimen. These data suggest that yoga and related practices result in rapid gene expression alterations which may be the basis for their longer term cell biological and higher level health effects.

  6. Cytosolic Hsp70/Hsc70 protein expression in lymphocytes exposed to low gamma-ray dose

    Energy Technology Data Exchange (ETDEWEB)

    Manzanares A, E.; Vega C, H.R.; Letechipia de Leon, C. [Unidades Academicas de Estudios Nucleares, UAZ, A.P. 336, 98000 Zacatecas (Mexico)]. E-mail: emanz@cantera.reduaz.mx; Guzman E, L.J. [Unidad Academica de Biologia Experimental, Guadalupe, Zacatecas (Mexico); Garcia T, M. [LIBRA, Centro I and D, Campus Miguel Delibes, Valladolid 47011 (Spain)

    2004-07-01

    The purpose of this study was to evaluate the effect of low gamma ray intensity upon Hsp70 expression in human Iymphocytes. The heat shock proteins (Hsp) family, are a group of proteins present in all living organism, therefore there are highly conserved and are related to adaptation and evolution. At cellular level these proteins acts as chaperones correcting denatured proteins. When a stress agent, such heavy metals, UV, heat, etc. is affecting a cell a response to this aggression is triggered only through over expression of Hsp. Several studies has been carried out in which the cellular effect are observed, mostly of these studies uses large doses, but very few studies are related with low doses. Blood of healthy volunteers was obtained and the Iymphocytes were isolated by ficoll- histopaque gradient. Experimental lots were irradiated in a {sup 137} Cs gamma-ray. Hsp70 expression was found since 0.5 c Gy, indicating a threshold to very low doses of gamma rays. (Author)

  7. Rapid gene expression changes in peripheral blood lymphocytes upon practice of a comprehensive yoga program.

    Science.gov (United States)

    Qu, Su; Olafsrud, Solveig Mjelstad; Meza-Zepeda, Leonardo A; Saatcioglu, Fahri

    2013-01-01

    One of the most common integrative medicine (IM) modalities is yoga and related practices. Previous work has shown that yoga may improve wellness in healthy people and have benefits for patients. However, the mechanisms of how yoga may positively affect the mind-body system are largely unknown. Here we have assessed possible rapid changes in global gene expression profiles in the peripheral blood mononuclear cells (PBMCs) in healthy people that practiced either a comprehensive yoga program or a control regimen. The experimental sessions included gentle yoga postures, breathing exercises, and meditation (Sudarshan Kriya and Related Practices--SK&P) compared with a control regimen of a nature walk and listening to relaxing music. We show that the SK&P program has a rapid and significantly greater effect on gene expression in PBMCs compared with the control regimen. These data suggest that yoga and related practices result in rapid gene expression alterations which may be the basis for their longer term cell biological and higher level health effects.

  8. Lymphocytes with Aberrant Expression of Fas or Fas-ligand Attenuate Immune Bone Marrow Failure in a Mouse Model

    Science.gov (United States)

    Omokaro, Stephanie O.; Desierto, Marie J.; Eckhaus, Michael A.; Ellison, Felicia M.; Chen, Jichun; Young, Neal S.

    2012-01-01

    Bone marrow (BM) and lymphocyte samples from aplastic anemia patients show up-regulated Fas and Fas-ligand (FasL) expression respectively, supporting a relationship between immune-mediated BM destruction and the Fas apoptotic pathway. Mice with spontaneous lymphoproliferation (lpr) and generalized lymphoproliferative disease (gld) mutations exhibit abnormal expression of Fas and FasL; serving as potential models to elucidate underlying mechanisms of BM failure. We examined cellular and functional characteristics of lpr and gld mutants on the C57BL/6 (B6) background. Lymph node (LN) cells from lpr and gld mice produced less apoptosis when co-incubated with C.B10-H2b/LilMcd (C.B10) BM cells in vitro. This functional difference was confirmed by infusing lpr, gld, and B6 LN cells into sub-lethally irradiated CB10 mice; all donor LN cells showed significant T cell expansion and activation but only B6 LN cells caused severe BM destruction. Mice infused with gld LN cells developed mild to moderate BM failure, despite receiving FasL-deficient effectors, thus suggesting the existence of alternative pathways or incomplete penetrance of the mutation. Paradoxically, mice that received Fas-deficient lpr LN cells also had reduced BM failure, likely due to down-regulation of pro-apoptotic genes, an effect that can be overcome by higher doses of lpr LN cells. Our model demonstrates that abnormal Fas or FasL expression interferes with the development of pancytopenia and marrow hypoplasia, validating a major role for the Fas/FasL cytotoxic pathway in immune-mediated BM failure, although disruption of this pathway does not completely abolish marrow destruction. PMID:19265119

  9. Lymphocytes with aberrant expression of Fas or Fas ligand attenuate immune bone marrow failure in a mouse model.

    Science.gov (United States)

    Omokaro, Stephanie O; Desierto, Marie J; Eckhaus, Michael A; Ellison, Felicia M; Chen, Jichun; Young, Neal S

    2009-03-15

    Bone marrow (BM) and lymphocyte samples from aplastic anemia patients show up-regulated Fas and Fas-ligand (FasL) expression, respectively, supporting a relationship between immune-mediated BM destruction and the Fas apoptotic pathway. Mice with spontaneous lymphoproliferation (lpr) and generalized lymphoproliferative disease (gld) mutations exhibit abnormal expression of Fas and FasL, serving as potential models to elucidate underlying mechanisms of BM failure. We examined cellular and functional characteristics of lpr and gld mutants on the C57BL/6 (B6) background. Lymph node (LN) cells from lpr and gld mice produced less apoptosis when coincubated with C.B10-H2(b)/LilMcd (C.B10) BM cells in vitro. This functional difference was confirmed by infusing lpr, gld, and B6 LN cells into sublethally irradiated CB10 mice. All donor LN cells showed significant T cell expansion and activation, but only B6 LN cells caused severe BM destruction. Mice infused with gld LN cells developed mild to moderate BM failure despite receiving FasL-deficient effectors, thus suggesting the existence of alternative pathways or incomplete penetrance of the mutation. Paradoxically, mice that received Fas-deficient lpr LN cells also had reduced BM failure, likely due to down-regulation of proapoptotic genes, an effect that can be overcome by higher doses of lpr LN cells. Our model demonstrates that abnormal Fas or FasL expression interferes with the development of pancytopenia and marrow hypoplasia, validating a major role for the Fas/FasL cytotoxic pathway in immune-mediated BM failure, although disruption of this pathway does not completely abolish marrow destruction.

  10. Phenotypic modulation of chronic lymphocytic leukemia cells by phorbol ester: induction of IgM secretion and changes in the expression of B cell-associated surface antigens.

    Science.gov (United States)

    Gordon, J; Mellstedt, H; Aman, P; Biberfeld, P; Klein, G

    1984-01-01

    Freshly explanted neoplastic populations from 22 cases of phenotypically well-characterized chronic type B lymphocytic leukemia were studied for their capacity to respond to the phorbol ester TPA in vitro. In all but four cases the secretion of IgM was either induced or increased, often to a high level. In contrast, the export of free immunoglobulin (Ig) light chains, an almost consistent feature of the B lymphocytic leukemias, remained relatively constant after TPA treatment. Parallel changes in leukemic cell surface phenotype were probed with both "conventional" and monoclonal antibodies, revealing some modulation of markers in every case investigated. A diminution in the level of surface Ig (preferentially IgD) and the accumulation of cytoplasmic Ig observed after phorbol ester treatment were accompanied by a corresponding reduction or loss of the B1 antigen and usually of B2 when present. The most consistent change induced by TPA was the appearance of BB-1, a marker of activated B lymphocytes, which was rarely expressed on fresh leukemic cells. Another marker of activated lymphocytes, LB-1, was also often induced or increased in its expression after exposure of the cells to TPA. The magnitude of the TPA response appeared to relate to the stage of maturation arrest of the individual leukemic clones rather than to any clinical parameter explored. The significance of the findings to normal B cell differentiation and their potential clinical utility are discussed.

  11. Characterization of a novel human scavenger receptor cysteine-rich molecule SCART1 expressed by lymphocytes

    DEFF Research Database (Denmark)

    Holm, D.; Fink, D. R.; Steffensen, M. A.;

    2013-01-01

    of hSCART1 in the small intestine and colon. An antibody raised against an N-terminal hSCART1 peptide stains a subset of cells in the small intestine, stomach, and gall bladder, and it also stains placental villi. In conclusion, the characterization of hSCART1 at the mRNA and protein level suggests......The scavenger receptor cysteine-rich (SRCR) superfamily is a group of membrane bound and secreted proteins expressed by cells of the immune system. Several members act as pattern recognition receptors that bind to conserved molecular structures of pathogens. We have previously characterized...

  12. ICOS-expressing lymphocytes promote resolution of CD8-mediated lung injury in a mouse model of lung rejection.

    Directory of Open Access Journals (Sweden)

    Qiang Wu

    Full Text Available Acute rejection, a common complication of lung transplantation, may promote obliterative bronchiolitis leading to graft failure in lung transplant recipients. During acute rejection episodes, CD8(+ T cells can contribute to lung epithelial injury but the mechanisms promoting and controlling CD8-mediated injury in the lung are not well understood. To study the mechanisms regulating CD8(+ T cell-mediated lung rejection, we used a transgenic model in which adoptively transferred ovalbumin (OVA-specific cytotoxic T lymphocytes (CTL induce lung injury in mice expressing an ovalbumin transgene in the small airway epithelium of the lungs (CC10-OVA mice. The lung pathology is similar to findings in humans with acute lung transplant. In the presence of an intact immune response the inflammation resolves by day 30. Using CC10-OVA.RAG(-/- mice, we found that CD4(+ T cells and ICOS(+/+ T cells were required for protection against lethal lung injury, while neutrophil depletion was not protective. In addition, CD4(+Foxp3 (+ ICOS(+ T cells were enriched in the lungs of animals surviving lung injury and ICOS(+/+ Tregs promoted survival in animals that received ICOS(-/- T cells. Direct comparison of ICOS(-/- Tregs to ICOS(+/+ Tregs found defects in vitro but no differences in the ability of ICOS(-/- Tregs to protect from lethal lung injury. These data suggest that ICOS affects Treg development but is not necessarily required for Treg effector function.

  13. Expression and purification of a soluble B lymphocyte stimulator mutant modified with the T-helper cell epitope.

    Science.gov (United States)

    Gao, Huiguang; Fu, Weiling; Li, Rongfen; Chen, Linfeng; Ji, Qing; Zhang, Li; Huang, Gang; He, Fengtian

    2006-10-01

    The DNA encoding soluble B lymphocyte stimulator (134-285 amino acids, sBLyS) mutant with residues 217-224 replaced by two glycines (named msBLyS) was constructed. The sequence encoding a foreign immunodominant T-helper epitope from ovalbumin (OVA) was then coupled to the 5'-end of msBLyS cDNA. After being sequenced, the recombinant DNA was ligated into the prokaryotic expression vector pQE-80L. The recombinant protein was produced in E. coli DH5alpha after induction with IPTG with the yield of more than 40% of total bacterial protein. The recombinant protein was purified with Ni-NTA chromatography and Sepharcryl S200 chromatography to a purity of more than 98%. The BALB/c mice, immunized with the recombinant protein, produced anti-BLyS antibodies at a high level, which indicated that the recombinant BLyS mutant modified with T-helper epitope elicited polyclonal antibodies with cross-reactivity with BLyS in vivo. This recombinant protein may therefore be used as immune inhibitor of BLyS for treating BLyS -associated autoimmune diseases.

  14. Different subtypes of GABA-A receptors are expressed in human, mouse and rat T lymphocytes.

    Directory of Open Access Journals (Sweden)

    Suresh K Mendu

    Full Text Available γ-Aminobutyric acid (GABA is the most prominent neuroinhibitory transmitter in the brain, where it activates neuronal GABA-A receptors (GABA-A channels located at synapses and outside of synapses. The GABA-A receptors are primary targets of many clinically useful drugs. In recent years, GABA has been shown to act as an immunomodulatory molecule. We have examined in human, mouse and rat CD4(+ and CD8(+ T cells which subunit isoforms of the GABA-A channels are expressed. The channel physiology and drug specificity is dictated by the GABA-A receptor subtype, which in turn is determined by the subunit isoforms that make the channel. There were 5, 8 and 13 different GABA-A subunit isoforms identified in human, mouse and rat CD4(+ and CD8(+ T cells, respectively. Importantly, the γ2 subunit that imposes benzodiazepine sensitivity on the GABA-A receptors, was only detected in the mouse T cells. Immunoblots and immunocytochemistry showed abundant GABA-A channel proteins in the T cells from all three species. GABA-activated whole-cell transient and tonic currents were recorded. The currents were inhibited by picrotoxin, SR95531 and bicuculline, antagonists of GABA-A channels. Clearly, in both humans and rodents T cells, functional GABA-A channels are expressed but the subtypes vary. It is important to bear in mind the interspecies difference when selecting the appropriate animal models to study the physiological role and pharmacological properties of GABA-A channels in CD4(+ and CD8(+ T cells and when selecting drugs aimed at modulating the human T cells function.

  15. Signal transducer and activator of transcription-3 induces microRNA-155 expression in chronic lymphocytic leukemia.

    Directory of Open Access Journals (Sweden)

    Ping Li

    Full Text Available MicroRNA (miR abnormalities play a key role in the pathogenesis of chronic lymphocytic leukemia (CLL. High levels of miR-155 have been detected in human neoplasms, and overexpression of miR-155 has been found to induce lymphoma in mice. High levels of miR-155 were detected in CLL cells and STAT3, which is known to induce miR-21 and miR-181b-1 expression, is constitutively activated in CLL. Given these findings, we hypothesized that STAT3 induces miR-155. Sequence analysis revealed that the miR-155 promoter harbors two putative STAT3 binding sites. Therefore, truncated miR-155 promoter constructs and STAT3 small interfering RNA (siRNA were co-transfected into MM1 cells. Of the two putative binding sites, STAT3-siRNA reduced the luciferase activity of the construct containing the 700-709 bp STAT3 binding site, suggesting that this site is involved in STAT3-induced transcription. Electrophoretic mobility shift assay confirmed that STAT3 bound to the miR-155 promoter in CLL cells, and chromatin immunoprecipitation and luciferase assay confirmed that STAT3 bound to the 700-709 bp but not the 615-624 bp putative STAT3 binding site in CLL cells. Finally, STAT3-small hairpin RNA downregulated miR-155 gene expression, suggesting that constitutively activated STAT3 binds to the miR-155 gene promoter. Together, these results suggest that STAT3 activates miR-155 in CLL cells.

  16. 27-Hydroxycholesterol and 7alpha-hydroxycholesterol trigger a sequence of events leading to migration of CCR5-expressing Th1 lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sun-Mi, E-mail: lala1647@hanmail.net [Department of Pharmacology, Pusan National University, School of Medicine, Yangsan, Gyeongnam 626-870 (Korea, Republic of); Kim, Bo-Young, E-mail: kimboyoung@pusan.ac.kr [Department of Pharmacology, Pusan National University, School of Medicine, Yangsan, Gyeongnam 626-870 (Korea, Republic of); Lee, Sae-A, E-mail: saeah486@nate.com [Department of Pharmacology, Pusan National University, School of Medicine, Yangsan, Gyeongnam 626-870 (Korea, Republic of); Eo, Seong-Kug, E-mail: vetvirus@chonbuk.ac.kr [Laboratory of Microbiology, College of Veterinary Medicine and Bio-Safety Research Institute, Chonbuk National University, Jeonju, Jeonbuk 561-756 (Korea, Republic of); Yun, Yungdae, E-mail: yunyung@ewha.ac.kr [Department of Life Science, Ewha Womans University, Seoul 120-750 (Korea, Republic of); Kim, Chi-Dae, E-mail: chidkim@pusan.ac.kr [Department of Pharmacology, Pusan National University, School of Medicine, Yangsan, Gyeongnam 626-870 (Korea, Republic of); Kim, Koanhoi, E-mail: koanhoi@pusan.ac.kr [Department of Pharmacology, Pusan National University, School of Medicine, Yangsan, Gyeongnam 626-870 (Korea, Republic of)

    2014-02-01

    Th1 lymphocytes are predominant in atherosclerotic lesions. However, mechanisms involved in the Th1 predominance are unknown. We have investigated the possibility of Th1 lymphocyte recruitment in a cholesterol-rich milieu. A high cholesterol diet resulted in enhanced expression of CCR5 ligands, including CCL3 and CCL4, but not of proatherogenic CXCR3 ligands, in atherosclerotic arteries of ApoE{sup −/−} mice. 27-Hydroxycholesterol and 7α-hydroxycholesterol, cholesterol oxides (oxysterols) detected in abundance in atherosclerotic lesions, greatly induced the transcription of CCL3 and CCL4 genes in addition to enhancing secretion of corresponding proteins by THP-1 monocytic cells. However, an identical or even higher concentration of cholesterol, 7β-hydroxycholesterol, and 7-ketocholsterol did not influence expression of these chemokines. Conditioned media containing the CCR5 ligands secreted from THP-1 cells induced migration of Jurkat T cells expressing CCR5, a characteristic chemokine receptor of Th1 cells, but not of Jurkat T cells that do not express CCR5. The migration of CCR5-expressing Jurkat T cells was abrogated in the presence of a CCR5-neutralizing antibody. 27-Hydroxycholesterol and 7α-hydroxycholesterol enhanced phosphorylation of Akt. Pharmacological inhibitors of phosphoinositide-3-kinase/Akt pathways blocked transcription as well as secretion of CCL3 and CCL4 in conjunction with attenuated migration of CCR5-expressing Jurkat T cells. This is the first report on the involvement of cholesterol oxides in migration of distinct subtype of T cells. We propose that 27-hydroxycholesterol and 7α-hydroxycholesterol can trigger a sequence of events that leads to recruitment of Th1 lymphocytes and phosphoinositide-3-kinase/Akt pathways play a major role in the process. - Graphical abstract: Th1 lymphocytes are predominant in atherosclerotic lesions. However, mechanisms involved in the Th1 predominance are unknown. We have investigated the possibility of

  17. The Avian Transcription Factor c-Rel is Expressed in Lymphocyte Precursor Cells and Antigen-Presenting Cells During Thymus Development

    OpenAIRE

    Huguet, C.; Bouali, F.; Enrietto, P. J.; Stehelin, D.; B. Vandenbunder; Abbadie, C

    1998-01-01

    Transcription factors of the Rel/NF-κB family are widely involved in the immune system. In this study, we investigate the in vivo expression of the avian protein c-Rel in the T-cell lineage during thymus development. The majority of thymocytes do not express the c-Rel protein. However, lymphocyte precursor cells that colonize the thymus express the c-Rel protein shortly after their homing in the organ and before they begin to differentiate, c-Rel is also detected in different subsets of,antig...

  18. Immunohistochemical study of IL-17, perforin and CD68 in colorectal cancer

    Directory of Open Access Journals (Sweden)

    Hassan Ali Al-Saadi

    2014-12-01

    Full Text Available The tumor development in colorectal cancer is one of the most investigated tumorigenesis in the world. This study aims to analyze the impact of presence of IL-17, perforin and CD68 on colorectal cancer. During the period October 1st 2013 – March 1st 2014, samples from fifty patients with colon cancer (14 female and 36 male, 7-75 years old from Al-Hussain Hospital City (Kerbala, Iraq, Hospital Medical City (Baghdad, Iraq and Teaching Oncology Hospital, Baghdad Medical City (Baghdad, Iraq were taken and analyzed. Immunohistochemical analyses of IL-17, perforin and CD68 of colorectal biopsies were performed by using Dako's Service (Denmark. The levels of IL-17 were significantly (p0.05 between the genders, whereas there was a highly significant (p<0.01 increase in age group 41-75 when compared with the others groups. Also, the results revealed a highly significant raise (p<0.01 in Duke C when compared with the TNM groups and highly significant (p<0.01 correlation among study parameters. These results demonstrated the critical role of IL-17, perforin and CD68 highlighting tumorigenesis and inflammation as pro-inflammatory factors in colorectal cancer.

  19. CD49d (ITGA4) expression is a predictor of time to first treatment in patients with chronic lymphocytic leukaemia and mutated IGHV status.

    Science.gov (United States)

    Baumann, Tycho; Delgado, Julio; Santacruz, Rodrigo; Martínez-Trillos, Alejandra; Rozman, María; Aymerich, Marta; López, Cristina; Costa, Dolors; Carrió, Anna; Villamor, Neus; Montserrat, Emili

    2016-01-01

    We investigated CD49d (also termed ITGA4) expression and its biological and clinical correlations in 415 patients with chronic lymphocytic leukaemia. CD49d expression was stable over the course of the disease. A high expression of CD49d (>30%) was found in 142/415 (34%) patients and was associated with progressive disease (advanced clinical stage, high serum lactate dehydrogenase or β2 -microglobulin levels; all p < 0·05) and aggressive disease biology (increased ZAP70 or CD38, unmutated IGHV, trisomy 12, mutations of NOTCH1 and SF3B1; all P < 0·05). A higher CD49d expression was also associated with a lower blood lymphocyte count and a higher number of lymphoid areas involved by the disease. Patients with high CD49d expression were treated more frequently (55% vs. 27%; P < 0·001) and earlier (median time to treatment [TTT] 65·4 months vs. not reached; P < 0·001) than those with low CD49d expression. However, no significant differences in response rates were observed. In the subgroup of patients with mutated IGHV, high CD49d expression was predictive of a shorter TTT while other markers, such as ZAP70 and CD38, were not. In conclusion, in this study CD49d expression correlated with high-risk CLL biomarkers and proved to be useful for separating patients with mutated IGHV into two different prognostic groups.

  20. Over-Expression of Dopamine D2 Receptor and Inwardly Rectifying Potassium Channel Genes in Drug-Naive Schizophrenic Peripheral Blood Lymphocytes as Potential Diagnostic Markers

    OpenAIRE

    Ágnes Zvara; György Szekeres; Zoltán Janka; Kelemen, János Z.; Csongor Cimmer; Miklós Sántha; Puskás, László G.

    2005-01-01

    Schizophrenia is one of the most common neuropsychiatric disorders affecting nearly 1% of the human population. Current diagnosis of schizophrenia is based on complex clinical symptoms. The use of easily detectable peripheral molecular markers could substantially help the diagnosis of psychiatric disorders. Recent studies showed that peripheral blood lymphocytes (PBL) express subtypes of D1 and D2 subclasses of dopamine receptors. Recently, dopamine receptor D3 (DRD3) was found to be over-exp...

  1. Effects of stress, mimicked by administration of corticosterone in drinking water, on the expression of chicken cytokine and chemokine genes in lymphocytes.

    Science.gov (United States)

    Shini, S; Kaiser, P

    2009-09-01

    In this study, we identify molecular mediators that participate in the regulation of the immune response during corticosterone-induced stress in chickens. At 7 weeks of age, 120 chickens were exposed for 1 week to corticosterone treatment. Cytokine and chemokine mRNA expression levels were evaluated in peripheral blood and splenic lymphocytes. Expression levels of interleukin (IL)-1beta, IL-6, IL-18 and transforming growth factor (TGF)-beta4 mRNA were significantly up-regulated in lymphocytes 3 h after first treatment with corticosterone. TGF-beta4 and IL-18 remained elevated 1 week post-initial treatment. Compared with controls, corticosterone-treated birds showed greater expression levels of chemokine (CC) mRNA, particularly for CCLi2, CCL5 (RANTES), CCL16 and CXCLi1, in peripheral and splenic lymphocytes 3 h post-initial exposure. CCLi2 mRNA was highly expressed in splenocytes at all time-points. Administration of corticosterone significantly increased circulating corticosterone concentrations and decreased total lymphocyte counts at 3, 24 h and 1 week post-initiation of corticosterone treatment. There was a positive correlation between plasma corticosterone concentrations and CCL5 and CCL16 mRNA at 3 h post-initial administration. At 1 week post-initial treatment, corticosterone concentrations correlated positively with CCL5 and negatively with IL-18 mRNA level. Conditions associated with significant changes in corticosterone levels might therefore affect the immune response by increasing pro-inflammatory responses, leading to potential modulation of the Th1/Th2 balance.

  2. Apoptotic depletion of infiltrating mucosal lymphocytes associated with Fas ligand expression by Helicobacter pylori-infected gastric mucosal epithelium: human glandular stomach as a site of immune privilege.

    Science.gov (United States)

    Koyama, S

    2000-04-01

    H. pylori infection almost invariably results in chronic gastritis, but only a proportion of patients develops severe destruction of epithelial glandular structure or peptic ulcer. To confirm the recent data obtained in testis and eye, showing that Fas ligand is involved in the phenomenon of "immune privilege," expression of Fas receptor and its ligand of the stomach was investigated in a panel of gastric biopsies obtained from patients H. pylori-positive (N = 42) and with H. pylori-negative (N = 18) by two-color flow cytometry. The results show that membrane-bound Fas ligand protein is constitutively expressed on freshly isolated human gastric mucosal epithelium coupled with infiltrating lymphocytes. There was significant overexpression of Fas receptor and its ligand, and a higher frequency of apoptotic cell death detected by TUNEL in epithelium and infiltrating lymphocytes in H. pylori-infected patients. These findings suggest that involvement of Fas receptor and its ligand system contributes to some extent to mucosal damage in H. pylori-associated gastritis. However, the more specific findings are apoptotic depletion of invading mucosal lymphocytes associated with Fas ligand expression by gastric epithelium. These provide the first direct quantitative evidence to support Fas receptor counterattack and/or paracrine fratricide as a mechanism of immune privilege in vivo in the H. pylori-infected glandular stomach.

  3. Different NK cell-activating receptors preferentially recruit Rab27a or Munc13-4 to perforin-containing granules for cytotoxicity

    DEFF Research Database (Denmark)

    Wood, Stephanie M; Meeths, Marie; Chiang, Samuel C C

    2009-01-01

    of perforin-containing lytic granules induced by signals for natural and antibody-dependent cellular cytotoxicity. We demonstrate here that these signals fail to induce degranulation in resting NK cells from Rab27a-deficient patients. In resting NK cells from healthy subjects, endogenous Rab27a and Munc13...... functional antigen-1, NKG2D, or 2B4 induced colocalization of Rab27a, but not Munc13-4, with perforin. Conversely, engagement of antibody-dependent cellular cytotoxicity receptor CD16 induced colocalization of Munc13-4, but not Rab27a, with perforin. Furthermore, colocalization of Munc13-4 with perforin...

  4. Molecular Mechanisms of Lymphocyte-Mediated Cytotoxicity

    Institute of Scientific and Technical Information of China (English)

    Zusen Fan; Qixiang Zhang

    2005-01-01

    Granule-mediated cytotoxicity is the major mechanism for lymphocytes to kill viruses, intracellular bacteria and tumors. The cytotoxic granules move to the immunological synapse by exocytosis after recognition of a killer cell.The contents of the granules are delivered into target cells with the help of perforin by endocytosis. A group of serine protease granzymes cleave their critical substrates to initiate DNA damage and cell death. The most abundant granzymes are granzyme A and B. They induce cell death through alternate and nonoverlapping pathways. The substrates and functions of the majority of the orphan granzymes have not yet been identified. It is possible that the diversity of granzymes provides fail-safe mechanisms for killing viruses and tumor cells.

  5. Melatonin may play a role in modulation of bax and bcl-2 expression levels to protect rat peripheral blood lymphocytes from gamma irradiation-induced apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Mohseni, Mehran [Department of Radiology and Medical Physics, Faculty of Paramedicine, Kashan University of Medical Sciences, Kashan (Iran, Islamic Republic of); Mihandoost, Ehsan, E-mail: mihandoost.e@gmail.com [Department of Medical Radiation Engineering, Science and Research Branch, Islamic Azad University, Tehran (Iran, Islamic Republic of); Shirazi, Alireza [Department of Medical Physics and Biomedical Engineering, Faculty of Medicine, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Sepehrizadeh, Zargham [Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Bazzaz, Javad Tavakkoly [Department of Medical Genetics, Faculty of Medicine, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Ghazi-khansari, Mahmoud [Department of Pharmacology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of)

    2012-10-15

    The close relationship between free radicals effects and apoptosis process has been proved. Melatonin has been reported as a direct free radical scavenger. We investigated the capability of melatonin in the modification of radiation-induced apoptosis and apoptosis-associated upstream regulators expression in rat peripheral blood lymphocytes. Rats were irradiated with a single whole body Cobalt 60-gamma radiation dose of 8 Gy at a dose rate of 101 cGy/min with or without melatonin pretreatments at different concentrations of 10 and 100 mg/kg body weight. The rats were divided into eight groups of control, irradiation-only, vehicle-only, vehicle plus irradiation, 10 mg/kg melatonin alone, 10 mg/kg melatonin plus irradiation, 100 mg/kg melatonin alone and 100 mg/kg melatonin plus irradiation. Rats were given an intraperitoneal (IP) injection of melatonin or the same volume of vehicle alone 1 h prior to irradiation. Blood samples were taken 4, 24, 48 and 72 h after irradiation for evaluation of flow cytometric analysis of apoptotic lymphocytes using Annexin V/PI assay and measurement of bax and bcl-2 expression using quantitative real-time PCR (RT{sup 2}qPCR). Irradiation-only and vehicle plus irradiation showed an increase in the percentage of apoptotic lymphocytes significantly different from control group (P < 0.01), while melatonin pretreatments in a dose-dependent manner reduced it as compared with the irradiation-only and vehicle plus irradiation groups (P < 0.01) in all time points. This reduced apoptosis by melatonin was related to the downregulation of bax, upregulation of bcl-2, and therefore reduction of bax/bcl-2 ratio. Our results suggest that melatonin in these doses may provide modulation of bax and bcl-2 expression as well as bax/bcl-2 ratio to protect rat peripheral blood lymphocytes from gamma irradiation-induced apoptosis.

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

    Directory of Open Access Journals (Sweden)

    Sha-Sha Huang

    2016-10-01

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

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

    Science.gov (United States)

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

    2016-10-01

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

  8. 27-Hydroxycholesterol and 7alpha-hydroxycholesterol trigger a sequence of events leading to migration of CCR5-expressing Th1 lymphocytes.

    Science.gov (United States)

    Kim, Sun-Mi; Kim, Bo-Young; Lee, Sae-A; Eo, Seong-Kug; Yun, Yungdae; Kim, Chi-Dae; Kim, Koanhoi

    2014-02-01

    Th1 lymphocytes are predominant in atherosclerotic lesions. However, mechanisms involved in the Th1 predominance are unknown. We have investigated the possibility of Th1 lymphocyte recruitment in a cholesterol-rich milieu. A high cholesterol diet resulted in enhanced expression of CCR5 ligands, including CCL3 and CCL4, but not of proatherogenic CXCR3 ligands, in atherosclerotic arteries of ApoE(-/-) mice. 27-Hydroxycholesterol and 7α-hydroxycholesterol, cholesterol oxides (oxysterols) detected in abundance in atherosclerotic lesions, greatly induced the transcription of CCL3 and CCL4 genes in addition to enhancing secretion of corresponding proteins by THP-1 monocytic cells. However, an identical or even higher concentration of cholesterol, 7β-hydroxycholesterol, and 7-ketocholsterol did not influence expression of these chemokines. Conditioned media containing the CCR5 ligands secreted from THP-1 cells induced migration of Jurkat T cells expressing CCR5, a characteristic chemokine receptor of Th1 cells, but not of Jurkat T cells that do not express CCR5. The migration of CCR5-expressing Jurkat T cells was abrogated in the presence of a CCR5-neutralizing antibody. 27-Hydroxycholesterol and 7α-hydroxycholesterol enhanced phosphorylation of Akt. Pharmacological inhibitors of phosphoinositide-3-kinase/Akt pathways blocked transcription as well as secretion of CCL3 and CCL4 in conjunction with attenuated migration of CCR5-expressing Jurkat T cells. This is the first report on the involvement of cholesterol oxides in migration of distinct subtype of T cells. We propose that 27-hydroxycholesterol and 7α-hydroxycholesterol can trigger a sequence of events that leads to recruitment of Th1 lymphocytes and phosphoinositide-3-kinase/Akt pathways play a major role in the process.

  9. ICOS, SLAM and PD-1 expression and regulation on T lymphocytes reflect the immune dysregulation in patients with HIV-related illness with pulmonary tuberculosis

    Science.gov (United States)

    Jurado, Javier Oscar; Pasquinelli, Virginia; Alvarez, Ivana Belén; Martínez, Gustavo Javier; Laufer, Natalia; Sued, Omar; Cahn, Pedro; Musella, Rosa María; Abbate, Eduardo; Salomón, Horacio; Quiroga, María Florencia

    2012-01-01

    Background Tuberculosis (TB) continues to be the most frequent cause of illness and death from an infectious agent globally, and its interaction with HIV is having devastating effects. To investigate how HIV alters the immune response to Mycobacterium tuberculosis (Mtb), we assessed basal and Mtb-induced proliferation, cytokine production, and expression of signalling lymphocytic activation molecule (SLAM), inducible costimulator (ICOS) and programmed death-1 (PD-1) on T lymphocytes from HIV-positive individuals coinfected with TB, HIV-positive subjects, TB patients and healthy donors (HD). Findings HIV-TB patients showed increased ICOS, SLAM and PD-1 basal levels on T lymphocytes, whereas HIV-positive individuals displayed elevated levels of SLAM and PD-1, TB patients high levels of SLAM, and HD low levels of the three proteins. Mtb-stimulation enhanced ICOS expression in the four groups, but only TB and HD increased SLAM and PD-1 levels. Conclusions These data show the immune deregulation that takes place during the immune response against TB in different study populations. PMID:22713261

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

    Institute of Scientific and Technical Information of China (English)

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

    2004-01-01

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

  11. ICOS, SLAM and PD-1 expression and regulation on T lymphocytes reflect the immune dysregulation in patients with HIV-related illness with pulmonary tuberculosis

    Directory of Open Access Journals (Sweden)

    Horacio Salomón

    2012-06-01

    Full Text Available Background: Tuberculosis (TB continues to be the most frequent cause of illness and death from an infectious agent globally, and its interaction with HIV is having devastating effects. To investigate how HIV alters the immune response to Mycobacterium tuberculosis (Mtb, we assessed basal and Mtb-induced proliferation, cytokine production, and expression of signalling lymphocytic activation molecule (SLAM, inducible costimulator (ICOS and programmed death-1 (PD-1 on T lymphocytes from HIV-positive individuals coinfected with TB, HIV-positive subjects, TB patients and healthy donors (HD. Findings: HIV-TB patients showed increased ICOS, SLAM and PD-1 basal levels on T lymphocytes, whereas HIV-positive individuals displayed elevated levels of SLAM and PD-1, TB patients high levels of SLAM, and HD low levels of the three proteins. Mtb-stimulation enhanced ICOS expression in the four groups, but only TB and HD increased SLAM and PD-1 levels. Conclusions: These data show the immune deregulation that takes place during the immune response against TB in different study populations.

  12. Neuroinflammation by cytotoxic T-lymphocytes impairs retrograde axonal transport in an oligodendrocyte mutant mouse.

    Directory of Open Access Journals (Sweden)

    Chi Wang Ip

    Full Text Available Mice overexpressing proteolipid protein (PLP develop a leukodystrophy-like disease involving cytotoxic, CD8+ T-lymphocytes. Here we show that these cytotoxic T-lymphocytes perturb retrograde axonal transport. Using fluorogold stereotactically injected into the colliculus superior, we found that PLP overexpression in oligodendrocytes led to significantly reduced retrograde axonal transport in retina ganglion cell axons. We also observed an accumulation of mitochondria in the juxtaparanodal axonal swellings, indicative for a disturbed axonal transport. PLP overexpression in the absence of T-lymphocytes rescued retrograde axonal transport defects and abolished axonal swellings. Bone marrow transfer from wildtype mice, but not from perforin- or granzyme B-deficient mutants, into lymphocyte-deficient PLP mutant mice led again to impaired axonal transport and the formation of axonal swellings, which are predominantly located at the juxtaparanodal region. This demonstrates that the adaptive immune system, including cytotoxic T-lymphocytes which release perforin and granzyme B, are necessary to perturb axonal integrity in the PLP-transgenic disease model. Based on our observations, so far not attended molecular and cellular players belonging to the immune system should be considered to understand pathogenesis in inherited myelin disorders with progressive axonal damage.

  13. Ontogenic development of kidney, thymus and spleen and phenotypic expression of CD3 and CD4 receptors on the lymphocytes of cobia (Rachycentroncanadum).

    Science.gov (United States)

    Klosterhoff, Marta C; Pereira Júnior, Joaber; Rodrigues, Ricardo V; Gusmão, Emeline P; Sampaio, Luís A; Tesser, Marcelo B; Romano, Luis A

    2015-01-01

    In the present study was evaluated the ontogenic of immunocompetent organs of cobia up to 53 days after hatching (dah) through histology and immunohistochemistry techniques. The kidney was the first lymphohematopoietic organ to appear, at 1 dah, followed by the spleen at 5 dah and the thymus at 7 dah. The first CD3 receptors on the lymphocytes were observed in 27% of the thymic tissue at 7 dah and in 99% at 53 dah. The phenotypic expression of CD3 receptors was registered in 10% of the kidney at 8 dah and in 32% at 53 dah. CD4 receptors were observed in 5% and 63% of the thymic area at 7 and 53 dah, respectively. In the kidney, T4 lymphocytes were first observed at 13 dah in 9% of the organ and in 28% at 53 dah, defining the functional development of the specific system associated with immunological memory capacity.

  14. Malignant transformation of CD4+ T lymphocytes mediated by oncogenic kinase NPM/ALK recapitulates IL-2-induced cell signaling and gene expression reprogramming.

    Science.gov (United States)

    Marzec, Michal; Halasa, Krzysztof; Liu, Xiaobin; Wang, Hong Y; Cheng, Mangeng; Baldwin, Donald; Tobias, John W; Schuster, Stephen J; Woetmann, Anders; Zhang, Qian; Turner, Suzanne D; Ødum, Niels; Wasik, Mariusz A

    2013-12-15

    Anaplastic lymphoma kinase (ALK), physiologically expressed only by nervous system cells, displays a remarkable capacity to transform CD4(+) T lymphocytes and other types of nonneural cells. In this study, we report that activity of nucleophosmin (NPM)/ALK chimeric protein, the dominant form of ALK expressed in T cell lymphomas (TCLs), closely resembles cell activation induced by IL-2, the key cytokine supporting growth and survival of normal CD4(+) T lymphocytes. Direct comparison of gene expression by ALK(+) TCL cells treated with an ALK inhibitor and IL-2-dependent ALK(-) TCL cells stimulated with the cytokine revealed a very similar, albeit inverse, gene-regulation pattern. Depending on the analysis method, up to 67% of the affected genes were modulated in common by NPM/ALK and IL-2. Based on the gene expression patterns, Jak/STAT- and IL-2-signaling pathways topped the list of pathways identified as affected by both IL-2 and NPM/ALK. The expression dependence on NPM/ALK and IL-2 of the five selected genes-CD25 (IL-2Rα), Egr-1, Fosl-1, SOCS3, and Irf-4-was confirmed at the protein level. In both ALK(+) TCL and IL-2-stimulated ALK(-) TCL cells, CD25, SOCS3, and Irf-4 genes were activated predominantly by the STAT5 and STAT3 transcription factors, whereas transcription of Egr-1 and Fosl-1 was induced by the MEK-ERK pathway. Finally, we found that Egr-1, a protein not associated previously with either IL-2 or ALK, contributes to the cell proliferation. These findings indicate that NPM/ALK transforms the target CD4(+) T lymphocytes, at least in part, by using the pre-existing, IL-2-dependent signaling pathways.

  15. Tumor-infiltrating lymphocytes expressing IOT-10 marker. An immunohistochemical study of a series of 185 brain tumors.

    Science.gov (United States)

    Zurita, M; Vaquero, J; Coca, S; Oya, S; Garcia, N

    1993-04-01

    The presence of IOT-10-positive lymphocytes among the tumor-infiltrating-lymphocyte (TIL) population was studied in a series of 185 brain tumors. In most of the tumors, IOT-10-positive lymphocytes were identified, but generally they were scarce and masked among the tumor cells, suggesting that NK-cells exercise a poor participation in the tissular response against brain tumors. Isolated tumor cells showing IOT-10-positivity were found in low-grade astrocytomas, neurinomas and medulloblastomas. IOT-10-positivity on both tumor neuropil and tumor cells was considered a characteristic finding in oligodendrogliomas. The number of IOT-10-positive NK-cells in brain metastases and in cerebellar hemangioblastomas was comparatively greater than in other types of brain tumor. Since in brain metastases, the presence of IOT-10-positive NK-cells can be related to the tissular response to an extracerebral malignancy, their considerable presence in cerebellar hemangioblastomas is an enigmatic finding that deserves further attention.

  16. Expression of Th17 and Treg lymphocyte subsets in hypertrophied adenoids of children and its clinical significance.

    Science.gov (United States)

    Sade, Kobi; Fishman, Gadi; Kivity, Shmuel; DeRowe, Ari; Langier, Sheila

    2011-01-01

    Adenoid hypertrophy is the most common cause of upper airway obstruction and sleep-disordered breathing in children, yet its pathogenesis remains unclear. The identification of the novel helper T cell subsets, Th17 cells and regulatory T cells (Tregs) could provide new insight into our understanding of the mechanisms involved in the development of this condition. The purpose of this study is to evaluate the adenoidal lymphocyte subsets to describe the percentage of various lymphocyte subsets in hypertrophied adenoids and correlate them with symptom severity. Twenty consecutive children undergoing adenoidectomy were included, and lymphocytes were isolated from their adenoids. T cell subpopulations were detected by flow cytometry using a fluoresceinated monoclonal antibody directed against a number of cell markers (CD4+, CD8+, CD25+, FOXP3 IL17+, and others). We found a significant negative linear correlation between the Th17/Treg ratio and the patients' clinical scores (R = -0.71 p adenoid hypertrophy.

  17. Expression of the glucocorticoid receptor from the 1A promoter correlates with T lymphocyte sensitivity to glucocorticoid-induced cell death.

    Science.gov (United States)

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

    2004-09-15

    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.

  18. Lymphocyte 'homing' and chronic inflammation.

    Science.gov (United States)

    Sakai, Yasuhiro; Kobayashi, Motohiro

    2015-07-01

    Chronic inflammation is a response to prolonged exposure to injurious stimuli that harm and destroy tissues and promote lymphocyte infiltration into inflamed sites. Following progressive accumulation of lymphocytes, the histology of inflamed tissue begins to resemble that of peripheral lymphoid organs, which can be referred to as lymphoid neogenesis or formation of tertiary lymphoid tissues. Lymphocyte recruitment to inflamed tissues is also reminiscent of lymphocyte homing to peripheral lymphoid organs. In the latter, under physiological conditions, homing receptors expressed on lymphocytes adhere to vascular addressin expressed on high endothelial venules (HEVs), initiating a lymphocyte migration process composed of sequential adhesive interactions. Intriguingly, in chronic inflammation, HEV-like vessels are induced de novo, despite the fact that the inflamed site is not originally lymphoid tissue, and these vessels contribute to lymphocyte recruitment in a manner similar to physiological lymphocyte homing. In this review, we first describe physiological lymphocyte homing mechanisms focusing on vascular addressins. We then describe HEV-like vessel-mediated pathogenesis seen in various chronic inflammatory disorders such as Helicobacter pylori gastritis, inflammatory bowel disease (IBD), autoimmune pancreatitis and sclerosing sialadenitis, as well as chronic inflammatory cell neoplasm MALT lymphoma, with reference to our work and that of others.

  19. Flow cytometric analysis of p21 protein expression on irradiated human lymphocytes; Analise por citometria de fluxo da expressao da proteina p21 em linfocitos humanos irradiados

    Energy Technology Data Exchange (ETDEWEB)

    Santos, N.F.G.; Amaral, A., E-mail: neyliane@gmail.com [Universidade Federal de Pernambuco (UFPE), Recife, PE (Brazil). Departamento de Energia Nuclear. Laboratorio de Modelagem e Biodosimetria Aplicada; Freitas-Silva, R. [Universidade Federal de Pernambuco (UFPE), Garanhuns, PE (Brazil). Departamento de Ciencias Naturais e Exatas; Pereira, V.R.A. [Fundacao Oswaldo Cruz (FIOCRUZ), Recife, PE (Brazil). Centro de Pesquisas Aggeu Magalhaes. Departamento de Imunologia. Lab. de Imunoparasitologia; Tasat, D.R. [Universidad Nacional de General San Martin, Buenos Aires (Argentina). Escuela de Ciencia y Tecnologia. Laboratorio de Biologia Celular del Pulmon

    2013-08-15

    Cell cycle blockage in G1 is a mechanism p21 protein-regulated and coupled to DNA damage response to permit genetic content analysis, damage repair and cell death. Analysis of proteins that participates of this response has progressed with new analytic tools, and data contributes to comprehension of radioinduced molecular events as well as to new approaches on practices that employ ionizing radiation. On this perspective, the aim of this research was to evaluate, by flow cytometry, p21 expression on irradiated human lymphocytes, maintained under different experimental conditions. Peripheral blood samples from 10 healthy subjects were irradiated with doses of 0 (non-irradiated), 1, 2 and 4 Gy. Lymphocytes were processed to analysis on ex vivo (no cultured) condition and after 24; 48 and 72 hours culture, with and without phytohemagglutinin stimulation. p21 protein expression levels were measured by flow cytometry, as percentage values. Results indicate that flow cytometric assay allows detection of changes on p21 expression, since it was detected significant increase on phytohemagglutinin-stimulated samples, for all times, against basal expression (ex vivo). However, it was not observed significant alterations on p21 protein radioinduced levels, for all doses, times and culture conditions analyzed. These results not indicate so p21 protein as bioindicator of ionizing radiation exposure. Nevertheless, data confirmation may to require analysis of a more numerous population. (author)

  20. REARRANGEMENT AND EXPRESSION OF T CELL RECEPTOR β GENE IN HUMAN HEMOPOIETIC CELL LINES AND PRIMARY CELLS FROM ACUTE LYMPHOCYTIC LEUKEMIAS

    Institute of Scientific and Technical Information of China (English)

    仇一华; 陈诗书

    1992-01-01

    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.

  1. MHC Expression on Spleen Lymphocyte Subsets in Genetically Resistant and Susceptible Chickens Infected with Marek's Disease Virus

    DEFF Research Database (Denmark)

    Dalgaard, Tina; Bøving, Mette K.; Handberg, Kurt

    2009-01-01

    Resistance and susceptibility to Marek's disease (MD) are strongly influenced by the chicken major histocompatibility complex (MHC). In this study, splenic lymphocytes from MD-resistant and MD-susceptible chickens of three MHC genotypes (B21/B21, B19/B21, and B19/B19) were analyzed by flow...

  2. Neuromyelitis optica, atypical hemophagocytic lymphohistiocytosis and heterozygous perforin A91V mutation.

    Science.gov (United States)

    Palterer, Boaz; Brugnolo, Francesca; Sieni, Elena; Barilaro, Alessandro; Parronchi, Paola

    2017-10-15

    Neuromyelitis optica is an autoimmune demyelinating inflammatory disease characterized by optic neuritis and myelitis with anti-aquaporin 4 antibodies. Hemophagocytic lymphohistiocytosis is a severe systemic inflammatory syndrome that can present in a genetic primary form or secondarily to infective, neoplastic or autoimmune diseases. Our case discusses the first reported case of atypical late-onset hemophagocytic lymphohistiocytosis in a patient with neuromyelitis optica, with multiple triggering factors and carrying the common A91V hypomorphic perforin mutation, that blurs the distinction between primary and secondary forms. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Increased lymphocyte apoptosis in mouse models of colitis upon ABT-737 treatment is dependent upon BIM expression.

    Science.gov (United States)

    Lutz, C; Mozaffari, M; Tosevski, V; Caj, M; Cippà, P; McRae, B L; Graff, C L; Rogler, G; Fried, M; Hausmann, M

    2015-08-01

    Exaggerated activation of lymphocytes contributes to the pathogenesis of inflammatory bowel disease (IBD). Medical therapies are linked to the BCL-2 family-mediated apoptosis. Imbalance in BCL-2 family proteins may cause failure in therapeutic responses. We investigated the role of BCL-2 inhibitor ABT-737 for lymphocyte apoptosis in mice under inflammatory conditions. B.6129P2-interleukin (IL)-10(tm1Cgn) /J (IL-10(-/-) ) weighing 25-30 g with ongoing colitis were used. Fifty mg/kg/day ABT-737 was injected intraperitoneally (i.p.). Haematological analyses were performed with an ADVIA 2120 flow cytometer and mass cytometry with a CyTOF 2. Following i.p. administration, ABT-737 was detected in both spontaneous and acute colitis in peripheral blood (PBL) and colon tissue. Treatment led to lymphopenia. CD4(+) CD44(+) CD62L(+) central memory and CD8(+) , CD44(+) CD62L(-) central memory T cells were decreased in PBL upon ABT-737 compared to vehicle-receiving controls. Increased apoptosis upon ABT-737 was determined in blood lymphocytes, splenocytes and Peyer's patches and was accompanied by a decrease in TNF and IL-1B. ABT-737 positively altered the colonic mucosa and ameliorated inflammation, as shown by colonoscopy, histology and colon length. A decreased BIM/BCL-2 ratio or absence of BIM in both Bim(-) (/) (-) and Il10(-) (/) (-) × Bim(-) (/) (-) impeded the protective effect of ABT-737. The BIM/BCL-2 ratio decreased with age and during the course of treatment. Thus, long-term treatment resulted in adapted TNF levels and macroscopic mucosal damage. ABT-737 was efficacious in diminishing lymphocytes and ameliorating colitis in a BIM-dependent manner. Regulation of inappropriate survival of lymphocytes by ABT-737 may provide a therapeutic strategy in IBD.

  4. Determination of CD30 Expression on Peripheral Blood T Lymphocyte Subsets in Patients with Hemorrhagic Fever with Renal Syndrome by FCM

    Institute of Scientific and Technical Information of China (English)

    XIONG; Lijuan; LUO; Duande; ZENG; Linglan; LI; Shuli

    2001-01-01

    To determine the CD30 expression on peripheral blood T lymphocyte subsets in patients with hemorrhagic fever with renal syndrome (HFRS) and its clinical implications, double immunofluorescence technique and flow cytometry were used. There was no significant difference among the severe group, mild-moderate group and normal control group in the CD4+CD30- T lymphocyte subset.While the CD4+CD30+ T cells of HFRS patients were increased and the difference between severe group and mild-moderate group or normal control group were very significant (P<0. 01) and the difference between the mild-moderate group and normal control group was also significant (P<0. 05). The CD8+CD30-T cells were increased while the CD8+CD30+ T cells decreased obviously in HFRS patients,and the differences among three groups in both subsets were very significant (P<0. 01). The results showed that the humoral immunity and cellular immunity are overactive in HFRS patients during acute phase. The loss of balance between T lymphocyte subsets may play an important role in the pathophysiology of HFRS and is closely correlated with the severity of the HFRS.

  5. Differential RNA expression between schizophrenic patients and controls of the dystrobrevin binding protein 1 and neuregulin 1 genes in immortalized lymphocytes.

    Science.gov (United States)

    Chagnon, Y C; Roy, M-A; Bureau, A; Mérette, C; Maziade, M

    2008-03-01

    The dystrobrevin binding protein 1 (DTNBP1) and neuregulin 1 (NRG1) genes have been related to schizophrenia (SZ) and bipolar disorder (BP) by several whole-genome linkage and associations studies. Few expression studies in post-mortem brains have also reported a lower or a higher expression of DTNBP1 and NRG1, respectively, in SZ. Since the difficulty to access post-mortem brains, we evaluated RNA expression of DTNBP1 and NRG1 in immortalized lymphocytes of SZ patients and unrelated-family controls. An antipsychotic stimulation was also used to challenge the genetic background of the subjects and enhance differential expression. Immortalized lymphocytes of twelve SZ and twelve controls were grown individually in the presence or not of the antipsychotic olanzapine (Zyprexa; EliLilly). RNA was extracted and pooled in four groups of three SZ and four groups of three controls, and used to probe Agilent 18K microchips. Mean gene expression values were contrasted between SZ and control groups using a T-test. For DTNBP1, RNA expression was lower in SZ than in controls before (-28%; p=0.02) and after (-30%; p=0.01) olanzapine stimulation. Similarly, NRG1 GGF2 isoform showed a lower expression in SZ before (-29%; p=0.04) and after (-33%; p=0.02) olanzapine stimulation. In contrast, NRG1 GGF isoform showed no significant difference between SZ and controls (-7%; p=0.61, +3%; p=0.86, respectively), but was slightly repressed by olanzapine in controls (-8%; p=0.008) but not in SZ (+1%; p=0.91). These results are in agreement with those observed in post-mortem brain when the isoforms involved are considered.

  6. Sphingosine-1-phosphate receptor-1 (S1P1) is expressed by lymphocytes, dendritic cells, and endothelium and modulated during inflammatory bowel disease

    Science.gov (United States)

    Karuppuchamy, Thangaraj; Behrens, En-hui; González-Cabrera, Pedro; Sarkisyan, Gor; Gima, Lauren; Boyer, Joshua D.; Bamias, Giorgos; Jedlicka, Paul; Veny, Marisol; Clark, David; Peach, Robert; Scott, Fiona; Rosen, Hugh; Rivera-Nieves, Jesús

    2016-01-01

    The sphingosine-1-phosphate receptor-1 (S1P1) agonist ozanimod ameliorates ulcerative colitis, yet its mechanism of action is unknown. Here we examine the cell subsets that express S1P1 in intestine using S1P1-eGFP mice, the regulation of S1P1 expression in lymphocytes after administration of DSS, after colitis induced by transfer of CD4+CD45RBhi cells and by crossing a mouse with TNF-driven ileitis with S1P1-eGFP mice. We then assayed the expression of enzymes that regulate intestinal S1P levels, and the effect of FTY720 on lymphocyte behavior and S1P1 expression. We found that not only T and B cells express S1P1, but also dendritic (DC) and endothelial cells. Furthermore, chronic but not acute inflammatory signals increased S1P1 expression, while the enzymes that control tissue S1P levels in mice and humans with IBD were uniformly dysregulated, favoring synthesis over degradation. Finally, we observed that FTY720 reduced T cell velocity and induced S1P1 degradation and retention of naïve but not effector T cells. Our data demonstrate that chronic inflammation modulates S1P1 expression and tissue S1P levels and suggests that the anti-inflammatory properties of S1PR agonists might not be solely due to their lymphopenic effects, but also due to potential effects on DC migration and vascular barrier function. PMID:27049060

  7. Improved cytotoxic T-lymphocyte immune responses to a tumor antigen by vaccines co-expressing the SLAM-associated adaptor EAT-2.

    Science.gov (United States)

    Aldhamen, Y A; Seregin, S S; Kousa, Y A; Rastall, D P W; Appledorn, D M; Godbehere, S; Schutte, B C; Amalfitano, A

    2013-10-01

    The signaling lymphocytic activation molecule-associated adaptor Ewing's sarcoma's-activated transcript 2 (EAT-2) is primarily expressed in dendritic cells, macrophages and natural killer cells. Including EAT-2 in a vaccination regimen enhanced innate and adaptive immune responses toward pathogen-derived antigens, even in the face of pre-existing vaccine immunity. Herein, we investigate whether co-vaccinations with two recombinant Ad5 (rAd5) vectors, one expressing the carcinoembryonic antigen (CEA) and one expressing EAT-2, can induce more potent CEA-specific cytotoxic T lymphocyte (CTL) and antitumor activity in the therapeutic CEA-expressing MC-38 tumor model. Our results suggest that inclusion of EAT-2 significantly alters the kinetics of Th1-biasing proinflammatory cytokine and chemokine responses, and enhances anti-CEA-specific CTL responses. As a result, rAd5-EAT2-augmented rAd5-CEA vaccinations are more efficient in eliminating CEA-expressing target cells as measured by an in vivo CTL assay. Administration of rAd5-EAT2 vaccines also reduced the rate of growth of MC-38 tumor growth in vivo. Also, an increase in MC-38 tumor cell apoptosis (as measured by hematoxylin and eosin staining, active caspase-3 and granzyme B levels within the tumors) was observed. These data provide evidence that more efficient, CEA-specific effector T cells are generated by rAd5 vaccines expressing CEA, when augmented by rAd5 vaccines expressing EAT-2, and this regimen may be a promising approach for cancer immunotherapy in general.

  8. 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.

    Science.gov (United States)

    Groma, Valerija; Kazanceva, Anna; Nora-Krukle, Zaiga; Murovska, Modra

    2012-09-15

    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.

  9. Vitamin D3 Suppresses Class II Invariant Chain Peptide Expression on Activated B-Lymphocytes: A Plausible Mechanism for Downregulation of Acute Inflammatory Conditions

    Directory of Open Access Journals (Sweden)

    Omar K. Danner

    2016-01-01

    Full Text Available Class II invariant chain peptide (CLIP expression has been demonstrated to play a pivotal role in the regulation of B cell function after nonspecific polyclonal expansion. Several studies have shown vitamin D3 helps regulate the immune response. We hypothesized that activated vitamin D3 suppresses CLIP expression on activated B-cells after nonspecific activation or priming of C57BL/6 mice with CpG. This study showed activated vitamin D3 actively reduced CLIP expression and decreased the number of CLIP+ B-lymphocytes in a dose and formulation dependent fashion. Flow cytometry was used to analyze changes in mean fluorescent intensity (MFI based on changes in concentration of CLIP on activated B-lymphocytes after treatment with the various formulations of vitamin D3. The human formulation of activated vitamin D (calcitriol had the most dramatic reduction in CLIP density at an MFI of 257.3 [baseline of 701.1 (P value = 0.01]. Cholecalciferol and alfacalcidiol had no significant reduction in MFI at 667.7 and 743.0, respectively. Calcitriol seemed to best reduce CLIP overexpression in this ex vivo model. Bioactive vitamin D3 may be an effective compliment to other B cell suppression therapeutics to augment downregulation of nonspecific inflammation associated with many autoimmune disorders. Further study is necessary to confirm these findings.

  10. Statins disrupt CCR5 and RANTES expression levels in CD4(+ T lymphocytes in vitro and preferentially decrease infection of R5 versus X4 HIV-1.

    Directory of Open Access Journals (Sweden)

    Alexey A Nabatov

    Full Text Available BACKGROUND: Statins have previously been shown to reduce the in vitro infection of human immunodeficiency virus type 1 (HIV-1 through modulation of Rho GTPase activity and lipid raft formation at the cell surface, as well as by disrupting LFA-1 incorporation into viral particles. PRINCIPLE FINDINGS: Here we demonstrate that treatment of an enriched CD4(+ lymphocyte population with lovastatin (Lov, mevastatin (Mev and simvastatin (activated and non-activated, Sim(A and Sim(N, respectively can reduce the cell surface expression of the CC-chemokine receptor CCR5 (P<0.01 for Sim(A and Lov. The lowered CCR5 expression was associated with down-regulation of CCR5 mRNA expression. The CC-chemokine RANTES protein and mRNA expression levels were slightly increased in CD4(+ enriched lymphocytes treated with statins. Both R5 and X4 HIV-1 were reduced for their infection of statin-treated cells; however, in cultures where statins were removed and where a decrease in CCR5 expression was observed, there was a preferential inhibition of infection with an R5 versus X4 virus. CONCLUSIONS: The results indicate that the modulation of CC-chemokine receptor (CCR5 and CC-chemokine (RANTES expression levels should be considered as contributing to the anti-viral effects of statins, preferentially inhibiting R5 viruses. This observation, in combination with the immunomodulatory activity exerted by statins, suggests they may possess more potent anti-HIV-1 activity when applied during the early stages of infection or in lowering viral transmission. Alternatively, statin treatment could be considered as a way to modulate immune induction such as during vaccination protocols.

  11. Expression of NY-ESO-1 in Triple-Negative Breast Cancer Is Associated with Tumor-Infiltrating Lymphocytes and a Good Prognosis.

    Science.gov (United States)

    Lee, Hee Jin; Kim, Joo Young; Song, In Hye; Park, In Ah; Yu, Jong Han; Gong, Gyungyub

    2015-01-01

    Accumulating evidence suggests that immunotherapy has great potential for treating triple-negative breast cancer (TNBC). We analyzed the expression of NY-ESO-1, which is a potent immunogenic cancer testis antigen, and its association with clinicopathological factors in large cohorts of breast cancer patients. A total of 623 consecutive breast cancer patients who underwent surgery between 1993 and 1998 and 612 TNBC patients who underwent surgery between 2004 and 2010 at Asan Medical Center were included. Immunohistochemical staining for NY-ESO-1 was performed using tissue microarrays. NY-ESO-1 was expressed in 2.6% of consecutive breast cancers, all of which were TNBC (p ESO-1 expression was identified in 9.7% of the TNBC cohort and was significantly correlated with a higher level of tumor-infiltrating lymphocytes (TIL; p = 0.026). In survival analyses, a lower level of TIL (all, p ESO-1 expression (p = 0.024) were significantly associated with poor disease-free survival. Additionally, positive NY-ESO-1 expression was an independent favorable prognostic factor in TNBC patients (p = 0.046). NY-ESO-1 is specifically expressed in TNBC, and NY-ESO-1 expression is an independent good prognostic factor in TNBC. Evaluation of NY-ESO-1 expression in TNBC might be useful for selecting patients who may benefit from vaccination therapy and also has a prognostic significance in TNBC. © 2015 S. Karger AG, Basel.

  12. Immunoregulation through IL-10 gene expression and the fate of cytotoxic T lymphocyte-mediated tumor immunotherapy

    Directory of Open Access Journals (Sweden)

    Chakraborty Nitya

    2009-01-01

    Full Text Available Gene analysis of tumor associated antigens revealed that tumor antigens are all normal gene product. Inducing tumor reactive cytotoxic T lymphocytes (CT in the patients is same as inducing autoimmunity in the patients. Immunosuppressive cytokine interleukin-10 (IL-10 plays an important role in maintaining homeostasis or tolerance. To break the tumor tolerance, blocking and IL-10 secretion or intervention in the pathways of IL-10 gene activation is indeed important.

  13. Increased Fas and Bcl-2 Expression on Peripheral Blood T and B Lymphocytes from Juvenile-Onset Systemic Lupus Erythematosus, but not from Juvenile Rheumatoid Arthritis and Juvenile Dermatomyositis

    Directory of Open Access Journals (Sweden)

    Bernadete L. Liphaus

    2006-01-01

    Full Text Available Defective regulation of apoptosis may play a role in the development of autoimmune diseases. Fas and Bcl-2 proteins are involved in the control of apoptosis. The aims of this study were to determine the expression of Fas antigen and Bcl-2 protein on peripheral blood T and B lymphocytes from patients with juvenile-onset systemic lupus erythematosus (JSLE, juvenile rheumatoid arthritis (JRA and juvenile dermatomyositis (JDM. Thirty-eight patients with JSLE, 19 patients with JRA, 10 patients with JDM and 25 healthy controls entered the study. Freshly isolated peripheral blood mononuclear cells (PBMC were stained for lymphocyte markers CD3, CD4, CD8, CD19 and for Fas and Bcl-2 molecules. Expressions were measured by three-color flow cytometry. Statistical analysis was performed using Kruskal–Wallis test. Percentages of freshly isolated T lymphocytes positively stained for Fas protein from JSLE patients were significantly increased compared to healthy controls, patients with JRA and patients with JDM. Percentages of B lymphocytes positive for Fas from JSLE patients were higher than healthy controls and JRA patients. In addition, Fas expression on T cells from patients with JRA was increased compared to JDM patients. Otherwise, Fas expression on T and B cells from JRA and JDM patients were similar to healthy controls. MFI of Bcl-2 positive T lymphocytes from JSLE patients were significantly increased compared to healthy controls and JRA patients. MFI of Bcl-2 protein on B lymphocytes from JSLE patients was similar to healthy controls and patients with JRA and JDM. Bcl-2 expression did not differ between JRA and JDM patients and healthy controls. In conclusion, increased expression of Fas and Bcl-2 proteins observed in circulating T and B lymphocytes from patients with JSLE, but not from patients with JRA and JDM, suggests that abnormalities of apoptosis may be related to the pathogenesis of JSLE and probably are not a result of chronic inflammation.

  14. Increased Fas and Bcl-2 expression on peripheral blood T and B lymphocytes from juvenile-onset systemic lupus erythematosus, but not from juvenile rheumatoid arthritis and juvenile dermatomyositis.

    Science.gov (United States)

    Liphaus, Bernadete L; Kiss, Maria H B; Carrasco, Solange; Goldenstein-Schainberg, Claudia

    2006-01-01

    Defective regulation of apoptosis may play a role in the development of autoimmune diseases. Fas and Bcl-2 proteins are involved in the control of apoptosis. The aims of this study were to determine the expression of Fas antigen and Bcl-2 protein on peripheral blood T and B lymphocytes from patients with juvenile-onset systemic lupus erythematosus (JSLE), juvenile rheumatoid arthritis (JRA) and juvenile dermatomyositis (JDM). Thirty-eight patients with JSLE, 19 patients with JRA, 10 patients with JDM and 25 healthy controls entered the study. Freshly isolated peripheral blood mononuclear cells (PBMC) were stained for lymphocyte markers CD3, CD4, CD8, CD19 and for Fas and Bcl-2 molecules. Expressions were measured by three-color flow cytometry. Statistical analysis was performed using Kruskal-Wallis test. Percentages of freshly isolated T lymphocytes positively stained for Fas protein from JSLE patients were significantly increased compared to healthy controls, patients with JRA and patients with JDM. Percentages of B lymphocytes positive for Fas from JSLE patients were higher than healthy controls and JRA patients. In addition, Fas expression on T cells from patients with JRA was increased compared to JDM patients. Otherwise, Fas expression on T and B cells from JRA and JDM patients were similar to healthy controls. MFI of Bcl-2 positive T lymphocytes from JSLE patients were significantly increased compared to healthy controls and JRA patients. MFI of Bcl-2 protein on B lymphocytes from JSLE patients was similar to healthy controls and patients with JRA and JDM. Bcl-2 expression did not differ between JRA and JDM patients and healthy controls. In conclusion, increased expression of Fas and Bcl-2 proteins observed in circulating T and B lymphocytes from patients with JSLE, but not from patients with JRA and JDM, suggests that abnormalities of apoptosis may be related to the pathogenesis of JSLE and probably are not a result of chronic inflammation.

  15. Altered cell cycle-related gene expression in brain and lymphocytes from a transgenic mouse model of Alzheimer's disease [amyloid precursor protein/presenilin 1 (PS1)].

    Science.gov (United States)

    Esteras, Noemí; Bartolomé, Fernando; Alquézar, Carolina; Antequera, Desireé; Muñoz, Úrsula; Carro, Eva; Martín-Requero, Ángeles

    2012-09-01

    Cumulative evidence indicates that aberrant re-expression of many cell cycle-related proteins and inappropriate neuronal cell cycle control are critical events in Alzheimer's disease (AD) pathogenesis. Evidence of cell cycle activation in post-mitotic neurons has also been observed in murine models of AD, despite the fact that most of these mice do not show massive loss of neuronal bodies. Dysfunction of the cell cycle appears to affect cells other than neurons, as peripheral cells, such as lymphocytes and fibroblasts from patients with AD, show an altered response to mitogenic stimulation. We sought to determine whether cell cycle disturbances are present simultaneously in both brain and peripheral cells from the amyloid precursor protein (APP)/presenilin 1 (PS1) mouse model of AD, in order to validate the use of peripheral cells from patients not only to study cell cycle abnormalities as a pathogenic feature of AD, but also as a means to test novel therapeutic approaches. By using cell cycle pathway-specific RT(2)Profiler™ PCR Arrays, we detected changes in a number of cell cycle-related genes in brain as well as in lymphocytes from APP/PS1 mice. Moreover, we found enhanced 5'-bromo-2'-deoxyuridine incorporation into DNA in lymphocytes from APP/PS1 mice, and increased expression of the cell proliferation marker proliferating cell nuclear antigen (PCNA), and the cyclin-dependent kinase (CDK) inhibitor Cdkn2a, as detected by immunohistochemistry in cortical neurons of the APP/PS1 mice. Taken together, the cell cycle-related changes in brain and blood cells reported here support the mitosis failure hypothesis in AD and validate the use of peripheral cells as surrogate tissue to study the molecular basis of AD pathogenesis.

  16. Expression of IL-2R on Peripheral Blood Lymphocytes of Patients with Colorectal Cancer and Its Clinical Significance

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective: To study expression of membrane receptors ofinterleukin-2 (CD25) on the peripheral blood lymphocytes (PBL) of patients with colorectal cancer and its clinical significance. Methods: CD25 percentages (CD25%) in PBL of 105 colorectal cancer patients before operation and 100 normal individuals were examined by flow cytometer, and the results were clinically and pathologically analyzed. Results: The mean of CD25% in PBL of the normal individuals was 17.24± 5.33, it was significantly lower (p<0.01) than that of the colon cancer patients (21.29± 7.95) or rectal cancer patients (21.62± 6.11). In contrast to the normal individuals, the means of CD25% in PBL in ulcer type (20.53± 6.50) or protruded type (21.56± 6.16) colorectal cancer patients were notably elevated (P<0.01). The significant difference (P<0.01) of means of CD25% in PBL was observed between the normal individuals and patients with less than 4 cm mass (22.10± 5.43) or 4cm- 8cm mass (20.90± 6.96). The significant difference (P<0.05) of means of CD25% in PBL was also observed between the normal individuals and patients with greater than 8 cm mass (21.56± 5.41). The mean of CD25% in PBL in patients with well differentiation colorectal cancer was 22.20± 5.50, it was significantly higher than that in normal individuals (P<0.05). The means of CD25% in PBL in patients with middle or poor differentiation colorectal cancer were 21.30± 6.89 and 22.15± 5.71 respectively, they were obviously higher than that in normal individuals (P<0.01). The significant difference (P<0.01) of means of CD25% in PBL was present between the colorectal cancer patients without metastatic lymph nodes (22.06± 6.90) and normal individuals. The significant difference (P<0.05) of means of CD25% in PBL was present between the colorectal cancer patients with metastatic lymph nodes (20.73± 6.40) and normal individuals. The means of CD25% in PBL in colorectal cancer patients in various clinic stages were significantly higher

  17. Chemokines, lymphocytes, and HIV

    Directory of Open Access Journals (Sweden)

    Farber J.M.

    1998-01-01

    Full Text Available Chemokines are members of a family of more than 30 human cytokines whose best-described activities are as chemotactic factors for leukocytes and that are presumed to be important in leukocyte recruitment and trafficking. While many chemokines can act on lymphocytes, the roles of chemokines and their receptors in lymphocyte biology are poorly understood. The recent discoveries that chemokines can suppress infection by HIV-1 and that chemokine receptors serve, along with CD4, as obligate co-receptors for HIV-1 entry have lent urgency to studies on the relationships between chemokines and lymphocytes. My laboratory has characterized Mig and Crg-2/IP-10, chemokines that are induced by IFN-g and that specifically target lymphocytes, particularly activated T cells. We have demonstrated that the genes for these chemokines are widely expressed during experimental infections in mice with protozoan and viral pathogens, but that the patterns of mig and crg-2 expression differed, suggesting non-redundant roles in vivo. Our related studies to identify new chemokine receptors from activated lymphocytes resulted in the cloning of STRL22 and STRL33. We and others have shown that STRL22 is a receptor for the CC chemokine MIP-3a, and STRL22 has been re-named CCR6. Although STRL33 remains an orphan receptor, we have shown that it can function as a co-receptor for HIV-1 envelope glycoproteins, and that it is active with a broader range of HIV-1 envelope glycoproteins than the major co-receptors described to date. The ability of STRL33 to function with a wide variety of envelope glycoproteins may become particularly important if therapies are instituted to block other specific co-receptors. We presume that investigations into the roles of chemokines and their receptors in lymphocyte biology will provide information important for understanding the pathogenesis of AIDS and for manipulating immune and inflammatory responses for clinical benefit

  18. Persistent expression and function of P-glycoprotein on peripheral blood lymphocytes identifies corticosteroid resistance in patients with systemic lupus erythematosus.

    Science.gov (United States)

    Kansal, Amit; Tripathi, Deepak; Rai, Mohit K; Agarwal, Vikas

    2016-02-01

    Corticosteroids (CS) are the mainstay of treatment in systemic lupus erythematosus (SLE) patients. However, some patients have poor response to CS treatment. Among the multiple mechanisms of CS resistance, overexpression of P-glycoprotein (P-gp) on peripheral blood lymphocytes (PBL) may be one of them as this result in efflux of CS from lymphocytes. Thus, we evaluated the role of P-gp protein on PBLs in patients with SLE in its response to CS therapy. SLE patients (n = 42) (fulfilling ACR revised criteria) who were naïve to CS and immunosuppressive drugs were enrolled. Disease activity was assessed using SLE disease activity index (SLEDAI) and expression, and function of P-gp was evaluated by flow cytometry at baseline and after 3 months of therapy with CS. At 3 months, patients with SLEDAI >4 and SLEDAI ≤4 were grouped as nonresponders and responders, respectively. P-gp expression was significantly increased on PBLs of SLE patients as compared to healthy controls (p < 0.001). P-gp expression and function correlated with SLEDAI (r = 0.49, p = 0.005; and r = 0.49, p = 0.001, respectively). P-gp expression and function were not different in responders and nonresponders at baseline. However, at 3 months of CS therapy, P-gp expression and function decreased in responders (p < 0.001 and p < 0.005, respectively), whereas in nonresponders, it remained unchanged. Persistent overexpression and activity of P-gp are associated with poor response to CS in CS naïve patients of SLE.

  19. Differential expression of adhesion molecules and chemokines between nasal and small intestinal mucosae: implications for T- and sIgA+ B-lymphocyte recruitment.

    Science.gov (United States)

    Bourges, Dorothée; Chevaleyre, Claire; Wang, CaiHong; Berri, Mustapha; Zhang, XiaoMei; Nicaise, Laetitia; Meurens, François; Salmon, Henri

    2007-12-01

    Nasal and small intestinal mucosae are the first sites of contact with infectious agents and the sites of T-cell-mediated and secreted immunoglobulin A (IgA)-mediated defences against pathogens. We investigated the factors controlling the infiltration of CD3(+) T lymphocytes and surface IgA(+) (sIgA(+)) B lymphocytes into swine epithelium and lamina propria (LP) within and between these two mucosal effector sites. Vascular addressins, vascular cell adhesion molecule 1 and mucosal addressin cell adhesion molecule-1 were reciprocally expressed in both mucosae. Strong expression of alpha(4)beta(1) relative to alpha(4)beta(7) was characteristic of CD3(+) T cells in nasal mucosa LP and epithelium and of sIgA(+) cells in nasal mucosa epithelium. The same profile was observed on corresponding blood cells. Conversely, higher levels of integrins beta(7) and alpha(4)beta(7) than alpha(4)beta(1) were characteristic of CD3(+) T cells and sIgA(+) cells in the small intestine. However, about 40% of the LP-activated sIgA(+) cells displayed sIgA(high), integrin alpha(4) and integrin alpha(4) expression. Whereas CCL19, CXCL12, CCL21 and CCL28 messenger RNAs were similarly expressed in both mucosae, CCL25 messenger RNA was only expressed in the small intestine. Thus, the nasal and small intestine mucosae represent separate compartments for infiltration by CD3(+) T cells and sIgA(+) effector cells, with the exception of a population of small intestine activated sIgA(+) cells, which may gain access to both mucosae.

  20. Antigen-specific cytotoxic T lymphocytes target airway CD103+ and CD11b+ dendritic cells to suppress allergic inflammation.

    Science.gov (United States)

    Daniels, N J; Hyde, E; Ghosh, S; Seo, K; Price, K M; Hoshino, K; Kaisho, T; Okada, T; Ronchese, F

    2016-01-01

    Allergic airway inflammation is driven by the recognition of inhaled allergen by T helper type 2 (Th2) cells in the airway and lung. Allergen-specific cytotoxic T lymphocytes (CTLs) can strongly reduce airway inflammation, however, the mechanism of their inhibitory activity is not fully defined. We used mouse models to show that allergen-specific CTLs reduced early cytokine production by Th2 cells in lung, and their subsequent accumulation and production of interleukin (IL)-4 and IL-13. In addition, treatment with specific CTLs also increased the proportion of caspase(+) dendritic cells (DCs) in mediastinal lymph node (MLN), and decreased the numbers of CD103(+) and CD11b(+) DCs in the lung. This decrease required expression of the cytotoxic mediator perforin in CTLs and of the appropriate MHC-antigen ligand on DCs, suggesting that direct CTL-DC contact was necessary. Lastly, lung imaging experiments revealed that in airway-challenged mice XCR1-GFP(+) DCs, corresponding to the CD103(+) DC subset, and XCR1-GFP(-) CD11c(+) cells, which include CD11b(+) DCs and alveolar macrophages, both clustered in the areas surrounding the small airways and were closely associated with allergen-specific CTLs. Thus, allergen-specific CTLs reduce allergic airway inflammation by depleting CD103(+) and CD11b(+) DC populations in the lung, and may constitute a mechanism through which allergic immune responses are regulated.

  1. Insulin growth factor 1 receptor expression is associated with NOTCH1 mutation, trisomy 12 and aggressive clinical course in chronic lymphocytic leukaemia.

    Directory of Open Access Journals (Sweden)

    Francesco Maura

    Full Text Available IGF1R is emerging as an important gene in the pathogenesis of many solid and haematological cancers and its over-expression has been reported as frequently associated with aggressive disease and chemotherapy resistance. In this study we performed an investigation of the role of IGF1R expression in a large and representative prospective series of 217 chronic lymphocytic leukaemia (CLL patients enrolled in the multicentre O-CLL1 protocol (clinicaltrial.gov #NCT00917540. High IGF1R gene expression was significantly associated with IGHV unmutated (IGHV-UM status (p<0.0001, high CD38 expression (p<0.0001, trisomy 12 (p<0.0001, and del(11(q23 (p=0.014. Interestingly, higher IGF1R expression (p=0.002 characterized patients with NOTCH1 mutation (c.7541_7542delCT, identified in 15.5% of cases of our series by next generation sequencing and ARMS-PCR. Furthermore, IGF1R expression has been proven as an independent prognostic factor associated with time to first treatment in our CLL prospective cohort. These data suggest that IGF1R may play an important role in CLL biology, in particular in aggressive CLL clones characterized by IGHV-UM, trisomy 12 and NOTCH1 mutation.

  2. Insulin Growth Factor 1 Receptor Expression Is Associated with NOTCH1 Mutation, Trisomy 12 and Aggressive Clinical Course in Chronic Lymphocytic Leukaemia

    Science.gov (United States)

    Maura, Francesco; Mosca, Laura; Fabris, Sonia; Cutrona, Giovanna; Matis, Serena; Lionetti, Marta; Agnelli, Luca; Barbieri, Marzia; D’Anca, Marianna; Manzoni, Martina; Colombo, Monica; Massucco, Carlotta; Reverberi, Daniele; Gentile, Massimo; Recchia, Anna Grazia; Bossio, Sabrina; Ilariucci, Fiorella; Musolino, Caterina; Di Raimondo, Francesco; Cortelezzi, Agostino; Morabito, Fortunato; Ferrarini, Manlio; Neri, Antonino

    2015-01-01

    IGF1R is emerging as an important gene in the pathogenesis of many solid and haematological cancers and its over-expression has been reported as frequently associated with aggressive disease and chemotherapy resistance. In this study we performed an investigation of the role of IGF1R expression in a large and representative prospective series of 217 chronic lymphocytic leukaemia (CLL) patients enrolled in the multicentre O-CLL1 protocol (clinicaltrial.gov #NCT00917540). High IGF1R gene expression was significantly associated with IGHV unmutated (IGHV-UM) status (p<0.0001), high CD38 expression (p<0.0001), trisomy 12 (p<0.0001), and del(11)(q23) (p=0.014). Interestingly, higher IGF1R expression (p=0.002) characterized patients with NOTCH1 mutation (c.7541_7542delCT), identified in 15.5% of cases of our series by next generation sequencing and ARMS-PCR. Furthermore, IGF1R expression has been proven as an independent prognostic factor associated with time to first treatment in our CLL prospective cohort. These data suggest that IGF1R may play an important role in CLL biology, in particular in aggressive CLL clones characterized by IGHV-UM, trisomy 12 and NOTCH1 mutation. PMID:25786252

  3. Soluble ions more than particulate cobalt-alloy implant debris induce monocyte costimulatory molecule expression and release of proinflammatory cytokines critical to metal-induced lymphocyte reactivity.

    Science.gov (United States)

    Caicedo, Marco S; Pennekamp, Peter H; McAllister, Kyron; Jacobs, Joshua J; Hallab, Nadim J

    2010-06-15

    Aseptic osteolysis has been associated with excessive immune reactivity to particulate implant debris; however, innate and adaptive immune mechanisms that underlie implant debris reactivity remain incompletely understood. Although particulate debris has been implicated as the major type of implant debris mediating macrophage-induced osteolysis, the degree to which metal ions affect a proinflammatory response (if at all) remains unknown. We hypothesized that both soluble and particulate metal implant debris will induce proinflammatory responses in human monocytes resulting in cytokine production and elevated expression of T cell costimulatory molecules, facilitating adaptive immune responses. We tested this hypothesis by characterizing the response of a human monocyte cell line (THP-1), isolated primary human monocytes and PBMCs challenged with Co-Cr-Mo alloy particles and soluble cobalt, chromium, molybdenum, and nickel ions. Our results indicate that soluble cobalt, nickel, and molybdenum can induce monocyte up-regulation of T cell costimulatory molecules (CD80, CD86, ICAM-1) in human monocytes/macrophages. Furthermore, cobalt, molybdenum ions, and Co-Cr-Mo alloy particles similarly induce elevated secretion of IL-1beta, TNFalpha, and IL-6. Antibody blockade of CD80 and CD86, crucial secondary molecules for adaptive responses, abrogated lymphocyte reactivity to metal challenge in metal reactive subjects. Also the addition of IL-1 receptor antagonist (IL-1ra), (which indirectly blocks pro-IL-1beta and thus IL-1beta release), significantly reduced lymphocyte reactivity in metal-reactive subjects. Thus, both soluble and particulate metal implant debris induce monocyte/macrophage proinflammatory responses that are metal and individual specific. This suggests metal-induced up-regulation of costimulatory molecules and proinflammatory cytokine production is necessary to induce lymphocyte activation/proliferation to metal implant debris.

  4. Endoplasmic reticulum membrane-sorting protein of lymphocytes (BAP31) is highly expressed in neurons and discrete endocrine cells.

    Science.gov (United States)

    Manley, H A; Lennon, V A

    2001-10-01

    BAP31 is a transmembrane protein that associates with nascent membrane proteins in transit between endoplasmic reticulum (ER) and cis-Golgi. Its C-terminal dilysine (KKEE) motif, mediating return to the ER, is consistent with a role in early sorting of membrane proteins. An initiator caspase-binding site in the C-terminal domain of BAP31 is implicated in cytoplasmic membrane fragmentation events of apoptosis. Although BAP31 RNA is ubiquitous, the protein's anatomic localization has not been determined. To gain further insight into its possible functions, we localized BAP31 in primate tissues using monoclonal antibodies. Immunoreactivity was prominent in T- and B-lymphocytes in blood and in thymus, in cerebellar Purkinje neuron bodies and dendrites, in gonadotrophs of the anterior pituitary, ovarian thecal and follicular cells, active but not quiescent thyroid epithelium, adrenal cortex more than medulla, and proximal more than distal renal tubules. Blood vessels and skeletal muscle were nonreactive. The anatomic distribution of BAP31 and the nature of proteins identified thus far as its cargo exiting the ER, suggest an interaction with proteins assembling in macromolecular complexes en route to selected sites of exocytotic and signaling activities. Apoptotic associations in mature tissues could be physiological (lymphocytes, endocrine cells) or pathological (Purkinje neurons, renal tubules).

  5. CD69 expression potentially predicts response to bendamustine and its modulation by ibrutinib or idelalisib enhances cytotoxic effect in chronic lymphocytic leukemia

    Science.gov (United States)

    Roldán, Jocabed; Jiménez, Laura; Cabezas, Sandra; Clot, Guillem; Pinyol, Magda; Xargay-Torrent, Sílvia; Rosich, Laia; Arimany-Nardí, Cristina; Aymerich, Marta; Villamor, Neus; López-Guillermo, Armando; Pérez-Galán, Patricia; Roué, Gaël; Pastor-Anglada, Marçal; Campo, Elías; López-Guerra, Mónica; Colomer, Dolors

    2016-01-01

    Clinical responses to bendamustine in chronic lymphocytic leukemia (CLL) are highly heterogeneous and no specific markers to predict sensitivity to this drug have been reported. In order to identify biomarkers of response, we analyzed the in vitro activity of bendamustine and the gene expression profile in primary CLL cells. We observed that mRNA expression of CD69 (CD69) and ITGAM (CD11b) constitute the most powerful predictor of response to bendamustine. When we interrogated the predictive value of the corresponding cell surface proteins, the expression of the activation marker CD69 was the most reliable predictor of sensitivity to bendamustine. Importantly, a multivariate analysis revealed that the predictive value of CD69 expression was independent from other clinico-biological CLL features. We also showed that when CLL cells were co-cultured with distinct subtypes of stromal cells, an upregulation of CD69 was accompanied by a reduced sensitivity to bendamustine. In agreement with this, tumor cells derived from lymphoid tumor niches harbored higher CD69 expression and were less sensitive to bendamustine than their peripheral blood counterparts. Furthermore, pretreatment of CD69 high CLL cases with the B-cell receptor (BCR) pathway inhibitors ibrutinib and idelalisib decreased CD69 levels and enhanced bendamustine cytotoxic effect. Collectively, our findings indicate that CD69 could be a predictor of bendamustine response in CLL patients and the combination of clinically-tested BCR signaling inhibitors with bendamustine may represent a promising strategy for bendamustine low responsive CLL cases. PMID:26701728

  6. Effect of acute and regular exercise on growth hormone secretagogue receptor-1a expression in human lymphocytes, T cell subpopulation and monocytes.

    Science.gov (United States)

    Bishop, Nicolette C; Hayashida, Harumi; Clark, Megan; Coombs, Charlotte; Miller, Sean; Stensel, David J

    2014-07-01

    The orexigenic peptide hormone ghrelin exerts potent inhibitory effects on pro-inflammatory cytokine release via the growth hormone secretagogue receptor-1a (GHS-R1a) on T cells and monocytes. As such, ghrelin is a promising therapeutic agent for the treatment of inflammatory conditions, but these effects depend on the availability of GHS-R1a. The aim of this study was to determine the effect of acute exercise on GHS-R1a expression on circulating CD14+ monocytes, total lymphocytes and CD3+ T cells. Nine male club-standard cyclists cycled for 1h at 75% V̇O2peak (EX) or rested (REST) in a randomised cross-over design. Compared with the equivalent times in REST, the concentration of circulating GHS-R1a+ lymphocytes and monocytes was higher in EX at immediately and 1 and 2h post-exercise (all pexercise only (258 (203)cellsμl(-1) vs. 62 (42)cellsμl(-1), pexercise. Given that the anti-inflammatory effects of ghrelin depend on the availability of GHS-R1a, the preferential recruitment of subpopulations with high anti-inflammatory potential found here add a novel aspect to the potential mechanisms by which exercise acts to reduce pro-inflammatory cytokine levels.

  7. T-cell population of primary and secondary cutaneous B-cell lymphomas does not express the cutaneous lymphocyte-associated antigen (CLA).

    Science.gov (United States)

    Marti, R M; Hausmann, G; Estrach, T; Cid, M C; Palou, J; Herrero, C; Mascaro, J M

    1997-05-01

    Primary cutaneous B-cell lymphomas (CBCL) are a group of malignant lymphomas with apparently distinct clinicopathological and immunophenotypical features. As in other B-cell lymphomas, the accompanying benign cell population in CBCL includes a variable number of T lymphocytes whose role is not well understood. In the present study we characterized the immunophenotype of these T cells and compared it with that of the reactive T-cell population in specific skin involvement by noncutaneous B-cell malignancies. Our results indicated that most T cells in both primary and secondary B-cell lymphomas were CLA+ memory/effector helper T cells which differed from the currently known CLA+ memory/effector helper T lymphocytes of the skin-associated lymphoid tissue (SALT) system. However, the endothelial CLA ligand, E-selectin, was expressed on dermal vessels. These results suggest that a B cell environment and/or a lack of epidermal involvement promote(s) the recruitment into the skin of a different, apparently less specific, subset of memory helper T cells from those seen in T-cell-mediated dermatoses.

  8. Type 1 Diabetes Prone NOD Mice Have Diminished Cxcr1 mRNA Expression in Polymorphonuclear Neutrophils and CD4+ T Lymphocytes.

    Directory of Open Access Journals (Sweden)

    Karine Haurogné

    Full Text Available In humans, CXCR1 and CXCR2 are two homologous proteins that bind ELR+ chemokines. Both receptors play fundamental roles in neutrophil functions such as migration and reactive oxygen species production. Mouse Cxcr1 and Cxcr2 genes are located in an insulin-dependent diabetes genetic susceptibility locus. The non obese diabetic (NOD mouse is a spontaneous well-described animal model for insulin-dependent type 1 diabetes. In this disease, insulin deficiency results from the destruction of insulin-producing beta cells by autoreactive T lymphocytes. This slow-progressing disease is dependent on both environmental and genetic factors. Here, we report descriptive data about the Cxcr1 gene in NOD mice. We demonstrate decreased expression of mRNA for Cxcr1 in neutrophils and CD4+ lymphocytes isolated from NOD mice compared to other strains, related to reduced NOD Cxcr1 gene promoter activity. Looking for Cxcr1 protein, we next analyze the membrane proteome of murine neutrophils by mass spectrometry. Although Cxcr2 protein is clearly found in murine neutrophils, we did not find evidence of Cxcr1 peptides using this method. Nevertheless, in view of recently-published experimental data obtained in NOD mice, we argue for possible Cxcr1 involvement in type 1 diabetes pathogenesis.

  9. CD4/CD8 double negative mycosis fungoides with PD-1 (CD279) expression--a disease of follicular helper T-cells?

    Science.gov (United States)

    Kempf, Werner; Kazakov, Dmitry V; Cipolat, Claudio; Kutzner, Heinz; Roncador, Giovanna; Tomasini, Dario

    2012-10-01

    CD4/CD8 double negative mycosis fungoides (MF) is a rare phenotypic variant of this epidermotropic cutaneous T-cell lymphoma. Clinically, this MF form manifests with unusual appearances such as annular lesions confined to one body region as in our patient in whom the lesions were found on the left lower leg. The cellular origin of CD4/CD8 double negative MF is unknown. In our case, the intraepidermal CD4/CD8 double negative clonal T-lymphocytes (CD2+, CD4-, CD8-, CD30-, beta-F1+) expressed programmed death-1 but were negative for CXCL-13 and cytotoxic molecules (TIA-1, granzyme B, perforin). Our observation may give an insight into the histogenesis of this unique MF variant and may also be of therapeutic significance because programmed death-1 may serve as a target for therapeutic intervention.

  10. LYMPHOCYTE APOPTOSIS IN PSORIASIS

    Directory of Open Access Journals (Sweden)

    О. M. Kapuler

    2006-01-01

    Full Text Available Abstract. Forty-two patients with progressive vulgar psoriasis (PASI = 19.7 ± 1.5 and 40 healthy volunteers were under investigation. Psoriatic patients were characterized by increased number of CD4+ CD95+ peripheral blood T lymphocytes, which correlates with clinical psoriatic score, and by increased levels of soluble Fas (sFas in serum, as compared to controls (resp., 1868.1 ± 186.8 pg/ml vs. 1281.4 ± 142.5 pg/ml, PLSD = 0.019. The levels of spontaneous lymphocyte apoptosis and anti-Fas (Mab-induced apoptosis in psoriatic patients did not differ from the controls. However, apoptosis induced by “oxidative stress” (50 M Н202, 4 hrs was depressed in the patients. Moreover, a simultaneous assessment of cell cycle structure (metachromatic staining with Acridine Orange, apoptosis and Fas receptor expression (AnnV-FITC/antiFas mAbs-PE staining following a short-term mitogenic stimulation (PHA-P, 5 µg/ml, 24 hrs were performed. We found no marked differences in mitogenic reactivity, activation-induced apoptosis, and activation-induced Fas receptor expression when studying lymphocytes from healthy donors and psoriatic patients. However, PHA-activated lymphocytes from psoriatic patients displayed a significantly decreased ratio of AnnV+CD95+ to the total AnnV+ subpopulation, thus suggesting a decreased role of Fas-dependent mechanisms of apoptosis during the cell activation. The data obtained confirm a view, that an abnormal lymphocyte “apoptotic reactivity”, which plays a crucial role in the mechanisms of autoimmunity, may also of importance in the pathogenesis of psoriasis.

  11. A different representation of natural T cells and natural killer cells between tumor-infiltrating and periphery lymphocytes in human hepatocellular carcinoma.

    Science.gov (United States)

    Li, Xiao-Feng; Dai, Dong; Song, Xiu-Yu; Liu, Jian-Jing; Zhu, Lei; Zhu, Xiang; Ma, Wenchao; Xu, Wengui

    2017-05-01

    Natural T cells [cluster of differentiation (CD) 3(+)CD56(+)] and natural killer (NK) cells (CD3(-)CD56(+)) are particularly abundant in the human liver and serve an important role in immune responses in the liver. The aim of the present study was to extensively determine the phenotypic and functional characteristics of natural T and NK cells in human hepatocellular carcinoma (HCC). Tumorous and non-tumorous tissue infiltrating lymphocytes (TILs and NILs, respectively) and peripheral blood mononuclear cells (PBMCs) from patients with hepatocellular carcinoma (HCC) were obtained to determine the frequency and phenotype of natural T/NK cells by a multicolor fluorescence activated cell sorting analysis. The abundance of natural T cells and NK cells was decreased in TILs vs. NILs (natural T cells, 6.315±1.002 vs. 17.16±1.804; NK cells, 6.324±1.559 vs. 14.52±2.336, respectively). However such results were not observed in PBMCs from HCC patients vs. that of healthy donors. Notably, a substantial fraction of the natural T cells (21.96±5.283) in TILs acquired forkhead box P3 (FOXP3) expression, and the FOXP3(+) natural T cells lost the expression of interferon-γ and perforin. Conversely, being similar to the conventional FOXP3(+) regulatory T cells, the FOXP3(+) natural T cells assumed a specific phenotype that was characteristic of CD25(+), CD45RO(+) and cytotoxic T-lymphocyte-associated protein 4(+). Consistent with the phenotypic conversion, the present functional results indicate that FOXP3 expression in natural T cells contributes to the acquisition of a potent immunosuppressive capability. In conclusion, the present study describes a different representation of natural T cells and NK cells in local tumor tissues and in the periphery blood of patients with HCC, and identified a new type of FOXP3-expressing natural T cell spontaneously arising in the TILs of HCC.

  12. The Fas counterattack in vivo: apoptotic depletion of tumor-infiltrating lymphocytes associated with Fas ligand expression by human esophageal carcinoma.

    LENUS (Irish Health Repository)

    Bennett, M W

    2012-02-03

    Various cancer cell lines express Fas ligand (FasL) and can kill lymphoid cells by Fas-mediated apoptosis in vitro. FasL expression has been demonstrated in several human malignancies in vivo. We sought to determine whether human esophageal carcinomas express FasL, and whether FasL expression is associated with increased apoptosis of tumor-infiltrating lymphocytes (TIL) in vivo, thereby contributing to the immune privilege of the tumor. Using in situ hybridization and immunohistochemistry, respectively, FasL mRNA and protein were colocalized to neoplastic esophageal epithelial cells in all esophageal carcinomas (squamous, n = 6; adenocarcinoma, n = 2). The Extent of FasL expression was variable, with both FasL-positive and FasL-negative neoplastic regions occurring within tumors. TIL were detected by immunohistochemical staining for the leukocyte common Ag, CD45. FasL expression was associated with a mean fourfold depletion of TIL when compared with FasL-negative areas within the same tumors (range 1.6- to 12-fold, n = 6,p < 0.05). Cell death of TIL was detected by dual staining of CD45 (immunohistochemistry) and DNA strand breaks (TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling). There was a mean twofold increase in detectable cell death among TIL in FasL-positive areas compared with FasL-negative areas (range 1.6- to 2.4-fold, n = 6, p < 0.05). In conclusion, we demonstrate a statistically significant, quantitative reduction of TIL concomitant with significantly increased TIL apoptosis within FasL-expressing areas of esophageal tumors. Our findings suggest Fas-mediated apoptotic depletion of TIL in response to FasL expression by esophageal cancers, and provide the first direct, quantitative evidence to support the Fas counterattack as a mechanism of immune privilege in vivo in human cancer.

  13. Over-Expression of Dopamine D2 Receptor and Inwardly Rectifying Potassium Channel Genes in Drug-Naive Schizophrenic Peripheral Blood Lymphocytes as Potential Diagnostic Markers

    Directory of Open Access Journals (Sweden)

    Ágnes Zvara

    2005-01-01

    Full Text Available Schizophrenia is one of the most common neuropsychiatric disorders affecting nearly 1% of the human population. Current diagnosis of schizophrenia is based on complex clinical symptoms. The use of easily detectable peripheral molecular markers could substantially help the diagnosis of psychiatric disorders. Recent studies showed that peripheral blood lymphocytes (PBL express subtypes of D1 and D2 subclasses of dopamine receptors. Recently, dopamine receptor D3 (DRD3 was found to be over-expressed in schizophrenic PBL and proposed to be a diagnostic and follow-up marker for schizophrenia. In this study we screened PBL of 13 drug-naive/drug-free schizophrenic patients to identify additional markers of schizophrenia. One of the benefits of our study is the use of blood samples of non-medicated, drug-naive patients. This excludes the possibility that changes detected in gene expression levels might be attributed to the medication rather than to the disorder itself. Among others, genes for dopamine receptor D2 (DRD2 and the inwardly rectifying potassium channel (Kir2.3 were found to be over-expressed in microarray analysis. Increased mRNA levels were confirmed by quantitative real-time PCR (QRT-PCR using the SybrGreen method and dual labeled TaqMan probes. The use of both molecular markers allows a more rapid and precise prediction of schizophrenia and might help find the optimal medication for schizophrenic patients.

  14. Over-expression of dopamine D2 receptor and inwardly rectifying potassium channel genes in drug-naive schizophrenic peripheral blood lymphocytes as potential diagnostic markers.

    Science.gov (United States)

    Zvara, Agnes; Szekeres, György; Janka, Zoltán; Kelemen, János Z; Cimmer, Csongor; Sántha, Miklós; Puskás, László G

    2005-01-01

    Schizophrenia is one of the most common neuropsychiatric disorders affecting nearly 1% of the human population. Current diagnosis of schizophrenia is based on complex clinical symptoms. The use of easily detectable peripheral molecular markers could substantially help the diagnosis of psychiatric disorders. Recent studies showed that peripheral blood lymphocytes (PBL) express subtypes of D1 and D2 subclasses of dopamine receptors. Recently, dopamine receptor D3 (DRD3) was found to be over-expressed in schizophrenic PBL and proposed to be a diagnostic and follow-up marker for schizophrenia. In this study we screened PBL of 13 drug-naive/drug-free schizophrenic patients to identify additional markers of schizophrenia. One of the benefits of our study is the use of blood samples of non-medicated, drug-naive patients. This excludes the possibility that changes detected in gene expression levels might be attributed to the medication rather than to the disorder itself. Among others, genes for dopamine receptor D2 (DRD2) and the inwardly rectifying potassium channel (Kir2.3) were found to be over-expressed in microarray analysis. Increased mRNA levels were confirmed by quantitative real-time PCR (QRT-PCR) using the SybrGreen method and dual labeled TaqMan probes. The use of both molecular markers allows a more rapid and precise prediction of schizophrenia and might help find the optimal medication for schizophrenic patients.

  15. A proline/arginine-rich end leucine-rich repeat protein (PRELP variant is uniquely expressed in chronic lymphocytic leukemia cells.

    Directory of Open Access Journals (Sweden)

    Eva Mikaelsson

    Full Text Available Proline/arginine-rich end leucine-rich repeat protein (PRELP belongs to the small leucine-rich proteoglycan (SLRP family, normally expressed in extracellular matrix of collagen-rich tissues. We have previously reported on another SLRP, fibromodulin (FMOD in patients with chronic lymphocytic leukemia (CLL. PRELP is structurally similar to FMOD with adjacent localization on chromosome 1 (1q32.1. As cluster-upregulation of genes may occur in malignancies, the aim of our study was to analyze PRELP expression in CLL. PRELP was expressed (RT-PCR in all CLL patients (30/30, as well as in some patients with mantle cell lymphoma (3/5, but not in healthy donor leukocytes (0/20 or tumor samples from other hematological malignancies (0/35. PRELP was also detected in CLL cell-lines (4/4 but not in cell-lines from other hematological tumors (0/9. PRELP protein was detected in all CLL samples but not in normal leukocytes. Deglycosylation experiments revealed a CLL-unique 38 kDa core protein, with an intact signal peptide. This 38 kDa protein was, in contrast to the normal 55 kDa size, not detected in serum which, in combination with the uncleaved signal peptide, suggests cellular retention. The unique expression of a 38 kDa PRELP in CLL cells may suggest involvement in the pathobiology of CLL and merits further studies.

  16. Identification of Multiple Antigens Recognized by Tumor-Infiltrating Lymphocytes From a Single Patient: Tumor Escape by Antigen Loss and Loss of MHC Expression

    Science.gov (United States)

    Khong, Hung T.; Wang, Qiong J.; Rosenberg, Steven A.

    2008-01-01

    The authors describe a patient who experienced recurrence of metastatic melanoma after an initial dramatic response to immunotherapy using peptides derived from gp100, MART-1, and tyrosinase emulsified in incomplete Freund’s adjuvant, and present data to support the hypothesis that the progression of disease in this patient was due to in vivo immunoselection for immunoresistant tumor variants. The authors previously demonstrated the existence of T-cell clones in this patient’s peripheral blood and tumor-infiltrating lymphocytes (TILs) reactive against multiple antigens, including gp100, the tyrosinase-related protein (TRP)-2, a novel TRP-2 isoform-TRP-2-6b, SOX10, and the melanoma antigen NY-ESO-1. In addition to the multiple HLA-A2 restricted T-cell clones, the authors have now identified additional HLA-B/C-restricted as well as class II (HLA-DP)-restricted anti-melanoma antigen T-cell clones from this patient’s TIL. One recurrent tumor showed loss of expression of multiple tumor antigens but retention of HLA class I expression. The other recurrent lesion showed total loss of HLA class I expression even though the tumor cells still expressed many melanoma antigens. This paper thus provides evidence for both the effectiveness of the immune destruction of cancer as well as problems associated with antigen-loss tumor escape mechanisms. PMID:15076135

  17. 5'-Ectonucleotidase/CD73 expression on lymph-circulating lymphocytes and lymphatic endothelial cells offers new paths to explore barrier function.

    Science.gov (United States)

    Sidibé, Adama; Imhof, Beat A

    2015-02-01

    5'-Nucleotidase/CD73 is a key enzyme in the regulation of purinergic signaling, hydrolyzing extracellular AMP to produce adenosine, which is critical in the blood vascular system and in immunosuppression. CD73 is expressed by both blood endothelial cells and lymphatic endothelial cells. Although the role of CD73 on blood endothelial cells in controlling vascular permeability and leukocyte trafficking has been studied, the role of lymphatic CD73 has thus far remained unknown. In this issue of European Journal of Immunology, Yegutkin et al. [Eur. J. Immunol. 2015. 45: 562-573] compare CD73 activity in the endothelia of lymphatics and blood vessels and investigate the CD73(+) lymphocyte subpopulations possibly involved in immunoregulation. This Commentary will discuss how the authors' work sheds light on the differential use of CD73 by these two cell populations to control endothelial permeability and sprouting.

  18. Glutaminase expression is a poor prognostic factor in node-positive triple-negative breast cancer patients with a high level of tumor-infiltrating lymphocytes.

    Science.gov (United States)

    Kim, Joo Young; Heo, Sun-Hee; Choi, Seul Ki; Song, In Hye; Park, In Ah; Kim, Young-Ae; Park, Hye Seon; Park, Suk Young; Bang, Won Seon; Gong, Gyungyub; Lee, Hee Jin

    2017-04-01

    Glutamine metabolism is emerging as one aspect of dysregulated metabolism of tumors. Triple-negative breast cancer (TNBC) cells are glutamine dependent, whereas luminal-type cells tend to be glutamine independent. Therefore, TNBC patients might benefit from therapies targeting glutamine metabolism. To investigate the clinical significance of glutamine metabolism, we examined expression and prognostic significance of glutaminase in tumor cells and tumor-infiltrating lymphocytes (TILs) in TNBC. We retrieved 658 surgically resected TNBCs and analyzed glutaminase expression in tumor cells and TILs by immunohistochemical staining. Glutaminase expression was observed in 237 cases (36.0%) in tumor cells and 104 cases (15.5%) in TILs. Although glutaminase expression in tumor cells was significantly associated with a low level of TILs (p = 0.018), glutaminase expression in TILs was significantly higher in cases with a high level of TILs (p = 0.031). Glutaminase expression in tumor cells was significantly associated with poor disease-free survival in patients with lymph node metastasis and high levels of TILs (p = 0.020). In addition, it was an independent poor prognostic factor (hazard ratio = 10.643, 95% confidence interval = 1.999-56.668; p = 0.006). Glutaminase expression in tumor cells was observed in a subset of TNBC patients. It was significantly associated with a low level of TILs and poor disease-free survival in TNBCs presenting with lymph node metastasis and high levels of TILs.

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

    Science.gov (United States)

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

    2003-01-01

    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.

  20. Analysis of CD95 and CCR7 expression on circulating CD4(+) lymphocytes revealed disparate immunoregulatory potentials in systemic lupus erythematosus.

    Science.gov (United States)

    Aldahlawi, Alia M; Elshal, Mohamed F; Damiaiti, Laila A; Damanhori, Laila H; Bahlas, Sami M

    2016-01-01

    Emerging data have implicated a critical role for CD4 in the pathogenesis of systemic lupus erythematosus (SLE). This study was designed to delineate the contribution of CD4(+) T cells in the pathogenesis of SLE disease. Forty-four patients (3 male: 41 female) and 20 healthy volunteers (4 male: 16 female) were included in the study. CD4(+) lymphocytes analysis was done using three-color flow cytometry with antibodies against human-CD95, a prototype cell death receptor, and the chemokine receptor-7 (CCR7) after gating for lymphocytes based on the forward and side scatter. Serum levels of IL-6, IL-12, IL-17, TNF-α and IL-10 cytokines were assayed using ELISA. Disease activity was assessed using the SLE disease activity index (SLEDAI). Based on the expression of CCR7 and CD95, CD4(+) lymphocytes were subdivided into three particular subsets; CD4(+)CD95(+)CCR7(+) cells, CD4(+)CD95(-)CCR7(+) cells and CD4(+)CD95(+)CCR7(-) cells. Percentage of CD4(+)CD95(+)CCR7(+) cell subset was significantly higher in patients with SLE with active disease (SLEDAI > 6) and inactive (SLEDAI CCR7(-) cell subset was significantly higher in active SLE patients in comparison to patients with inactive disease and controls (P = 0.05, P = 0.005 respectively), and it correlates positively with SLEDAI, IL-6 and IL-17 levels (P = 0.001, 0.05, 0.01 respectively), and negatively with blood WBCs counts (P = 0.001). The third CD4(+)CD95(-)CCR7(+)cell subset was found significantly lower in SLE patients compared with controls, and it was found negatively correlated with IL-10, IL-6, and IL-17. The results show that CD4(+)CD95(+)subset lacking expression of CCR7 is associated with cell mediated inflammatory response as manifested by its correlation with signs of inflammation, inflammatory cytokines and disease activity index. Whereas, CD4(+)CD95(+)CCR7(+) correlate more with antibody immune responses as manifested by association with serum ANA. These data suggest disparate roles of these

  1. Polyclonal, newly derived T cells with low expression of inhibitory molecule PD-1 in tonsils define the phenotype of lymphocytes in children with Periodic Fever, Aphtous Stomatitis, Pharyngitis and Adenitis (PFAPA) syndrome.

    Science.gov (United States)

    Dytrych, Petra; Krol, Petra; Petra, Dytrych; Petra, Krol; Kotrova, Michaela; Kuzilkova, Daniela; Michaela, Kotrova; Daniela, Kuzilkova; Hubacek, Petr; Krol, Ladislav; Petr, Hubacek; Ladislav, Krol; Katra, Rami; Hrusak, Ondrej; Rami, Katra; Ondrej, Hrusak; Kabelka, Zdenek; Dolezalova, Pavla; Zdenek, Kabelka; Pavla, Dolezalova; Kalina, Tomas; Fronkova, Eva; Tomas, Kalina; Eva, Fronkova

    2015-05-01

    PFAPA syndrome is a benign, recurrent inflammatory disease of childhood. Tonsillectomy is one of the therapeutic options with a yet unexplained biological mechanism. We tested whether specific lymphocyte subsets recruited from blood to human tonsils participate in PFAPA pathogenesis. Paired tonsils/peripheral blood (PB) samples were investigated (a) from children with PFAPA that successfully resolved after tonsillectomy (n=10) (b) from children with obstructive sleep apnoea syndrome as controls (n=10). The lymphocyte profiles were analysed using 8-colour flow cytometry, immunoglobulin (IGH) and T-cell receptor (TCR) gene rearrangements via PCR and next generation sequencing; a TREC/KREC analysis was performed using qPCR. The PFAPA tonsils in the asymptomatic phase had a lower percentage of B-lymphocytes than controls; T-lymphocyte counts were significantly higher in PB. The percentages of cytotoxic CD8pos T-lymphocytes were approximately 2-fold higher in PFAPA tonsils; the transitional B cells and naïve stages of both the CD4pos and CD8pos T-lymphocytes with a low expression of PD-1 molecule and high numbers of TREC were also increased. With the exception of elevated plasmablasts, no other differences were significant in PB. The expression levels of CXCL10, CXCL9 and CCL19 genes were significantly higher in PFAPA tonsils. The IGH/TCR pattern showed no clonal/oligoclonal expansion. DNA from the Epstein-Barr virus, Human Herpervirus-6 or adenovirus was detected in 7 of 10 PFAPA tonsils but also in 7 of 9 controls. Our findings suggest that the uninhibited, polyclonal response of newly derived lymphocytes participate in the pathogenesis of PFAPA. Because most of the observed changes were restricted to tonsils and were not present in PB, they partly explain the therapeutic success of tonsillectomy in PFAPA syndrome. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Viral hemorrhagic septicaemia virus (VHSV) up-regulates the cytotoxic activity and the perforin/granzyme pathway in the rainbow trout RTS11 cell line.

    Science.gov (United States)

    Ordás, M C; Cuesta, A; Mercado, L; Bols, N C; Tafalla, C

    2011-08-01

    A survey of immune-relevant genes that might be up-regulated in response to viral hemorrhagic septicaemia virus (VHSV) in the rainbow trout monocyte-macrophage cell line, RTS11, unexpectedly revealed an increased expression of perforin (PRF) and granzyme (GRZ) genes, which represent components of the major cytotoxic pathway. The natural killer-enhancing factor (NKEF), also known to modulate cytotoxic activity, was up-regulated at the gene but strikingly down-regulated at protein level. The expression of these genes was not affected in head kidney leukocytes (HKLs) infected with VHSV, leading us to evaluate the potential cytotoxic activity of RTS11 and HKLs. For the first time, the cytotoxic activity of RTS11 against xenogeneic targets has been demonstrated, although this was modest relative to HKLs. Yet the activity in RTS11 was significantly increased by VHSV, as in HKLs. This cytotoxic activity elicited by viral infection appeared to require viral gene expression because inactivated VHSV failed to increase RTS11 cytotoxic activity. As for other immune functions, RTS11 cells provide a model for further studying cytotoxic activities of fish monocyte-macrophages.

  3. Strong Correlation of Indoleamine 2,3-Dioxygenase 1 Expression with Basal-Like Phenotype and Increased Lymphocytic Infiltration in Triple-Negative Breast Cancer

    Science.gov (United States)

    Kim, Sewha; Park, Sanghui; Cho, Min Sun; Lim, Woosung; Moon, Byung-In; Sung, Sun Hee

    2017-01-01

    Indoleamine 2,3-dioxygenase 1 (IDO1) is an immunosuppressive enzyme involved in tumor immune escape. Blockade of the IDO1 pathway is an emerging modality of cancer immunotherapy. Triple-negative breast cancer (TNBC) lacks established therapeutic targets and may be a good candidate for this novel immunotherapeutic agent. The purpose of this study was to evaluate the clinicopathologic characteristics of the IDO1-expressing TNBC subset. A tissue microarray was constructed from 200 patients with TNBC. Immunohistochemistry (IHC) for IDO1 and TNBC molecular subtype-surrogate markers (AR, GCDFP-15, claudin-3, E-cadherin, CK5/6, and EGFR) was performed using this tissue microarray. Real-time polymerase chain reaction was performed to confirm the IDO1 mRNA expression level in 16 fresh-frozen TNBC samples. Two hundred TNBCs were classified into four subtypes based on surrogate IHC results: 22 luminal androgen receptor type (11.0%), 23 claudin-low type (11.4%), 103 basal-like type (51.5%), and 52 mixed type (26.0%). IDO1 positivity (defined as expression of >10% tumor cells) was observed in 37% of all TNBCs. IDO1 IHC expression was well correlated with mRNA expression. IDO1 positivity was significantly associated with smaller tumor size, dense stromal lymphocytic infiltration, and basal-like phenotype; however, it did not affect the patients' prognosis. IDO1 expression in basal-like TNBCs is considered an immune inhibitory signal that counterbalances active immunity and may reflect the high mutational load of these tumors. Our results suggest which patients with TNBC would be more efficaciously treated with IDO1 blockade. PMID:28123606

  4. Strong Correlation of Indoleamine 2,3-Dioxygenase 1 Expression with Basal-Like Phenotype and Increased Lymphocytic Infiltration in Triple-Negative Breast Cancer.

    Science.gov (United States)

    Kim, Sewha; Park, Sanghui; Cho, Min Sun; Lim, Woosung; Moon, Byung-In; Sung, Sun Hee

    2017-01-01

    Indoleamine 2,3-dioxygenase 1 (IDO1) is an immunosuppressive enzyme involved in tumor immune escape. Blockade of the IDO1 pathway is an emerging modality of cancer immunotherapy. Triple-negative breast cancer (TNBC) lacks established therapeutic targets and may be a good candidate for this novel immunotherapeutic agent. The purpose of this study was to evaluate the clinicopathologic characteristics of the IDO1-expressing TNBC subset. A tissue microarray was constructed from 200 patients with TNBC. Immunohistochemistry (IHC) for IDO1 and TNBC molecular subtype-surrogate markers (AR, GCDFP-15, claudin-3, E-cadherin, CK5/6, and EGFR) was performed using this tissue microarray. Real-time polymerase chain reaction was performed to confirm the IDO1 mRNA expression level in 16 fresh-frozen TNBC samples. Two hundred TNBCs were classified into four subtypes based on surrogate IHC results: 22 luminal androgen receptor type (11.0%), 23 claudin-low type (11.4%), 103 basal-like type (51.5%), and 52 mixed type (26.0%). IDO1 positivity (defined as expression of >10% tumor cells) was observed in 37% of all TNBCs. IDO1 IHC expression was well correlated with mRNA expression. IDO1 positivity was significantly associated with smaller tumor size, dense stromal lymphocytic infiltration, and basal-like phenotype; however, it did not affect the patients' prognosis. IDO1 expression in basal-like TNBCs is considered an immune inhibitory signal that counterbalances active immunity and may reflect the high mutational load of these tumors. Our results suggest which patients with TNBC would be more efficaciously treated with IDO1 blockade.

  5. Ly49E expression on CD8αα-expressing intestinal intraepithelial lymphocytes plays no detectable role in the development and progression of experimentally induced inflammatory bowel diseases.

    Directory of Open Access Journals (Sweden)

    Aline Van Acker

    Full Text Available The Ly49E NK receptor is a unique inhibitory receptor, presenting with a high degree of conservation among mouse strains and expression on both NK cells and intraepithelial-localised T cells. Amongst intraepithelial-localised T cells, the Ly49E receptor is abundantly expressed on CD8αα-expressing innate-like intestinal intraepithelial lymphocytes (iIELs, which contribute to front-line defense at the mucosal barrier. Inflammatory bowel diseases (IBDs, encompassing Crohn's disease and ulcerative colitis, have previously been suggested to have an autoreactive origin and to evolve from a dysbalance between regulatory and effector functions in the intestinal immune system. Here, we made use of Ly49E-deficient mice to characterize the role of Ly49E receptor expression on CD8αα-expressing iIELs in the development and progression of IBD. For this purpose we used the dextran sodium sulphate (DSS- and trinitrobenzenesulfonic-acid (TNBS-induced colitis models, and the TNFΔARE ileitis model. We show that Ly49E is expressed on a high proportion of CD8αα-positive iIELs, with higher expression in the colon as compared to the small intestine. However, Ly49E expression on small intestinal and colonic iIELs does not influence the development or progression of inflammatory bowel diseases.

  6. Calcium-activated potassium channels sustain calcium signaling in T lymphocytes. Selective blockers and manipulated channel expression levels.

    Science.gov (United States)

    Fanger, C M; Rauer, H; Neben, A L; Miller, M J; Rauer, H; Wulff, H; Rosa, J C; Ganellin, C R; Chandy, K G; Cahalan, M D

    2001-04-13

    To maintain Ca(2+) entry during T lymphocyte activation, a balancing efflux of cations is necessary. Using three approaches, we demonstrate that this cation efflux is mediated by Ca(2+)-activated K(+) (K(Ca)) channels, hSKCa2 in the human leukemic T cell line Jurkat and hIKCa1 in mitogen-activated human T cells. First, several recently developed, selective and potent pharmacological inhibitors of K(Ca) channels but not K(V) channels reduce Ca(2+) entry in Jurkat and in mitogen-activated human T cells. Second, dominant-negative suppression of the native K(Ca) channel in Jurkat T cells by overexpression of a truncated fragment of the cloned hSKCa2 channel decreases Ca(2+) influx. Finally, introduction of the hIKCa1 channel into Jurkat T cells maintains rapid Ca(2+) entry despite pharmacological inhibition of the native small conductance K(Ca) channel. Thus, K(Ca) channels play a vital role in T cell Ca(2+) signaling.

  7. Transcriptome and Proteome Expressions Involved in Insulin Resistance in Muscle and Activated T-Lymphocytes of Patients with Type 2 Diabetes

    Institute of Scientific and Technical Information of China (English)

    Frankie; B.; Stentz; Abbas; E.; Kitabchi

    2007-01-01

    We analyzed the genes expressed (transcriptomes) and the proteins translated (pro- teomes) in muscle tissues and activated CD4+ and CD8+ T-lymphocytes (T-cells) of five Type 2 diabetes (T2DM) subjects using Affymetrix microarrays and mass spectrometry, and compared them with matched non-diabetic controls. Gene ex- pressions of insulin receptor (INSR), vitamin D receptor, insulin degrading enzyme, Akt, insulin receptor substrate-1 (IRS-1), IRS-2, glucose transporter 4 (GLUT4), and enzymes of the glycolytic pathway were decreased at least 50% in T2DM than in controls. However, there was greater than two-fold gene upregulation of plasma cell glycoprotein-1, tumor necrosis factor α (TNFα), and gluconeogenic enzymes in T2DM than in controls. The gene silencing for INSR or TNFα resulted in the inhibition or stimulation of GLUT4, respectively. Proteome profiles correspond- ing to molecular weights of the above translated transcriptomes showed different patterns of changes between T2DM and controls. Meanwhile, changes in tran- scriptomes and proteomes between muscle and activated T-cells of T2DM were comparable. Activated T-cells, analogous to muscle cells, expressed insulin sig- naling and glucose metabolism genes and gene products. In conclusion, T-cells and muscle in T2DM exhibited differences in expression of certain genes and gene products relative to non-diabetic controls. These alterations in transcriptomes and proteomes in T2DM may be involved in insulin resistance.

  8. Hyperoxia activates NF-kappaB and increases TNF-alpha and IFN-gamma gene expression in mouse pulmonary lymphocytes.

    Science.gov (United States)

    Shea, L M; Beehler, C; Schwartz, M; Shenkar, R; Tuder, R; Abraham, E

    1996-11-01

    Hyperoxia-associated production of reactive oxygen species leads to neutrophil infiltration into the lungs and increased pulmonary proinflammatory cytokine expression. However, the initial events induced by hyperoxia, and leading to acute inflammatory lung injury, remain incompletely characterized. To explore this issue, we examined nuclear transcriptional regulatory factor (NF-kappaB and NF-IL-6) activation and cytokine expression in the lungs following 12 to 48 h of hyperoxia exposure. No increases in cytokine (IL-1beta, IL-6, IL-10, TGF-beta, TNF-alpha, IFN-gamma) expression nor in NF-kappaB activation were found after 12 h of hyperoxia. Following 24 h of hyperoxia, NF-kappaB activation and increased levels of TNF-alpha mRNA were present in pulmonary lymphocytes. By 48 h of hyperoxia, amounts of IFN-gamma and TNF-alpha protein as well as mRNA were increased in the lungs, and NF-kappaB continued to show activation, even though no histologic abnormalities were present. These results show that hyperoxia activates NF-kappaB in the lungs before any increase in proinflammatory cytokine protein occurs, and suggest that NF-kappaB activation may represent an initial event in the proinflammatory sequence induced by hyperoxia.

  9. Global Gene Expression Profiling in Interleukin-12-Induced Activation of CD8+ Cytotoxic T Lymphocytes against MouseMammary Carcinoma

    Institute of Scientific and Technical Information of China (English)

    ShanjinCao; ZhaoyingXiang; XiaojingMa

    2004-01-01

    Interleukin-12 (IL-12) is a critical cytokine representing the link between the cellular and humoral branches of host immune defense apparatus. IL-12-induced cytotoxic lymphocyte (CTL) development is a central mechanism in immune responses against intracellular infectious agents as well as malignant growth. However,the molecular basis of tumor-specific CTL responses mediated by IL-12 remains poorly defined. In this study,we addressed this issue in a comprehensive manner to probe into IL-12-induced anti-tumor responses by global gene expression profiling of mRNA expression in CD8+T cells in a transplantable syngeneic mouse mammarycarcinoma model treated or not with recombinant IL-12. A strong tumor regression was induced by the IL-12 treatment. An introspection of differential gene expression at an early stage of the IL-12-initiated CTL activation reveals interesting genes and molecular pathways that may account for the marked tumor regression,and is likely to provide a rich source of potential targets for further research and development of effective therapeutic modalities. Cellular & Molecular Immunology. 2004;1(5):357-366.

  10. Identification of gene profiles of CD4~+ and CD8~+ T lymphocyte in systemic lupus erythematosus by generation of longer cDNA fragments from serial analysis of gene expression tags for gene identification

    Institute of Scientific and Technical Information of China (English)

    王惠琳

    2006-01-01

    Objective To identify LongSAGE Tags in systemic lupus erythematosus (SLE) by generation of longer cDNA fragments from serial analysis of gene expression (SAGE) tags for gene identification (GLGI). Methods CD4+ and CD8+ T lymphocytes were collected from the PBMCs of 25 patients with SLE and 10 healthy controls. Then the total RNA was extracted and reversely

  11. Antigen receptors on immature, but not mature, B and T cells are coupled to cytosolic phospholipase A2 activation: expression and activation of cytosolic phospholipase A2 correlate with lymphocyte maturation.

    Science.gov (United States)

    Gilbert, J J; Stewart, A; Courtney, C A; Fleming, M C; Reid, P; Jackson, C G; Wise, A; Wakelam, M J; Harnett, M M

    1996-03-15

    The Ag receptors on mature B and T cells are not coupled to the activation of cytosolic phospholipase A2 (cPLA2) and arachidonic acid release. Moreover, phorbol esters such as PMA, which can activate cPLA2 via mitogen-activated protein (MAP) kinase in most cell types, also failed to induce the release of arachidonate from mature cells, suggesting that the cPLA2 pathway may not be functional in mature lymphocytes. Interestingly, Western blot analysis revealed that cPLA2, which had previously been thought to be expressed ubiquitously, is not expressed in mature B or T cells and that cytosolic phospholipase A2 expression could not be up-regulated in lymphocytes following culture with a range of cytokines most likely to be involved in an immune response such as IL-1 alpha, IL-3, or TNF-alpha. In contrast, cPLA2 was shown to be expressed and activated in thymocytes and immature B cells under conditions in which ligation of the Ag receptors led to growth arrest and/or apoptosis. Taken together, these data suggest that cPLA2 does not play a role in Ag receptor-mediated lymphocyte activation, but may be involved in the molecular mechanisms underlying lymphocyte maturation and/or self tolerance by clonal deletion.

  12. Expression of HLA-G in patients with B-cell chronic lymphocytic leukemia (B-CLL.

    Directory of Open Access Journals (Sweden)

    M Schmitt

    2009-01-01

    Full Text Available The expression of HLA-G was reported in certain malignancies and its role in escaping from immunosurveillance in cancers was proposed since HLA-G is a nonconventional HLA class I molecule that protects fetus from immunorecognition during pregnancy. Recent studies proposed HLA-G as novel prognostic marker for patients with B-CLL. HLA-G was showed to bear even better prognostic information compared to Zeta-chain associated protein of 70kDa (ZAP-70 and CD38 although some other authors did not find HLA-G expression in CLL. Therefore in this study we characterized the expression of HLA-G on both RNA and protein level. In most of 20 B-CLL patients we were able to detect signal from HLA-G using flow cytometry analysis. The expression of HLA-G was confirmed on messenger level by real-time RT-PCR experiments. No correlation between HLA-G expression and expression of well established prognostic factors such as ZAP-70 and CD38 was detected. These results confirm that HLA-G is expressed on CLL leukemic cells. Furthermore the expression of HLA-G on CLL cells suggests that this molecule might be involved in escaping of CLL cells from immunosurveillance.

  13. Complement receptor expression and activation of the complement cascade on B lymphocytes from patients with systemic lupus erythematosus (SLE)

    DEFF Research Database (Denmark)

    Marquart, H V; Svendsen, Anders Jørgen; Rasmussen, J M

    1995-01-01

    It has previously been reported that the expression of the complement receptors, CR1 on erythrocytes and blood leucocytes and CR2 on B cells, is reduced in patients with SLE, and that the reduced expression of CR1 on erythrocytes is related to disease activity. We have earlier demonstrated that n...

  14. Complement receptor expression and activation of the complement cascade on B lymphocytes from patients with systemic lupus erythematosus (SLE)

    DEFF Research Database (Denmark)

    Marquart, H V; Svendsen, A; Rasmussen, J M

    1995-01-01

    It has previously been reported that the expression of the complement receptors, CR1 on erythrocytes and blood leucocytes and CR2 on B cells, is reduced in patients with SLE, and that the reduced expression of CR1 on erythrocytes is related to disease activity. We have earlier demonstrated that n...

  15. IFNγ and perforin cooperate to control infection and prevent fatal pathology during persistent gammaherpesvirus infection in mice

    DEFF Research Database (Denmark)

    Bartholdy, Christina; Høgh-Petersen, Mette; Storm, Pernille

    2014-01-01

    Infection with murine gammaherpes virus 68 has become an accepted model for studying the virus/host interactions with regard to gammaherpes virus infections. Previous studies using gene deficient mice have revealed that neither IFNγ nor perforin are essential in controlling the course of infectio...... is tipped in favour of the virus causing chronic immune activation and fatal disease. This article is protected by copyright. All rights reserved....

  16. Selective role of mevalonate pathway in regulating perforin but not FasL and TNFalpha release in human Natural Killer cells.

    Directory of Open Access Journals (Sweden)

    Alessandro Poggi

    Full Text Available We have analyzed the effects of fluvastatin, an inhibitor of the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA reductase involved in mevalonate synthesis, on human NK cell-mediated anti-tumor cytolysis. Fluvastatin inhibited the activation of the small guanosin triphosphate binding protein (GTP RhoA and the consequent actin redistribution induced by ligation of LFA1 involved in NK-tumor target cell adhesion. Also, fluvastatin reduced ganglioside M1 rafts formation triggered through the engagement of NK cell activating receptors as FcγRIIIA (CD16, NKG2D and DNAM1. Cytolysis of tumor targets was inhibited up to 90% when NK cells were cultured with fluvastatin by affecting i receptor-mediated increase of the intracellular free calcium concentration, ii activation of akt1/PKB and iii perforin and granzyme release. Fluvastatin displayed a stronger inhibiting effect on NKG2D, DNAM1, 2B4, NKp30, NKp44 and NKp46 than on CD16-mediated NK cell triggering. This was in line with the impairment of surface expression of all these receptors but not of CD16. Remarkably, fluvastatin did not affect the expression of the inhibiting receptors CD94, KIR2D and LAIR1. FasL release elicited by either NK-tumor cell interaction or CD16 or NKG2D engagement, as well as FasL-mediated killing, were not sensitive to fluvastatin. Moreover, TNFα secretion triggered in NK cells upon incubation with tumor target cells or engagement of NKG2D receptor was not impaired in fluvastatin-treated NK cells. Likewise, antibody dependent cellular cytotoxicity (ADCC triggered through FcγRIIIA engagement with the humanized monoclonal antibody rituximab or trastuzumab was only marginally affected in fluvastatin-treated NK cells. Altogether these findings suggest that interference with mevalonate synthesis impairs activation and assembly of cytoskeleton, degranulation and cytotoxic effect of perforins and granzyme but not FasL- and TNFα-mediated cytotoxicity.

  17. Zinc-finger protein ZFP318 is essential for expression of IgD, the alternatively spliced Igh product made by mature B lymphocytes

    Science.gov (United States)

    Enders, Anselm; Short, Alanna; Miosge, Lisa A.; Bergmann, Hannes; Sontani, Yovina; Bertram, Edward M.; Whittle, Belinda; Balakishnan, Bhavani; Yoshida, Kaoru; Sjollema, Geoff; Field, Matthew A.; Andrews, T. Daniel; Hagiwara, Hiromi; Goodnow, Christopher C.

    2014-01-01

    IgD and IgM are produced by alternative splicing of long primary RNA transcripts from the Ig heavy chain (Igh) locus and serve as the receptors for antigen on naïve mature B lymphocytes. IgM is made selectively in immature B cells, whereas IgD is coexpressed with IgM when the cells mature into follicular or marginal zone B cells, but the transacting factors responsible for this regulated change in splicing have remained elusive. Here, we use a genetic screen in mice to identify ZFP318, a nuclear protein with two U1-type zinc fingers found in RNA-binding proteins and no known role in the immune system, as a critical factor for IgD expression. A point mutation in an evolutionarily conserved lysine-rich domain encoded by the alternatively spliced Zfp318 exon 10 abolished IgD expression on marginal zone B cells, decreased IgD on follicular B cells, and increased IgM, but only slightly decreased the percentage of B cells and did not decrease expression of other maturation markers CD21, CD23, or CD62L. A targeted Zfp318 null allele extinguished IgD expression on mature B cells and increased IgM. Zfp318 mRNA is developmentally regulated in parallel with IgD, with little in pro-B cells, moderate amounts in immature B cells, and high levels selectively in mature follicular B cells. These findings identify ZFP318 as a crucial factor regulating the expression of the two major antibody isotypes on the surface of most mature B cells. PMID:24616512

  18. Allergen-stimulated T lymphocytes from allergic patients induce vascular cell adhesion molecule-1 (VCAM-1) expression and IL-6 production by endothelial cells.

    Science.gov (United States)

    Delneste, Y; Jeannin, P; Gosset, P; Lassalle, P; Cardot, E; Tillie-Leblond, I; Joseph, M; Pestel, J; Tonnel, A B

    1995-01-01

    Adhesion of inflammatory cells to endothelium is a critical step for their transvascular migration to inflammatory sites. To evaluate the relationship between T lymphocytes (TL) and vascular endothelium, supernatants from allergen-stimulated TL obtained from patients sensitive to Dermatophagoides pteronyssinus (Dpt) versus healthy subjects were added to endothelial cell (EC) cultures. TL were stimulated by autologous-activated antigen-presenting cells (APC) previously fixed in paraformaldehyde to prevent monokine secretion. Two parameters were measured: the expression of adhesion molecule and the production of IL-6. Related allergen-stimulated TL supernatants from allergic patients induced an increase of VCAM-1 and intercellular adhesion molecule-1 (ICAM-1) expression when supernatants of the control groups (TL exposed to an unrelated allergen or not stimulated or TL obtained from healthy subjects) did not. E-selectin expression was not modulated whatever the supernatant added to EC culture. IL-6 production by EC was significantly enhanced after activation with related allergen-stimulated TL supernatants from allergics compared with control supernatants. Induction of VCAM-1 expression was inhibited by adding neutralizing antibodies against IL-4, whereas IL-6 production and ICAM-1 expression were inhibited by anti-interferon-gamma (IFN-gamma) antibodies. Enhanced production of IL-4 and IFN-gamma was detected in related allergen-stimulated TL supernatants from allergic subjects compared with the different supernatants. These data suggest that allergen-specific TL present in the peripheral blood of allergic patients are of Th1 and Th2 subtypes. Their stimulation in allergic patients may lead to the activation of endothelial cells and thereby participate in leucocyte recruitment towards the inflammatory site. PMID:7542574

  19. Integration Analysis of MicroRNA and mRNA Expression Profiles in Human Peripheral Blood Lymphocytes Cultured in Modeled Microgravity

    Directory of Open Access Journals (Sweden)

    C. Girardi

    2014-01-01

    Full Text Available We analyzed miRNA and mRNA expression profiles in human peripheral blood lymphocytes (PBLs incubated in microgravity condition, simulated by a ground-based rotating wall vessel (RWV bioreactor. Our results show that 42 miRNAs were differentially expressed in MMG-incubated PBLs compared with 1 g incubated ones. Among these, miR-9-5p, miR-9-3p, miR-155-5p, miR-150-3p, and miR-378-3p were the most dysregulated. To improve the detection of functional miRNA-mRNA pairs, we performed gene expression profiles on the same samples assayed for miRNA profiling and we integrated miRNA and mRNA expression data. The functional classification of miRNA-correlated genes evidenced significant enrichment in the biological processes of immune/inflammatory response, signal transduction, regulation of response to stress, regulation of programmed cell death, and regulation of cell proliferation. We identified the correlation of miR-9-3p, miR-155-5p, miR-150-3p, and miR-378-3p expression with that of genes involved in immune/inflammatory response (e.g., IFNG and IL17F, apoptosis (e.g., PDCD4 and PTEN, and cell proliferation (e.g., NKX3-1 and GADD45A. Experimental assays of cell viability and apoptosis induction validated the results obtained by bioinformatics analyses demonstrating that in human PBLs the exposure to reduced gravitational force increases the frequency of apoptosis and decreases cell proliferation.

  20. Interleukin 21 Controls mRNA and MicroRNA Expression in CD40-Activated Chronic Lymphocytic Leukemia Cells

    Science.gov (United States)

    De Cecco, Loris; Capaia, Matteo; Zupo, Simona; Cutrona, Giovanna; Matis, Serena; Brizzolara, Antonella; Orengo, Anna Maria; Croce, Michela; Marchesi, Edoardo; Ferrarini, Manlio; Canevari, Silvana; Ferrini, Silvano

    2015-01-01

    Several factors support CLL cell survival in the microenvironment. Under different experimental conditions, IL21 can either induce apoptosis or promote CLL cell survival. To investigate mechanisms involved in the effects of IL21, we studied the ability of IL21 to modulate gene and miRNA expressions in CD40-activated CLL cells. IL21 was a major regulator of chemokine production in CLL cells and it modulated the expression of genes involved in cell movement, metabolism, survival and apoptosis. In particular, IL21 down-regulated the expression of the chemokine genes CCL4, CCL3, CCL3L1, CCL17, and CCL2, while it up-regulated the Th1-related CXCL9 and CXCL10. In addition, IL21 down-regulated the expression of genes encoding signaling molecules, such as CD40, DDR1 and PIK3CD. IL21 modulated a similar set of genes in CLL and normal B-cells (e.g. chemokine genes), whereas other genes, including MYC, TNF, E2F1, EGR2 and GAS-6, were regulated only in CLL cells. An integrated analysis of the miRNome and gene expression indicated that several miRNAs were under IL21 control and these could, in turn, influence the expression of potential target genes. We focused on hsa-miR-663b predicted to down-regulate several relevant genes. Transfection of hsa-miR-663b or its specific antagonist showed that this miRNA regulated CCL17, DDR1, PIK3CD and CD40 gene expression. Our data indicated that IL21 modulates the expression of genes mediating the crosstalk between CLL cells and their microenvironment and miRNAs may take part in this process. PMID:26305332

  1. Interleukin 21 Controls mRNA and MicroRNA Expression in CD40-Activated Chronic Lymphocytic Leukemia Cells.

    Directory of Open Access Journals (Sweden)

    Loris De Cecco

    Full Text Available Several factors support CLL cell survival in the microenvironment. Under different experimental conditions, IL21 can either induce apoptosis or promote CLL cell survival. To investigate mechanisms involved in the effects of IL21, we studied the ability of IL21 to modulate gene and miRNA expressions in CD40-activated CLL cells. IL21 was a major regulator of chemokine production in CLL cells and it modulated the expression of genes involved in cell movement, metabolism, survival and apoptosis. In particular, IL21 down-regulated the expression of the chemokine genes CCL4, CCL3, CCL3L1, CCL17, and CCL2, while it up-regulated the Th1-related CXCL9 and CXCL10. In addition, IL21 down-regulated the expression of genes encoding signaling molecules, such as CD40, DDR1 and PIK3CD. IL21 modulated a similar set of genes in CLL and normal B-cells (e.g. chemokine genes, whereas other genes, including MYC, TNF, E2F1, EGR2 and GAS-6, were regulated only in CLL cells. An integrated analysis of the miRNome and gene expression indicated that several miRNAs were under IL21 control and these could, in turn, influence the expression of potential target genes. We focused on hsa-miR-663b predicted to down-regulate several relevant genes. Transfection of hsa-miR-663b or its specific antagonist showed that this miRNA regulated CCL17, DDR1, PIK3CD and CD40 gene expression. Our data indicated that IL21 modulates the expression of genes mediating the crosstalk between CLL cells and their microenvironment and miRNAs may take part in this process.

  2. Myostatin expression, lymphocyte population, and potential cytokine production correlate with predisposition to high-fat diet induced obesity in mice.

    Directory of Open Access Journals (Sweden)

    Jeri-Anne Lyons

    Full Text Available A strong relationship exists between increased inflammatory cytokines and muscle insulin resistance in obesity. This study focused on identifying a relationship between metabolic propensity and myostatin expression in muscle and spleen cells in response to high-fat diet intake. Using a comparative approach, we analyzed the effects of high-fat diet intake on myostatin and follistatin expression, spleen cell composition, and potential cytokine expression in high-fat diet induced obesity (HFDIO resistant (SWR/J and susceptible (C57BL/6 mice models. Results demonstrated overall increased myostatin expression in muscle following high-fat diet intake in HFDIO-susceptible mice, while myostatin expression levels decreased initially in muscle from high-fat diet fed resistant mice. In HFDIO-resistant mice, myostatin expression decreased in spleen, while myostatin increased in spleen tissue from HFDIO-susceptible mice. Proinflammatory cytokine (IL-17, IL-1β, and IFNγ potential increased in splenocytes from HFDIO-susceptible mice. In comparison, C57BL/6 mice fed a high-fat diet exhibited higher frequencies of CD4(+/CD44(hi and CD8(+/CD44(hi cells in the spleen compared to control fed mice. Together, these results suggest that susceptibility to high-fat diet induced obesity could be influenced by local myostatin activity in a tissue-specific manner and that splenocytes exhibit differential cytokine production in a strain-dependent manner. This study sets the stage for future investigations into the interactions between growth, inflammation, and metabolism.

  3. Expression of apoptosis markers on peripheral blood lymphocytes from patients with cutaneous T-cell lymphoma during extracorporeal photochemotherapy.

    Science.gov (United States)

    Osella-Abate, S; Zaccagna, A; Savoia, P; Quaglino, P; Salomone, B; Bernengo, M G

    2001-01-01

    The mechanisms of extracorporeal photochemotherapy (ExP) therapeutic activity in cutaneous T-cell lymphomas (CTCLs) are not yet well understood, even though it has been suggested that a major mechanism may be induction of apoptosis. In vitro studies demonstrate that UVA-induced apoptosis is mediated by CD95-Fas expression and inhibited by Bcl-2 up-regulation and that UVA irradiation is able to down-regulate Bcl-2 expression. High-resolution multiparameter flow-cytometric analyses were used to evaluate Bcl-2/CD95-Fas expression on phenotypically identifiable circulating clonal T cells from 7 patients with CTCL (4 with Sézary syndrome and 3 with mycosis fungoides with peripheral involvement) before and during ExP, in an attempt to ascertain whether Bcl-2/CD95-Fas status can be related to the hematologic response. A Bcl-2 normal phenotype before ExP or a normalization in Bcl-2 expression during ExP were related to a better clinical response, whereas a persistent Bcl-2 high expression was a negative prognostic factor. On the other hand, no response was found in patients with a CD95-Fas-negative phenotype, whereas the expression of CD95-Fas was associated with hematologic remission. Although further studies are needed to confirm these preliminary results, this study suggests that Bcl-2 and CD95-Fas expression could be evaluated, together with the other known clinical and immunologic factors, as additional parameters related to clinical response in patients with CTCL undergoing ExP.

  4. HIV慢性感染者Vδ2T淋巴细胞的CD25表达与其凋亡及疾病进展的关系研究%Correlations between CD25 expression on Vδ2 T lymphocytes and the apoptosis of T lymphocytes and disease progression in chronically HIV-infected patients

    Institute of Scientific and Technical Information of China (English)

    许文; 黄磊; 谢杨新; 秦恩强; 石磊; 武晓丽; 杨俊连; 杨欣欣; 赵敏

    2013-01-01

    Objective To investigate the correlations between CD25 expression on Vδ2 T lymphocytes and the frequency and apoptosis of Vδ2 T lymphocytes and disease progression in chronically HIV-infected patients.Methods Thirty-one chronically HIV-infected outpatients treated in our hospital from Mar.2012 to Apr.2013 and 10 healthy controls (HC) were enrolled in the study.Of 31 chronically HIV-infected patients,10 didn't receive highly active antiretroviral therapy (HAART) (without HAART group),10 received 3 months of HAART (3-month HAART group),and 11 received 6 months of HAART (6-month HAART group).The frequency and apoptosis of Vδ2 T lymphocytes and CD25 expression on Vδ2 T lymphocytes were detected by flow cytometry.The changes of the above-mentioned indicators of without HAART group,3-month HAART group and 6-month HAART group were observed respectively,and the correlations between these indicators and CD4+ T lymphocyte counts and viral loads of each group were analyzed respectively.The correlations between CD25 expression and the frequency and apoptosis of Vδ2 T lymphocytes were analyzed as well.Results CD25 expression on Vδ2 T lymphocytes in chronically HIV-infected patients increased significantly as compared with HC.Though 6 months of HAART could down-regulate it,the expression was still higher than that of HC.CD25 expression on Vδ2 T lymphocytes decreased along with the increase in CD4+ T lymphocyte counts,but the correlation was not significant.The correlation between CD25 expression on Vδ2 T lymphocytes and viral loads in the patients not receiving HAART was not significant.The frequency of Vδ2 T lymphocytes was negatively correlated with CD25 expression in chronically HIV-infected patients,but the correlation between the apoptosis of and CD25 expression on Vδ2 T lymphocytes was not significant.Conclusions Vδ2 T lymphocytes are activated in chronically HIV-infected patients,which may be correlated with the decline in the frequency of Vδ2 T lymphocytes

  5. Artificial antigen-presenting cells expressing AFP(158-166) peptide and interleukin-15 activate AFP-specific cytotoxic T lymphocytes.

    Science.gov (United States)

    Sun, Longhao; Guo, Hao; Jiang, Ruoyu; Lu, Li; Liu, Tong; Zhang, Zhixiang; He, Xianghui

    2016-04-05

    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.

  6. Serglycin determines secretory granule repertoire and regulates natural killer cell and cytotoxic T lymphocyte cytotoxicity.

    Science.gov (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

    2016-03-01

    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.

  7. Use of Gene Expression Profiles of Peripheral Blood Lymphocytes to Distinguish BRCA1 Mutation Carriers in High Risk Breast Cancer Families

    Directory of Open Access Journals (Sweden)

    Marie-Laure Vuillaume

    2009-01-01

    Full Text Available Mutations in two major genes, BRCA1 and BRCA2, account for up to 30% of families with hereditary breast cancer. Unfortunately, in most families there is little to indicate which gene should be targeted first for mutation screening, which is labor intensive, time consuming and often prohibitively expensive. As BRCA1 is a tumor suppressor gene involved in various cellular processes, heterozygous mutations could deregulate dependent pathways, such as DNA damage response, and disturb transcriptional activity of genes involved in the downstream signaling cascade. We investigated gene expression profiling in peripheral blood lymphocytes to evaluate this strategy for distinguishing BRCA1 mutation carriers from non-carriers. RNA from whole blood samples of 15 BRCA1 mutation carriers and 15 non-carriers from BRCA1 or BRCA2 families were hybridized to Agilent Technologies Whole Human Genome OligoMicroarrays (4 × 44 K multiplex format containing 41,000 unique human genes and transcripts. Gene expression data were analyzed with Welch’s t-tests and submitted to hierarchical clustering (GeneSpring GX software, Agilent Technologies. Statistical analysis revealed a slight tendency for 133 genes to be differentially expressed between BRCA1 mutation carriers and non-carriers. However, hierarchical clustering of these genes did not accurately discriminate BRCA1 mutation carriers from non-carriers. Expression variation for these genes according to BRCA1 mutation status was weak. In summary, microarray profiling of untreated whole blood does not appear to be informative in identifying breast cancer risk due to BRCA1 mutation.

  8. CX3CR1 is expressed by human B lymphocytes and mediates [corrected] CX3CL1 driven chemotaxis of tonsil centrocytes.

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    Anna Corcione

    Full Text Available BACKGROUND: Fractalkine/CX(3CL1, a surface chemokine, binds to CX(3CR1 expressed by different lymphocyte subsets. Since CX(3CL1 has been detected in the germinal centres of secondary lymphoid tissue, in this study we have investigated CX(3CR1 expression and function in human naïve, germinal centre and memory B cells isolated from tonsil or peripheral blood. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate unambiguously that highly purified human B cells from tonsil and peripheral blood expressed CX(3CR1 at mRNA and protein levels as assessed by quantitative PCR, flow cytometry and competition binding assays. In particular, naïve, germinal centre and memory B cells expressed CX(3CR1 but only germinal centre B cells were attracted by soluble CX(3CL1 in a transwell assay. CX(3CL1 signalling in germinal centre B cells involved PI3K, Erk1/2, p38, and Src phosphorylation, as assessed by Western blot experiments. CX(3CR1(+ germinal centre B cells were devoid of centroblasts and enriched for centrocytes that migrated to soluble CX(3CL1. ELISA assay showed that soluble CX(3CL1 was secreted constitutively by follicular dendritic cells and T follicular helper cells, two cell populations homing in the germinal centre light zone as centrocytes. At variance with that observed in humans, soluble CX(3CL1 did not attract spleen B cells from wild type mice. OVA immunized CX(3CR1(-/- or CX(3CL1(-/- mice showed significantly decreased specific IgG production compared to wild type mice. CONCLUSION/SIGNIFICANCE: We propose a model whereby human follicular dendritic cells and T follicular helper cells release in the light zone of germinal centre soluble CX(3CL1 that attracts centrocytes. The functional implications of these results warrant further investigation.

  9. The B7-H1 (PD-L1 T Lymphocyte-Inhibitory Molecule Is Expressed in Breast Cancer Patients with Infiltrating Ductal Carcinoma: Correlation with Important High-Risk Prognostic Factors

    Directory of Open Access Journals (Sweden)

    Hazem Ghebeh

    2006-03-01

    Full Text Available B7-H1 molecule increases the apoptosis of tumorreactive T lymphocytes and reduces their immunogenicity. Breast cancer is the second most common cause of mortality after lung cancer. Direct evidence linking B7-H1 with cancer has been shown in several malignancies; however, its expression in breast cancer has not been investigated. We used immunohistochemistry to investigate the expression of the B7-H1 molecule in 44 breast cancer specimens and to study its correlation with patients' clinicopathological parameters. The expression of B7-H1 was shown in 22 of 44 patients and was not restricted to the tumor epithelium (15 of 44, 34% in tumor cells, but was also expressed by tumor-infiltrating lymphocytes (TIL; 18 of 44, 41%. Interestingly, intratumor expression of B7-H1 was significantly associated with histologic grade IIInegative (P = .012, estrogen receptor-negative (P = .036, and progesterone receptor-negative (P = .040 patients. In addition, the expression of B7-H1 in TIL was associated with large tumor size (P = .042, histologic grade III (P=.015, positivity of Her2/neu status (P=.019, and severe tumor lymphocyte infiltration (P = .001. Taken together, these data suggest that B7-H1 may be an important risk factor in breast cancer patients and may represent a potential immunotherapeutic target using monoclonal antibody against the B7-H1 molecule.

  10. The B7-H1 (PD-L1)T Lymphocyte-Inhibitory Molecule Is Expressed in Breast Cancer Patients with Infiltrating Ductal Carcinoma: Correlation with Important High-Risk Prognostic Factors1

    Science.gov (United States)

    Ghebeh, Hazem; Mohammed, Shamayel; Al-Omair, Abeer; Qattan, Amal; Lehe, Cynthia; Al-Qudaihi, Ghofran; Elkum, Naser; Alshabanah, Mohamed; Amer, Suad Bin; Tulbah, Asma; Ajarim, Dahish; Al-Tweigeri, Taher; Dermime, Said

    2006-01-01

    Abstract B7-H1 molecule increases the apoptosis of tumor-reactive T lymphocytes and reduces their immunogenicity. Breast cancer is the second most common cause of mortality after lung cancer. Direct evidence linking B7-H1 with cancer has been shown in several malignancies; however, its expression in breast cancer has not been investigated. We used immunohistochemistry to investigate the expression of the B7-H1 molecule in 44 breast cancer specimens and to study its correlation with patients' clinicopathological parameters. The expression of B7-H1 was shown in 22 of 44 patients and was not restricted to the tumor epithelium (15 of 44, 34% in tumor cells), but was also expressed by tumor-infiltrating lymphocytes (TIL; 18 of 44, 41%). Interestingly, intratumor expression of B7-H1 was significantly associated with histologic grade III-negative (P = .012), estrogen receptor-negative (P = .036), and progesterone receptor-negative (P = .040) patients. In addition, the expression of B7-H1 in TIL was associated with large tumor size (P = .042), histologic grade III (P = .015), positivity of Her2/neu status (P = .019), and severe tumor lymphocyte infiltration (P = .001). Taken together, these data suggest that B7-H1 may be an important risk factor in breast cancer patients and may represent a potential immunotherapeutic target using monoclonal antibody against the B7-H1 molecule. PMID:16611412

  11. The B7-H1 (PD-L1) T lymphocyte-inhibitory molecule is expressed in breast cancer patients with infiltrating ductal carcinoma: correlation with important high-risk prognostic factors.

    Science.gov (United States)

    Ghebeh, Hazem; Mohammed, Shamayel; Al-Omair, Abeer; Qattan, Amal; Lehe, Cynthia; Al-Qudaihi, Ghofran; Elkum, Naser; Alshabanah, Mohamed; Bin Amer, Suad; Tulbah, Asma; Ajarim, Dahish; Al-Tweigeri, Taher; Dermime, Said

    2006-03-01

    B7-H1 molecule increases the apoptosis of tumor-reactive T lymphocytes and reduces their immunogenicity. Breast cancer is the second most common cause of mortality after lung cancer. Direct evidence linking B7-H1 with cancer has been shown in several malignancies; however, its expression in breast cancer has not been investigated. We used immunohistochemistry to investigate the expression of the B7-H1 molecule in 44 breast cancer specimens and to study its correlation with patients' clinicopathological parameters. The expression of B7-H1 was shown in 22 of 44 patients and was not restricted to the tumor epithelium (15 of 44, 34% in tumor cells), but was also expressed by tumor-infiltrating lymphocytes (TIL; 18 of 44, 41%). Interestingly, intratumor expression of B7-H1 was significantly associated with histologic grade III-negative (P = .012), estrogen receptor-negative (P = .036), and progesterone receptor-negative (P = .040) patients. In addition, the expression of B7-H1 in TIL was associated with large tumor size (P = .042), histologic grade III (P = .015), positivity of Her2/neu status (P = .019), and severe tumor lymphocyte infiltration (P = .001). Taken together, these data suggest that B7-H1 may be an important risk factor in breast cancer patients and may represent a potential immunotherapeutic target using monoclonal antibody against the B7-H1 molecule.

  12. Stonefish toxin defines an ancient branch of the perforin-like superfamily.

    Science.gov (United States)

    Ellisdon, Andrew M; Reboul, Cyril F; Panjikar, Santosh; Huynh, Kitmun; Oellig, Christine A; Winter, Kelly L; Dunstone, Michelle A; Hodgson, Wayne C; Seymour, Jamie; Dearden, Peter K; Tweten, Rodney K; Whisstock, James C; McGowan, Sheena

    2015-12-15

    The lethal factor in stonefish venom is stonustoxin (SNTX), a heterodimeric cytolytic protein that induces cardiovascular collapse in humans and native predators. Here, using X-ray crystallography, we make the unexpected finding that SNTX is a pore-forming member of an ancient branch of the Membrane Attack Complex-Perforin/Cholesterol-Dependent Cytolysin (MACPF/CDC) superfamily. SNTX comprises two homologous subunits (α and β), each of which comprises an N-terminal pore-forming MACPF/CDC domain, a central focal adhesion-targeting domain, a thioredoxin domain, and a C-terminal tripartite motif family-like PRY SPla and the RYanodine Receptor immune recognition domain. Crucially, the structure reveals that the two MACPF domains are in complex with one another and arranged into a stable early prepore-like assembly. These data provide long sought after near-atomic resolution insights into how MACPF/CDC proteins assemble into prepores on the surface of membranes. Furthermore, our analyses reveal that SNTX-like MACPF/CDCs are distributed throughout eukaryotic life and play a broader, possibly immune-related function outside venom.

  13. A mouse model for chronic lymphocytic leukemia based on expression of the SV40 large T antigen

    DEFF Research Database (Denmark)

    ter Brugge, Petra J; Ta, Van B T; de Bruijn, Marjolein J W;

    2009-01-01

    The simian virus 40 (SV40) T antigen is a potent oncogene able to transform many cell types and has been implicated in leukemia and lymphoma. In this report, we have achieved sporadic SV40 T-antigen expression in mature B cells in mice, by insertion of a SV40 T antigen gene in opposite transcript...

  14. Prostaglandin E-2 differentially modulates IL-5 gene expression in activated human T lymphocytes depending on the costimulatory signal

    NARCIS (Netherlands)

    Borger, P; Vellenga, E; Gringhuis, SI; Timmerman, JAB; Lummen, C; Postma, DS; Kauffman, HF

    1998-01-01

    Background: Protein kinase A (PKA) activation is documented to be inhibitory for T helper cell (T-H1)-like cytokines (IL-2, IFN-gamma), whereas T-H2-like cytokines (IL-4, IL-5) are not affected or upregulated. We have recently shown that IL-4 gene expression can be inhibited by PKA activation but de

  15. Differing activation status and immune effector molecule expression profiles of neonatal and maternal lymphocytes in an African population.

    NARCIS (Netherlands)

    Engelmann, I.; Moeller, U.; Santamaria, A.; Kremsner, P.G.; Luty, A.J.F.

    2006-01-01

    Higher susceptibility of newborns to infections has been attributed to the hypo-responsiveness of their cellular immune system. Here we compared the activation status and expression of cytokines and cytotoxic molecules of cord versus maternal peripheral blood mononuclear cells in an African

  16. [Effect of stimulation with the measles virus on expression of early activation markers on CD4+ T lymphocytes].

    Science.gov (United States)

    Siennicka, Joanna; Cześcik, Agnieszka; Dunal, Milena; Trzcińska, Agnieszka

    2011-01-01

    Elimination of measles is one of the priority plans of WHO. The success of this plan depends on the development of long lasting, postvaccinal immune response. The aim of this study was to present the effect of stimulation with different strains of measles virus on the expression of T-helper cell (CD4+ T) early activation markers in people with different history of measles infection and to determine the correlation between the activation and dose of virus used for stimulation. The study was conducted using material derived from two patients: one seropositive due to natural infection and one vaccinated, with traces of anti-MeV IgG antibodies. In the CD4 T helper cells, the expression of CD69 receptor and the ability of the cells to produce INF after stimulation with the vaccine-derived or wild-type strain of measles virus was determined. For antigen-specific stimulation the virus suspension containing about 100 infectious particle, its tenfold and hundredfold dilutions was used. We found that the expression of T-helper cells early activation markers depended on the strain of the measles virus used for the stimulation, type of the immune response (postvaccinal, natural infection), and in the case of CD69 expression also on the dose of the virus used for the stimulation.

  17. B-lymphocyte subpopulations are equally susceptible to Epstein-Barr virus infection, irrespective of immunoglobulin isotype expression.

    Science.gov (United States)

    Ehlin-Henriksson, Barbro; Gordon, John; Klein, George

    2003-04-01

    While Epstein-Barr virus (EBV) is known to establish latency in the memory B-cell compartment, there is controversy as to whether the memory or the naïve B cell is the initial target for infection. Here we have explored the infectability of the B-cell subsets contained in peripheral blood and tonsils, as distinguished by their surface expression of the immunoglobulin isotypes that help to define naïve and memory pools. First we show that both CD21 and major histocompatibility complex (MHC) class II molecules--respectively, the major receptor and co-receptor for EBV on B cells--are expressed at similar levels on blood and tonsillar B cells, irrespective of surface immunoglobulin class, indicating that each of the subsets demonstrate an equal potential, at least for infection. Then, following in vitro infection of total tonsillar B cells, we found that the relative frequencies of immunoglobulin (Ig)M-, IgG- and IgA-positive cells containing EBV-encoded Epstein-Barr virus nuclear antigen 5 (EBNA5) protein at 48 hr were similar to those of the starting population. However, IgD expression was uniformly decreased, probably as a consequence of cellular activation. These data indicate that recirculating B cells have both the potential for, and susceptibility to, initial infection by EBV, irrespective of the immunoglobulin isotype expressed.

  18. Expression of PD-1 Molecule on Regulatory T Lymphocytes in Patients with Insulin-Dependent Diabetes Mellitus

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    Valentina Perri

    2015-09-01

    Full Text Available Type 1 diabetes is caused by autoreactive T cells that destroy pancreatic beta cells. Animal models suggested that a CD4+CD25+ population has a regulatory function capable of preventing activation and effector functions of autoreactive T cells. However, the role of CD4+CD25high T cells in autoimmunity and their molecular mechanisms remain the subject of investigation. We therefore evaluated T regulatory cell frequencies and their PD-1 expression in the peripheral blood of long-standing diabetics under basal conditions and after CD3/CD28 stimulation. Under basal conditions, the percentages of T regulatory cells were significantly higher while that of T effector cells were significantly lower in patients than in controls. The ratio of regulatory to effector T cells was higher in patients than that in controls, suggesting that T regulatory cells were functional in patients. Percentages of total PD-1+, PD-1low and PD-1high expressing T regulatory cells did not change in patients and in controls. After stimulation, a defect in T regulatory cell proliferation was observed in diabetics and the percentages of total PD-1+, PD-1low and PD-1high expressing cells were lower in patients. Our data suggest a defective activation of T regulatory cells in long-standing diabetics due to a lower expression of PD-1 on their surface.

  19. Variation of ATM protein expression in response to irradiation of lymphocytes in lung cancer patients and controls.

    Science.gov (United States)

    Jianlin, Lou; Jiliang, He; Lifen, Jin; Wei, Zheng; Zhijian, Chen; Shijie, Chen; Shijie, Xu

    2006-07-05

    The aim of this research work was to study the cellular response to ionizing radiation (IR) and its relationship with the ATM protein expression levels in lung cancer patients. Heparinized blood samples were collected from 22 controls and 22 lung cancer patients. Each sample was divided into two parts: non-irradiated sample and irradiated sample, which was exposed to 3 Gy X-ray. The spontaneous and IR-induced genetic damage in both lung cancer patients and controls was measured with comet assay and micronucleus (MN) assay, and the ATM protein expression levels of non-irradiated samples in lung cancer patients and controls were detected by Western blotting. The results indicated that the baseline values of average mean tail moment (MTM) and micronucleus rate (MNR) in lung cancer patients were 0.86 and 11.41 per thousand, respectively, which was significantly higher than those (0.64 and 6.77 per thousand) of controls (P<0.05 for MTM, P<0.01 for MNR). The IR-induced average MTM and MNR in lung cancer patients were 1.23 and 77.64 per thousand, respectively, which was also significantly higher than those (0.71 and 66.05 per thousand) of controls (P<0.05 for MTM, P<0.01 for MNR). The results of Western blotting showed that the ATM protein expression levels in lung cancer patients and controls were 0.64 and 1.71, respectively, and there was significant (P<0.01) difference between lung cancer patients and controls. In present investigation, it was found that the genetic instability measured with comet assay and MN assay in lung cancer patients were significantly higher than those in controls, on the contrary, ATM protein expression level in lung cancer patients were significantly lower than that in controls. However, no good correlation was found either between ATM protein expression and IR-induced MTM or between ATM protein expression and IR-induced MNR in lung cancer patients.

  20. The human fetal lymphocyte lineage: identification by CD27 and LIN28B expression in B cell progenitors

    Science.gov (United States)

    McWilliams, Laurie; Su, Kuei-Ying; Liang, Xiaoe; Liao, Dongmei; Floyd, Serina; Amos, Joshua; Moody, M. Anthony; Kelsoe, Garnett; Kuraoka, Masayuki

    2013-01-01

    CD27, a member of the TNFR superfamily, is used to identify human memory B cells. Nonetheless, CD27+ B cells are present in patients with HIGM1 syndrome who are unable to generate GCs or memory B cells. CD27+IgD+ fetal B cells are present in umbilical cord blood, and CD27 may also be a marker of the human B1-like B cells. To define the origin of naïve CD27+IgD+ human B cells, we studied B cell development in both fetal and adult tissues. In human FL, most CD19+ cells coexpressed CD10, a marker of human developing B cells. Some CD19+CD10+ B cells expressed CD27, and these fetal CD27+ cells were present in the pro-B, pre-B, and immature/transitional B cell compartments. Lower frequencies of phenotypically identical cells were also identified in adult BM. CD27+ pro-B, pre-B, and immature/transitional B cells expressed recombination activating gene-1, terminal deoxynucleotidyl transferase and Vpre-B mRNA comparably to their CD27− counterparts. CD27+ and CD27− developing B cells showed similar Ig heavy chain gene usage with low levels of mutations, suggesting that CD27+ developing B cells are distinct from mutated memory B cells. Despite these similarities, CD27+ developing B cells differed from CD27− developing B cells by their increased expression of LIN28B, a transcription factor associated with the fetal lymphoid lineages of mice. Furthermore, CD27+ pro-B cells efficiently generated IgM+IgD+ immature/transitional B cells in vitro. Our observations suggest that CD27 expression during B cell development identifies a physiologic state or lineage for human B cell development distinct from the memory B cell compartment. PMID:23901121

  1. Expression of Hsp27 and Hsp70 in Lymphocytes and Plasma in Healthy Workers and Coal Miners with Lung Cancer

    Institute of Scientific and Technical Information of China (English)

    王海椒; 邢景才; 王峰; 韩文慧; 任侯卯; 邬堂春; 陈卫红

    2010-01-01

    In coal mines, main occupational hazard is coal-mine dust, which can cause health problem including coal workers' pneumoconiosis and lung cancer. Some heat shock proteins (Hsps) have been reported as an acute response to a wide variety of stressful stimuli. Whether Hsps protect against chronic environmental coal-mine dust over years is unknown. It is also interesting to know that whether the expression of Hsp27 and Hsp70 proteins as a marker for exposure is associated risk of lung cancer among coal miners. ...

  2. The selenium metabolite methylselenol regulates the expression of ligands that trigger immune activation through the lymphocyte receptor NKG2D

    DEFF Research Database (Denmark)

    Hagemann-Jensen, Michael Henrik; Uhlenbrock, Franziska Katharina; Kehlet, Stephanie;

    2014-01-01

    For decades Selenium (Se) research has been focused on the identification of active metabolites, which are crucial for Se chemoprevention of cancer. In this context, the metabolite methylselenol (CH3SeH) is known for its action to selectively kill transformed cells through mechanisms that include...... ligands. A balanced cell-surface expression of NKG2D ligands is considered as an innate barrier against tumor development. Our work therefore indicates that the application of selenium compounds, which are metabolized to CH3SeH, could improve NKG2D-based immune therapy....

  3. RPR 106541, a novel, airways-selective glucocorticoid: effects against antigen-induced CD4+ T lymphocyte accumulation and cytokine gene expression in the Brown Norway rat lung.

    Science.gov (United States)

    Underwood, S L; Raeburn, D; Lawrence, C; Foster, M; Webber, S; Karlsson, J A

    1997-10-01

    1. The effects of a novel 17-thiosteroid, RPR 106541, were investigated in a rat model of allergic airway inflammation. 2. In sensitized Brown Norway rats, challenge with inhaled antigen (ovalbumin) caused an influx of eosinophils and neutrophils into the lung tissue and airway lumen. In the lung tissue there was also an accumulation of CD4+ T lymphocytes and increased expression of mRNA for interleukin-4 (IL-4) and IL-5, but not interferon-gamma (IFN-gamma). These findings are consistent with an eosinophilia orchestrated by activated Th2-type cells. 3. RPR 106541 (10-300 microg kg[-1]), administered by intratracheal instillation into the airways 24 h and 1 h before antigen challenge, dose-dependently inhibited cell influx into the airway lumen. RPR 106541 (100 microg kg[-1]) caused a significant (PRPR 106541 was approximately 7 times and 4 times more potent than budesonide and fluticasone propionate, respectively. 4. When tested at a single dose (300 microg kg[-1]), RPR 106541 and fluticasone each caused a significant (PRPR 106541 and fluticasone (300 microg kg[-1]), but not budesonide (300 microg kg[-1]), significantly (PRPR 106541 (300 microg kg[-1]) also significantly (PRPR 106541 in this model, which mimics important aspects of airway inflammation in human allergic asthmatics, suggests that this glucocorticoid may be useful in the treatment of bronchial asthma.

  4. B Lymphocyte Stimulator (BLyS) is expressed in human adipocytes in vivo and is related to obesity but not to insulin resistance.

    Science.gov (United States)

    Müller, Nike; Schulte, Dominik M; Hillebrand, Susann; Türk, Kathrin; Hampe, Jochen; Schafmayer, Clemens; Brosch, Mario; von Schönfels, Witigo; Ahrens, Markus; Zeuner, Rainald; Schröder, Johann O; Blüher, Matthias; Gutschow, Christian; Freitag-Wolf, Sandra; Stelmach-Mardas, Marta; Saggau, Carina; Schreiber, Stefan; Laudes, Matthias

    2014-01-01

    Inflammation and metabolism have been shown to be evolutionary linked and increasing evidence exists that pro-inflammatory factors are involved in the pathogenesis of obesity and type 2 diabetes. Until now, most data suggest that within adipose tissue these factors are secreted by cells of the innate immune system, e. g. macrophages. In the present study we demonstrate that B lymphocyte stimulator (BLyS) is increased in human obesity. In contrast to several pro-inflammatory factors, we found the source of BLyS in human adipose tissue to be the adipocytes rather than immune cells. In grade 3 obese human subjects, expression of BLyS in vivo in adipose tissue is significantly increased (pr = 0.43, panti-BLyS antibody belimumab. Since BLyS is known to promote B-cell proliferation and immunoglobulin secretion, the present data suggest that adipocytes of grade 3 obese human subjects are able to activate the adaptive immune system, suggesting that in metabolic inflammation in humans both, innate and adaptive immunity, are of pathophysiological relevance.

  5. Isolation and expression of the full-length cDNA encoding CD59 antigen of human lymphocytes.

    Science.gov (United States)

    Sawada, R; Ohashi, K; Anaguchi, H; Okazaki, H; Hattori, M; Minato, N; Naruto, M

    1990-04-01

    To identify the primary structure of CD59 antigen and to elucidate its function, a full-length cDNA clone of CD59 was isolated. The cDNA sequence contained an open reading frame that encodes an 128-amino-acid peptide. The amino-terminal 25 amino acids represented a typical signal peptide sequence and the carboxy-terminal hydrophobic amino acids were characteristic for phosphatidylinositol-anchored proteins. The predicted mature protein sequence showed 35% homology with murine Ly-6C.1 and 31% with Ly-6A.2. The number and the distribution of cysteine residues were conserved, implying that the CD59 represented a human homologue of murine Ly-6. RNA blot hybridization analysis revealed the expression of CD59 mRNA in placental, lung, and pancreatic tissues. The mRNA was not only expressed in T-cell lines but in some of monocytic, myeloid, and B-cell lines. In all of these tissues and cell lines, at least four mRNA species were detected. DNA blot hybridization analysis revealed a rather simple genomic structure, which suggested a single gene as compared with the complex multigene family of murine Ly-6.

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

    Science.gov (United States)

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

    2016-01-01

    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.

  7. Activation of nuclear factor-kappa B and cell adhesion molecule mRNA expression in duodenal mucosa of dogs with lymphocytic-plasmacytic enteritis.

    Science.gov (United States)

    Okanishi, Hiroki; Kabeya, Hidenori; Maruyama, Soichi; Kagawa, Yumiko; Watari, Toshihiro

    2013-08-15

    Lymphocytic-plasmacytic enteritis (LPE) is the most common form of inflammatory bowel disease (IBD) affecting the canine small intestine; however, the molecular basis of the pathogenesis remains unclear. It has recently been hypothesized that the primary defect is impaired innate immune function, as is the case for human IBD. Nuclear factor-kappa B (NFkappaB) plays a central role in innate immunity, and is a major transcriptional regulator of several proinflammatory cytokines, pattern recognition receptors (PRRs) such as toll-like receptors (TLRs), nucleotide-binding oligomerization domain-like receptors and cell adhesion molecules (CAMs). The purpose of this study was to evaluate, in the duodenal mucosa of 21 dogs with LPE and 8 control dogs, the degree of NFkappaB activity and the mRNA expression of two selected cytokines, nucleotide oligomerization domain two (NOD2) receptor and three selected CAMs, all of which are regulated by NFkappaB, using the electrophoretic mobility shift assay and real-time reverse transcription PCR. NFkappaB binding activity was significantly higher in the inflamed duodenal mucosa of the LPE dogs as compared to healthy controls. Furthermore, expression of mRNA for intercellular cell adhesion molecule 1 (ICAM-1) and mucosal addressin cell adhesion molecule 1 (MAdCAM-1) was significantly higher and vascular cell adhesion molecule 1 (VCAM-1) mRNA significantly lower in LPE dogs than in healthy controls. However, there was no significant difference in the mRNA levels for TNFα, IL1β and NOD2 between the two groups. These results suggest that NFkappaB and CAMs may play important roles in the pathogenesis of canine LPE. Copyright © 2013 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2001-05-01

    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.

  9. Assessment of expression of selected Bcl-2 family proteins in lymphoid infiltration in patients with B-cell chronic lymphocytic leukaemia treated with nucleoside analogues.

    Directory of Open Access Journals (Sweden)

    Janusz Kłoczko

    2008-12-01

    Full Text Available B-cell chronic lymphocytic leukaemia (B-CLL is characterized by clonal growth and accumulation of mature lymphoid cells due to disturbance in genetically regulated form of cell death called apoptosis. The intrinsic mechanism of apoptosis is controlled by Bcl-2 family proteins. Purine nucleoside analogues induce the apoptosis in cells in a state of quiescence. The aim of the study was to assess expression of selected Bcl-2 family proteins in neoplastic infiltration in bone marrow in patients with B-CLL treated with nucleoside analogues. The study comprised examination of bone marrow obtained routinely by trephine biopsy from 18 patients with B-CLL diagnosed before administration of purine nucleoside analogues treatment and after its completion. Expression of Bcl-2, Bcl-x and Bax proteins was examined. Lymphoid cells in bone marrow were present in all patients before administration of treatment. After treatment in two patients bone marrow was infiltrated in diffuse pattern, whereas other patients presented nodular pattern of infiltration. The difference between stage of infiltration before and after treatment was statistically significant (p<0.002. High percentage of infiltration cells with positive anti Bcl-2 reaction from 42.0% in one patient to 85.33+/-3.06% in four patients before treatment was observed. After treatment percentage of infiltration cells with positive anti Bcl-2 antibody reaction was from 33.0+/-18.38% in two patients to 99.0% in one patient. Positive correlation between stage of infiltration and expression of Bcl-2 protein was confirmed before and after treatment. Such correlations were not observed in case of Bax and Bcl-x. Strong staining of immunohistochemical reaction of cells in lymphoid infiltration with Bcl-2 antibody was confirmed. There was a difference between Bcl-/Bax ratio before and after treatment. Immunohistochemical assessment of expression of Bcl-2 family proteins in cells of lymphoid infiltration in bone

  10. Cytochrome P450 1A1 and 1B1 in human blood lymphocytes are not suitable as biomarkers of exposure to dioxin-like compounds: polymorphisms and interindividual variation in expression and inducibility.

    Science.gov (United States)

    van Duursen, Majorie B M; Sanderson, J Thomas; van den Berg, Martin

    2005-05-01

    Cytochrome P450 1A1 (CYP1A1) and 1B1 (CYP1B1) are phase I enzymes, the expression of which can be affected by many environmental compounds, including dioxins and dioxin-like compounds. Because CYP1A1 and CYP1B1 expression can easily be determined in peripheral blood lymphocytes, it is often suggested as biomarker of exposure to these compounds. In this study we investigated the interindividual differences in constitutive and induced CYP1A1-catalyzed ethoxyresorufin-O-deethylase (EROD) activity and CYP1A1 and CYP1B1 gene expression in human blood lymphocytes in a group of ten non-smoking females. Freshly isolated lymphocytes were cultured in medium containing the mitogen PHA and were exposed to the most potent dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or the less potent dioxin-like polychlorinated biphenyl 126 (PCB126). In addition, we determined the occurrence of the CYP1A1 MspI and CYP1B1 Leu432Val polymorphisms. All individuals showed a concentration-dependent increase of EROD activity by TCDD, which was significantly correlated with an increase in CYP1A1, but not CYP1B1 expression. The maximum induced EROD activity by TCDD was very different among the individuals, but the EC50 values were about the same. PCB126 also caused a concentration-dependent increase of EROD activity, but was a factor 100-1000 less potent than TCDD among the individuals. The allele frequencies for CYP1A1 MspI and CYP1B1 Leu432Val reflected a normal Caucasian population and in this study the polymorphisms had no apparent effect on the expression and activity of these enzymes. Our study shows a large interindividual variability in constitutive and induced EROD activity, and CYP1A1 and CYP1B1 expression in human lymphocytes. In addition, dioxin concentrations at which effects were observed in our in vitro study are about 10-fold higher than the human blood levels found in vivo, indicating that EROD activity and CYP1A1 and CYP1B1 expression in human lymphocytes might not be

  11. CXC chemokine receptor 4 expression and stromal cell-derived factor-1alpha-induced chemotaxis in CD4+ T lymphocytes are regulated by interleukin-4 and interleukin-10

    DEFF Research Database (Denmark)

    Jinquan, T; Quan, S; Jacobi, H H

    2000-01-01

    We report that interleukin (IL)-4 and IL-10 can significantly up- or down-regulate CXC chemokine receptor 4 (CXCR4) expression on CD4+ T lymphocytes, respectively. Stromal cell-derived factor-1alpha (SDF-1alpha)-induced CD4+ T-lymphocyte chemotaxis was also correspondingly regulated by IL-4 and IL......,000 SDF-1alpha-binding sites per cell, among freshly isolated CD4+ T lymphocytes, and two types of CXCR4 with different affinities (Kd1 approximately 4.4 nM and Kd2 approximately 14.6 nM), and a total of approximately 130,000 SDF-1alpha-binding sites per cell, among IL-4-stimulated CD4+ T lymphocytes......-mobilization stimulation. These results indicate that the effects of IL-4 and IL-10 on the CXCR4-SDF-1 receptor-ligand pair may be of particular importance in the cytokine/chemokine environment concerning the inflammatory processes and in the progression of human immunodeficiency virus (HIV) infection....

  12. Adipose tissue lymphocytes: types and roles.

    Science.gov (United States)

    Caspar-Bauguil, S; Cousin, B; Bour, S; Casteilla, L; Castiella, L; Penicaud, L; Carpéné, C

    2009-12-01

    Besides adipocytes, specialized in lipid handling and involved in energy balance regulation, white adipose tissue (WAT) is mainly composed of other cell types among which lymphocytes represent a non-negligible proportion. Different types of lymphocytes (B, alphabetaT, gammadeltaT, NK and NKT) have been detected in WAT of rodents or humans, and vary in their relative proportion according to the fat pad anatomical location. The lymphocytes found in intra-abdominal, visceral fat pads seem representative of innate immunity, while those present in subcutaneous fat depots are part of adaptive immunity, at least in mice. Both the number and the activity of the different lymphocyte classes, except B lymphocytes, are modified in obesity. Several of these modifications in the relative proportions of the lymphocyte classes depend on the degree of obesity, or on leptin concentration, or even fat depot anatomical location. Recent studies suggest that alterations of lymphocyte number and composition precede the macrophage increase and the enhanced inflammatory state of WAT found in obesity. Lymphocytes express receptors to adipokines while several proinflammatory chemokines are produced in WAT, rendering intricate crosstalk between fat and immune cells. However, the evidences and controversies available so far are in favour of an involvement of lymphocytes in the control of the number of other cells in WAT, either adipocytes or immune cells and of their secretory and metabolic activities. Therefore, immunotherapy deserves to be considered as a promising approach to treat the endocrino-metabolic disorders associated to excessive fat mass development.

  13. СHANGES OF Glut1, mTOR AND AMPK1α GENE EXPRESSION IN PANCREATIC LYMPH NODE LYMPHOCYTES OF RATS WITH EXPERIMENTAL DIABETES MELLITUS

    Directory of Open Access Journals (Sweden)

    D. A. Putilin

    2016-01-01

    Full Text Available With the help of molecular genetic method we have investigated the level of mRNA gene expressions Glut1, mTOR and AMPK1α in PLN in pancreatic lymph nodes (PLN of rats with streptozotocininduced diabetes mellitus (SIDM and after administration of metformin. The levels of Glut1, mTOR and AMPK1α mRNA were determined by means of RT-PCR using CFX96™ thermocycler (Real-Time PCR Detection Systems, Bio-Rad, USA. Relative gene expression levels were calculated as ratios to GAPDH reference gene using ΔΔCt method. Statistical analysis was performed with available “Bio-Rad СFX Manager 3.1” software (Bio-Rad, USA. The mTOR+ positive lymphocytes were identified by means of monoclonal antibodies, using an indirect immunofluorescence method. Hyperglycemia was accompanied by transcriptional induction of glucose transporter Glut1 gene (9.9 to 28.9-fold, p < 0.05, and mTOR protein kinase gene (5.3 to 3.3-fold, p < 0.05 in PLN. Development of diabetes was also associated with increase by 24-34% in total mTOR+ cell numbers in PLN at the 5th week of developing diabetes (p <  0.05 and increased concentrations of rapamycin target in the immunopositive cells. Metformin administration to diabetic rats was followed by increased AMPK1α mRNA level of by 87% (p < 0,05 at the 3rd week, and 38-fold (p < 0,05, at the 5th week of SIDM development and inhibition of mTOR expression in PLN (3 to 14.7-fold, p < 0.05 accompanied by a 40 per cent decrease (p < 0.05 in total density of mTOR+ cells in PLN lymph cords of the rats following 5 weeks of SIDM.

  14. B Lymphocyte Stimulator (BLyS is expressed in human adipocytes in vivo and is related to obesity but not to insulin resistance.

    Directory of Open Access Journals (Sweden)

    Nike Müller

    Full Text Available Inflammation and metabolism have been shown to be evolutionary linked and increasing evidence exists that pro-inflammatory factors are involved in the pathogenesis of obesity and type 2 diabetes. Until now, most data suggest that within adipose tissue these factors are secreted by cells of the innate immune system, e. g. macrophages. In the present study we demonstrate that B lymphocyte stimulator (BLyS is increased in human obesity. In contrast to several pro-inflammatory factors, we found the source of BLyS in human adipose tissue to be the adipocytes rather than immune cells. In grade 3 obese human subjects, expression of BLyS in vivo in adipose tissue is significantly increased (p<0.001. Furthermore, BLyS serum levels are elevated in grade 3 human obesity (862.5+222.0 pg/ml vs. 543.7+60.7 pg/ml in lean controls, p<0.001 and are positively correlated to the BMI (r = 0.43, p<0.0002. In the present study, bariatric surgery significantly altered serum BLyS concentrations. In contrast, weight loss due to a very-low-calorie-formula-diet (800 kcal/d had no such effect. To examine metabolic activity of BLyS, in a translational research approach, insulin sensitivity was measured in human subjects in vivo before and after treatment with the human recombinant anti-BLyS antibody belimumab. Since BLyS is known to promote B-cell proliferation and immunoglobulin secretion, the present data suggest that adipocytes of grade 3 obese human subjects are able to activate the adaptive immune system, suggesting that in metabolic inflammation in humans both, innate and adaptive immunity, are of pathophysiological relevance.

  15. Expression of costimulatory molecule CD86 in HL-60 cells induced by MG132 and its effect on allogeneic mixed lymphocyte reaction.

    Science.gov (United States)

    Yu, Mei-Xia; Liu, Xun; Zhou, Yong-Ming; Cheng, Yan-Xiang; Cheng, Jing; Qiu, Yu-Zhen; Xing, Xiao-Lei; Yao, Chun-Hong; Bai, Ru-Jun

    2014-10-01

    This study was aimed to elucidate the expression of costimulatory molecule CD80 and CD86 in HL-60 cells induced by proteasome inhibitor MG132 and its effect on allogeneic mixed lymphocyte reaction. Acute myelocytic leukemia cell line HL-60 and chronic myelocytic leukemia cell line K562 were cultured. The viability of the cells was measured by flow cytometry. Proteasome inhibitor MG132 at the concentrations of 2 or 3 µmol/L was used to stimulate the HL-60 cell cultured for 24 h and 48 h respectively, and the Annexin V/7-AAD staining and flow cytomotry were used to detect the apoptosis of the HL-60 cells. HL-60 and K562 cells were treated with 1 µmol/L MG132 for 24 h and 48 h respectively, then CD80 and CD86 antibodies were added, finally the expression of CD80 and CD86 was analysed by flow cytomery. The mRNA expression of CD86 in the HL-60 cells treated with 1 µmol/L MG132 was detected by RT-PCR. HL-60 and K562 cells were treated by 1 µmol/L MG132 and then underwent irradiation of 75 Gy (60)Co to kill the cells with their antigenicity preserved. Peripheral blood mononuclear cells (PBMNCs) of healthy volunteers, as reactive cells, were isolated and inoculated into the (60)Co irradiated HL-60 cells of different concentrations, as stimulating cells, CCK-8 was added and then the A value of absorbance was measured at the wave length of 450 nm in an enzyme labeling instrument. The results showed that the cell viability of the HL-60 cells treated with 1 µmol/L MG132 for 24 h an d 48 h was 92.95% and 85.87% respectively. The apoptotic rates of the HL-60 cells treated with MG132 increased in dose-and time-dependent manner. High-concentration of MG132 directly killed HL-60 cells. Before MG132 treatment K562 cells did not express CD86, but the CD86 expression of the HL-60 cells was up-regulated time-dependently after MG132 treatment (P HL-60 treated with MG132 was up-regulated time-dependently (P HL-60 cells treated with MG132 and reached its peak when the concentration

  16. Conformational changes during pore formation by the perforin-related protein pleurotolysin.

    Directory of Open Access Journals (Sweden)

    Natalya Lukoyanova

    2015-02-01

    Full Text Available Membrane attack complex/perforin-like (MACPF proteins comprise the largest superfamily of pore-forming proteins, playing crucial roles in immunity and pathogenesis. Soluble monomers assemble into large transmembrane pores via conformational transitions that remain to be structurally and mechanistically characterised. Here we present an 11 Å resolution cryo-electron microscopy (cryo-EM structure of the two-part, fungal toxin Pleurotolysin (Ply, together with crystal structures of both components (the lipid binding PlyA protein and the pore-forming MACPF component PlyB. These data reveal a 13-fold pore 80 Å in diameter and 100 Å in height, with each subunit comprised of a PlyB molecule atop a membrane bound dimer of PlyA. The resolution of the EM map, together with biophysical and computational experiments, allowed confident assignment of subdomains in a MACPF pore assembly. The major conformational changes in PlyB are a ∼70° opening of the bent and distorted central β-sheet of the MACPF domain, accompanied by extrusion and refolding of two α-helical regions into transmembrane β-hairpins (TMH1 and TMH2. We determined the structures of three different disulphide bond-trapped prepore intermediates. Analysis of these data by molecular modelling and flexible fitting allows us to generate a potential trajectory of β-sheet unbending. The results suggest that MACPF conformational change is triggered through disruption of the interface between a conserved helix-turn-helix motif and the top of TMH2. Following their release we propose that the transmembrane regions assemble into β-hairpins via top down zippering of backbone hydrogen bonds to form the membrane-inserted β-barrel. The intermediate structures of the MACPF domain during refolding into the β-barrel pore establish a structural paradigm for the transition from soluble monomer to pore, which may be conserved across the whole superfamily. The TMH2 region is critical for the release of

  17. Conformational changes during pore formation by the perforin-related protein pleurotolysin.

    Science.gov (United States)

    Lukoyanova, Natalya; Kondos, Stephanie C; Farabella, Irene; Law, Ruby H P; Reboul, Cyril F; Caradoc-Davies, Tom T; Spicer, Bradley A; Kleifeld, Oded; Traore, Daouda A K; Ekkel, Susan M; Voskoboinik, Ilia; Trapani, Joseph A; Hatfaludi, Tamas; Oliver, Katherine; Hotze, Eileen M; Tweten, Rodney K; Whisstock, James C; Topf, Maya; Saibil, Helen R; Dunstone, Michelle A

    2015-02-01

    Membrane attack complex/perforin-like (MACPF) proteins comprise the largest superfamily of pore-forming proteins, playing crucial roles in immunity and pathogenesis. Soluble monomers assemble into large transmembrane pores via conformational transitions that remain to be structurally and mechanistically characterised. Here we present an 11 Å resolution cryo-electron microscopy (cryo-EM) structure of the two-part, fungal toxin Pleurotolysin (Ply), together with crystal structures of both components (the lipid binding PlyA protein and the pore-forming MACPF component PlyB). These data reveal a 13-fold pore 80 Å in diameter and 100 Å in height, with each subunit comprised of a PlyB molecule atop a membrane bound dimer of PlyA. The resolution of the EM map, together with biophysical and computational experiments, allowed confident assignment of subdomains in a MACPF pore assembly. The major conformational changes in PlyB are a ∼70° opening of the bent and distorted central β-sheet of the MACPF domain, accompanied by extrusion and refolding of two α-helical regions into transmembrane β-hairpins (TMH1 and TMH2). We determined the structures of three different disulphide bond-trapped prepore intermediates. Analysis of these data by molecular modelling and flexible fitting allows us to generate a potential trajectory of β-sheet unbending. The results suggest that MACPF conformational change is triggered through disruption of the interface between a conserved helix-turn-helix motif and the top of TMH2. Following their release we propose that the transmembrane regions assemble into β-hairpins via top down zippering of backbone hydrogen bonds to form the membrane-inserted β-barrel. The intermediate structures of the MACPF domain during refolding into the β-barrel pore establish a structural paradigm for the transition from soluble monomer to pore, which may be conserved across the whole superfamily. The TMH2 region is critical for the release of both TMH clusters

  18. Structure of a membrane-attack complex/perforin (MACPF) family protein from the human gut symbiont Bacteroides thetaiotaomicron.

    Science.gov (United States)

    Xu, Qingping; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L; Bakolitsa, Constantina; Cai, Xiaohui; Carlton, Dennis; Chen, Connie; Chiu, Hsiu Ju; Clayton, Thomas; Das, Debanu; Deller, Marc C; Duan, Lian; Ellrott, Kyle; Farr, Carol L; Feuerhelm, Julie; Grant, Joanna C; Grzechnik, Anna; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K; Klock, Heath E; Knuth, Mark W; Kozbial, Piotr; Krishna, S Sri; Kumar, Abhinav; Lam, Winnie W; Marciano, David; Miller, Mitchell D; Morse, Andrew T; Nigoghossian, Edward; Nopakun, Amanda; Okach, Linda; Puckett, Christina; Reyes, Ron; Tien, Henry J; Trame, Christine B; van den Bedem, Henry; Weekes, Dana; Wooten, Tiffany; Yeh, Andrew; Zhou, Jiadong; Hodgson, Keith O; Wooley, John; Elsliger, Marc André; Deacon, Ashley M; Godzik, Adam; Lesley, Scott A; Wilson, Ian A

    2010-10-01

    Membrane-attack complex/perforin (MACPF) proteins are transmembrane pore-forming proteins that are important in both human immunity and the virulence of pathogens. Bacterial MACPFs are found in diverse bacterial species, including most human gut-associated Bacteroides species. The crystal structure of a bacterial MACPF-domain-containing protein BT_3439 (Bth-MACPF) from B. thetaiotaomicron, a predominant member of the mammalian intestinal microbiota, has been determined. Bth-MACPF contains a membrane-attack complex/perforin (MACPF) domain and two novel C-terminal domains that resemble ribonuclease H and interleukin 8, respectively. The entire protein adopts a flat crescent shape, characteristic of other MACPF proteins, that may be important for oligomerization. This Bth-MACPF structure provides new features and insights not observed in two previous MACPF structures. Genomic context analysis infers that Bth-MACPF may be involved in a novel protein-transport or nutrient-uptake system, suggesting an important role for these MACPF proteins, which were likely to have been inherited from eukaryotes via horizontal gene transfer, in the adaptation of commensal bacteria to the host environment.

  19. An NK Cell Perforin Response Elicited via IL-18 Controls Mucosal Inflammation Kinetics during Salmonella Gut Infection.

    Directory of Open Access Journals (Sweden)

    Anna A Müller

    2016-06-01

    Full Text Available Salmonella Typhimurium (S.Tm is a common cause of self-limiting diarrhea. The mucosal inflammation is thought to arise from a standoff between the pathogen's virulence factors and the host's mucosal innate immune defenses, particularly the mucosal NAIP/NLRC4 inflammasome. However, it had remained unclear how this switches the gut from homeostasis to inflammation. This was studied using the streptomycin mouse model. S.Tm infections in knockout mice, cytokine inhibition and -injection experiments revealed that caspase-1 (not -11 dependent IL-18 is pivotal for inducing acute inflammation. IL-18 boosted NK cell chemoattractants and enhanced the NK cells' migratory capacity, thus promoting mucosal accumulation of mature, activated NK cells. NK cell depletion and Prf-/- ablation (but not granulocyte-depletion or T-cell deficiency delayed tissue inflammation. Our data suggest an NK cell perforin response as one limiting factor in mounting gut mucosal inflammation. Thus, IL-18-elicited NK cell perforin responses seem to be critical for coordinating mucosal inflammation during early infection, when S.Tm strongly relies on virulence factors detectable by the inflammasome. This may have broad relevance for mucosal defense against microbial pathogens.

  20. An NK Cell Perforin Response Elicited via IL-18 Controls Mucosal Inflammation Kinetics during Salmonella Gut Infection.

    Science.gov (United States)

    Müller, Anna A; Dolowschiak, Tamas; Sellin, Mikael E; Felmy, Boas; Verbree, Carolin; Gadient, Sandra; Westermann, Alexander J; Vogel, Jörg; LeibundGut-Landmann, Salome; Hardt, Wolf-Dietrich

    2016-06-01

    Salmonella Typhimurium (S.Tm) is a common cause of self-limiting diarrhea. The mucosal inflammation is thought to arise from a standoff between the pathogen's virulence factors and the host's mucosal innate immune defenses, particularly the mucosal NAIP/NLRC4 inflammasome. However, it had remained unclear how this switches the gut from homeostasis to inflammation. This was studied using the streptomycin mouse model. S.Tm infections in knockout mice, cytokine inhibition and -injection experiments revealed that caspase-1 (not -11) dependent IL-18 is pivotal for inducing acute inflammation. IL-18 boosted NK cell chemoattractants and enhanced the NK cells' migratory capacity, thus promoting mucosal accumulation of mature, activated NK cells. NK cell depletion and Prf-/- ablation (but not granulocyte-depletion or T-cell deficiency) delayed tissue inflammation. Our data suggest an NK cell perforin response as one limiting factor in mounting gut mucosal inflammation. Thus, IL-18-elicited NK cell perforin responses seem to be critical for coordinating mucosal inflammation during early infection, when S.Tm strongly relies on virulence factors detectable by the inflammasome. This may have broad relevance for mucosal defense against microbial pathogens.

  1. DNA and Modified Vaccinia Virus Ankara Vaccines Encoding Multiple Cytotoxic and Helper T-Lymphocyte Epitopes of Human Immunodeficiency Virus Type 1 (HIV-1) Are Safe but Weakly Immunogenic in HIV-1-Uninfected, Vaccinia Virus-Naive Adults

    Science.gov (United States)

    Newman, Mark J.; deCamp, Allan; Hay, Christine Mhorag; De Rosa, Stephen C.; Noonan, Elizabeth; Livingston, Brian D.; Fuchs, Jonathan D.; Kalams, Spyros A.; Cassis-Ghavami, Farah L.

    2012-01-01

    We evaluated a DNA plasmid-vectored vaccine and a recombinant modified vaccinia virus Ankara vaccine (MVA-mBN32), each encoding cytotoxic and helper T-lymphocyte epitopes of human immunodeficiency virus type 1 (HIV-1) in a randomized, double-blinded, placebo-controlled trial in 36 HIV-1-uninfected adults using a heterologous prime-boost schedule. HIV-1-specific cellular immune responses, measured as interleukin-2 and/or gamma interferon production, were induced in 1 (4%) of 28 subjects after the first MVA-mBN32 immunization and in 3 (12%) of 25 subjects after the second MVA-mBN32 immunization. Among these responders, polyfunctional T-cell responses, including the production of tumor necrosis factor alpha and perforin, were detected. Vaccinia virus-specific antibodies were induced to the MVA vector in 27 (93%) of 29 and 26 (93%) of 28 subjects after the first and second immunizations with MVA-mBN32. These peptide-based vaccines were safe but were ineffective at inducing HIV-1-specific immune responses and induced much weaker responses than MVA vaccines expressing the entire open reading frames of HIV-1 proteins. PMID:22398243

  2. Cyclosporin A suppresses the expression of the interleukin 2 gene by inhibiting the binding of lymphocyte-specific factors to the IL-2 enhancer.

    OpenAIRE

    Randak, C; Brabletz, T; Hergenröther, M; Sobotta, I; Serfling, E

    1990-01-01

    Cyclosporin A (CsA), a powerful immunosuppressive drug, inhibits the synthesis of lymphokines in T lymphocytes at the level of gene transcription. Using protein extracts from El4 lymphoma cells we show that the binding of lymphocyte-specific factors interacting with the two so-called purine boxes (Pu-boxes) of the interleukin 2 (IL-2) enhancer are missing in CsA-treated cells. The CsA-sensitive factors are newly synthesized upon induction. The most prominent factor consists of 45 kd polypepti...

  3. Domain analyses of the Runx1 transcription factor responsible for modulating T-cell receptor-β/CD4 and interleukin-4/interferon-γ expression in CD4+ peripheral T lymphocytes

    Science.gov (United States)

    Uchino, Ryuji

    2009-01-01

    The Runx1 transcription factor is one of the master regulators of T-lymphocyte differentiation. There have been several reports trying to assign a domain within the Runx1 protein that is responsible for gene expression in thymocytes. The Runx1 domains involved in regulating the expression of several genes in peripheral CD4+ T cells were analysed. It was observed that Runx1 over-expression enhanced the surface expression of CD4 and CD69 molecules via its activation domain and VWRPY domain, and decreased that of T-cell receptor-β via its activation domain. Runx1 over-expression enhanced interferon-γ expression via its activation and VWRPY domains, and abolished interleukin-4 expression through its activation domain. Transduction of Runx1 did not down-regulate CD4 expression until 72 hr of culture, but the repression of CD4 expression became evident after 96 hr. The main region responsible for repressing CD4 expression was the inhibitory domain of Runx1. Taken together, these results lead to a proposal that the regions in Runx1 responsible for modulating gene expression are distinct in thymocytes and in peripheral CD4+ T cells. PMID:19689732

  4. SAMHD1 restricts HIV-1 infection in dendritic cells (DCs by dNTP depletion, but its expression in DCs and primary CD4+ T-lymphocytes cannot be upregulated by interferons

    Directory of Open Access Journals (Sweden)

    St Gelais Corine

    2012-12-01

    Full Text Available Abstract Background SAMHD1 is an HIV-1 restriction factor in non-dividing monocytes, dendritic cells (DCs, macrophages, and resting CD4+ T-cells. Acting as a deoxynucleoside triphosphate (dNTP triphosphohydrolase, SAMHD1 hydrolyzes dNTPs and restricts HIV-1 infection in macrophages and resting CD4+ T-cells by decreasing the intracellular dNTP pool. However, the intracellular dNTP pool in DCs and its regulation by SAMHD1 remain unclear. SAMHD1 has been reported as a type I interferon (IFN-inducible protein, but whether type I IFNs upregulate SAMHD1 expression in primary DCs and CD4+ T-lymphocytes is unknown. Results Here, we report that SAMHD1 significantly blocked single-cycle and replication-competent HIV-1 infection of DCs by decreasing the intracellular dNTP pool and thereby limiting the accumulation of HIV-1 late reverse transcription products. Type I IFN treatment did not upregulate endogenous SAMHD1 expression in primary DCs or CD4+ T-lymphocytes, but did in HEK 293T and HeLa cell lines. When SAMHD1 was over-expressed in these two cell lines to achieve higher levels than that in DCs, no HIV-1 restriction was observed despite partially reducing the intracellular dNTP pool. Conclusions Our results suggest that SAMHD1-mediated reduction of the intracellular dNTP pool in DCs is a common mechanism of HIV-1 restriction in myeloid cells. Endogenous expression of SAMHD1 in primary DCs or CD4+ T-lymphocytes is not upregulated by type I IFNs.

  5. Inactivation of TP53 correlates with disease progression and low miR-34a expression in previously treated chronic lymphocytic leukemia patients

    DEFF Research Database (Denmark)

    Dufour, Annika; Palermo, Giuseppe; Zellmeier, Evelyn;

    2013-01-01

    in a large cohort of previously treated and relapsed CLL patients. Here, we present the results of TP53 gene sequencing and fluorescence in situ hybridization for del17p in a phase 3 clinical trial (REACH [Rituximab in the Study of Relapsed Chronic Lymphocytic Leukemia]). Of the 457 patients, 52 had TP53...

  6. Chronic Brucellosis Patients Retain Low Frequency of CD4+ T-Lymphocytes Expressing CD25 and CD28 after Escherichia coli LPS Stimulation of PHA-Cultured PBMCs

    Directory of Open Access Journals (Sweden)

    Panagiotis Skendros

    2008-01-01

    Full Text Available Chronic brucellosis patients display a defective Th1 response to PHA. We have previously shown that heat-killed B. abortus (HKBA can downregulate the PHA-induced increase of CD4+/CD25+ and CD14+/CD80+ cells of brucellosis patients. In the present study, we investigate the effect of E. coli LPS, as a potent stimulant of monocytes and autologous T-lymphocytes, on the PHA-cultured PBMCs of the same groups of patients. Thirteen acute brucellosis (AB patients, 22 chronic brucellosis (CB patients, 11 “cured” subjects, and 15 healthy volunteers were studied. The percentage of CD4+/CD25+ and CD4+/CD28+ T-lymphocytes as well as CD14+/CD80+ monocytes were analyzed by flow cytometry after PBMCs culture with PHA plus E. coli LPS. A significant decrease in the percentage of CD4+/CD25+ and CD4+/CD28+ T-lymphocytes was observed in CB compared to AB. In HKBA cultures, compared to E. coli LPS-cultures, there was a significant reduction of CD4+/CD25+ T-lymphocytes in all groups and CD14+/CD80+ in patients groups. We suggest that Brucella can modulate host immune response, leading to T-cell anergy and chronic infection.

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

    Directory of Open Access Journals (Sweden)

    Thomas J. Sayers

    2012-05-01

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

  8. Cryptotanshinone induces inhibition of breast tumor growth by cytotoxic CD4+ T cells through the JAK2/STAT4/ perforin pathway.

    Science.gov (United States)

    Zhou, Jun; Xu, Xiao-Zhen; Hu, Yao-Ren; Hu, Ai-Rong; Zhu, Cheng-Liang; Gao, Guo-Sheng

    2014-01-01

    Cryptotanshinone (CPT), is a quinoid diterpene isolated from the root of the Asian medicinal plant, Salvia miotiorrhiza bunge. Numerous researchers have found that it could work as a potent antitumor agent to inhibit tumor growth in vitro, buith there has been much less emphasis on its in vivo role against breast tumors. Using a mouse tumor model of MCF7 cells, we showed that CPT strongly inhibited MCF7 cell growth in vivo with polarization of immune reactions toward Th1-type responses, stimulation of naive CD4+ T cell proliferation, and also increased IFN-γ and perforin production of CD4+ T cells in response to tumor-activated splenocytes. Furthermore, data revealed that the cytotoxic activity of CD4+ T cells induced by CPT was markedly abrogated by concanamycin A(CMA), a perforin inhibitor, but not IFN-γ Ab. On the other hand, after depletion of CD4+ T cells or blocked perforin with CMA in a tumor-bearing model, CPT could not effectively suppress tumor growth, but this phenomenon could be reversed by injecting naive CD4+ T cells. Thus, our results suggested that CPT mainly inhibited breast tumor growth through inducing cytotoxic CD4+ T cells to secrete perforin. We further found that CPT enhanced perforin production of CD4+ T cells by up-regulating JAK2 and STAT4 phosphorylation. These findings suggest a novel potential therapeutic role for CPT in tumor therapy, and demonstrate that CPT performs its antitumor functions through cytotoxic CD4+ T cells.

  9. Chronic lymphocytic leukemia nurse-like cells express hepatocyte growth factor receptor (c-MET) and indoleamine 2,3-dioxygenase and display features of immunosuppressive type 2 skewed macrophages

    Science.gov (United States)

    Giannoni, Paolo; Pietra, Gabriella; Travaini, Giorgia; Quarto, Rodolfo; Shyti, Genti; Benelli, Roberto; Ottaggio, Laura; Mingari, Maria Cristina; Zupo, Simona; Cutrona, Giovanna; Pierri, Ivana; Balleari, Enrico; Pattarozzi, Alessandra; Calvaruso, Marco; Tripodo, Claudio; Ferrarini, Manlio; de Totero, Daniela

    2014-01-01

    Hepatocyte growth factor, produced by stromal and follicular dendritic cells, and present at high concentrations in the sera of patients with chronic lymphocytic leukemia, prolongs the survival of leukemic B cells by interacting with their receptor, c-MET. It is, however, unknown whether hepatocyte growth factor influences microenvironmental cells, such as nurse-like cells, which deliver survival signals to the leukemic clone. We evaluated the expression of c-MET on nurse-like cells and monocytes from patients with chronic lymphocytic leukemia and searched for phenotypic/functional features supposed to be influenced by the hepatocyte growth factor/c-MET interaction. c-MET is expressed at high levels on nurse-like cells and at significantly higher levels than normal on monocytes from patients. Moreover, the hepatocyte growth factor/c-MET interaction activates STAT3TYR705 phosphorylation in nurse-like cells. Indoleamine 2,3-dioxygenase, an enzyme modulating T-cell proliferation and induced on normal monocytes after hepatocyte growth factor treatment, was detected together with interleukin-10 on nurse-like cells, and on freshly-prepared patients’ monocytes. Immunohistochemical/immunostaining analyses demonstrated the presence of c-MET+ and indoleamine 2,3-dioxygenase+ cells in lymph node biopsies, co-expressed with CD68 and vimentin. Furthermore nurse-like cells and chronic lymphocytic monocytes significantly inhibited T-cell proliferation, prevented by anti-transforming growth factor beta and interleukin-10 antibodies and indoleamine 2,3-dioxygenase inhibitors, and supported CD4+CD25high+/FOXP3+ T regulatory cell expansion. We suggest that nurse-like cells display features of immunosuppressive type 2 macrophages: higher hepatocyte growth factor levels, produced by leukemic or other microenvironmental surrounding cells, may cooperate to induce M2 polarization. Hepatocyte growth factor may thus have a dual pathophysiological role: directly through enhancement of

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

    Science.gov (United States)

    2014-10-30

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

  11. Prenatal ontogeny of lymphocyte subpopulations in pigs.

    Science.gov (United States)

    Sinkora, M; Sinkora, J; Reháková, Z; Splíchal, I; Yang, H; Parkhouse, R M; Trebichavsk, I

    1998-12-01

    Although porcine lymphocytes have been classified into numerous subpopulations in postnatal animals, little is known about the ontogeny of these complex cell subsets. Using double- and triple-colour flow cytometry (FCM), we investigated the surface phenotype of fetal lymphoid cells in the thymus, cord blood, spleen and mesenteric lymph nodes at different stages of gestation. It was found that the major lymphocyte subpopulations started to appear at the beginning of the second third of the gestation period, with B cells being the earliest lymphocyte subpopulation to appear in the periphery. The T-cell receptor (TCR) gamma delta+ cells were the earliest detectable T-cell subset, developing first in the thymus and subsequently arriving in the periphery. Later in ontogeny, however, the number of TCRalpha beta+ lymphocytes rapidly increased, becoming the predominant T cells both in the thymus and in the periphery. Cells with the phenotype of adult natural killer cells were also identified in pig fetuses, though their nature and functional roles remain to be investigated. In addition, CD2 was expressed on most B cells whilst very few CD4+ TCRalpha beta+ cells or CD2+ TCRgamma delta+ cells expressed CD8, suggesting that the expression of CD2 and CD8 may reflect the functional status of the cells in postnatal animals. Taken together, this study has provided a systematic analysis of fetal porcine lymphocyte subpopulations and may provide the base for studies to establish the physiological roles of these lymphocyte subsets.

  12. Analysis of mRNA expression for genes associated with regulatory T lymphocytes (CD25, FoxP3, CTLA4, and IDO) after experimental infection with bovine viral diarrhea virus of low or high virulence in beef calves.

    Science.gov (United States)

    Palomares, Roberto A; Hurley, David J; Woolums, Amelia R; Parrish, Jacqueline E; Brock, Kenny V

    2014-12-01

    Immunosuppression caused by bovine viral diarrhea virus (BVDV) has been associated with lymphocyte depletion, leukopenia and impairment of leukocyte function; however, no work has been done on the relationship between BVDV and regulatory T lymphocytes (Tregs). The objective of this study was to compare the mRNA expression of genes associated with Tregs (CD25, FoxP3, CTLA4, and IDO), after experimental infection of beef calves with low (LV) or high (HV) virulence BVDV. Thirty BVDV-naïve calves were randomly assigned to three groups. Calves were intra-nasally inoculated with LV (n=10, strain SD-1) or HV (n=10, strain 1373) BVDV or BVDV-free cell culture medium (control, n=10). Quantitative RT-PCR was used to determine the expression of target genes in tracheo-bronchial lymph nodes and spleen on day 5 post-infection. The mRNA expression of CD25 was up-regulated in tracheo-bronchial lymph nodes of LV (Pviral strains, or differences in viral infectivity of the host cells.

  13. B Lymphocyte Stimulator (BLyS) Is Expressed in Human Adipocytes In Vivo and Is Related to Obesity but Not to Insulin Resistance

    OpenAIRE

    Nike Müller; Schulte, Dominik M.; Susann Hillebrand; Kathrin Türk; Jochen Hampe; Clemens Schafmayer; Mario Brosch; Witigo von Schönfels; Markus Ahrens; Rainald Zeuner; Schröder, Johann O.; Matthias Blüher; Christian Gutschow; Sandra Freitag-Wolf; Marta Stelmach-Mardas

    2014-01-01

    Inflammation and metabolism have been shown to be evolutionary linked and increasing evidence exists that pro-inflammatory factors are involved in the pathogenesis of obesity and type 2 diabetes. Until now, most data suggest that within adipose tissue these factors are secreted by cells of the innate immune system, e. g. macrophages. In the present study we demonstrate that B lymphocyte stimulator (BLyS) is increased in human obesity. In contrast to several pro-inflammatory factors, we found ...

  14. Cyclosporin A suppresses the expression of the interleukin 2 gene by inhibiting the binding of lymphocyte-specific factors to the IL-2 enhancer.

    Science.gov (United States)

    Randak, C; Brabletz, T; Hergenröther, M; Sobotta, I; Serfling, E

    1990-08-01

    Cyclosporin A (CsA), a powerful immunosuppressive drug, inhibits the synthesis of lymphokines in T lymphocytes at the level of gene transcription. Using protein extracts from El4 lymphoma cells we show that the binding of lymphocyte-specific factors interacting with the two so-called purine boxes (Pu-boxes) of the interleukin 2 (IL-2) enhancer are missing in CsA-treated cells. The CsA-sensitive factors are newly synthesized upon induction. The most prominent factor consists of 45 kd polypeptides and contacts both Pu-boxes at the two central G residues within the identical core sequence AAGAGGAAAA. The CsA-mediated suppression of factor binding to the Pu-boxes correlates well with functional studies in which the inducible, T cell-restricted proto-enhancer activity of Pu-boxes was selectively repressed by CsA. These observations support the conclusion that the suppression of factor binding to the Pu-boxes by CsA impairs the activity of IL-2 and of further lymphokine genes, thereby inhibiting the synthesis of lymphokines in T lymphocytes.

  15. Activation of Natural Killer Cells in Patients with Chronic Bone and Joint Infection due to Staphylococci Expressing or Not the Small Colony Variant Phenotype

    Directory of Open Access Journals (Sweden)

    Sébastien Viel

    2014-01-01

    Full Text Available Chronic bone and joint infections (BJI are devastating diseases. Relapses are frequently observed, as some pathogens, especially staphylococci, can persist intracellularly by expressing a particular phenotype called small colony variant (SCV. As natural killer (NK cells are lymphocytes specialized in the killing of host cells infected by intracellular pathogens, we studied NK cells of patients with chronic BJI due to staphylococci expressing or not SCVs (10 patients in both groups. Controls were patients infected with other bacteria without detectable expression of SCVs, and healthy volunteers. NK cell phenotype was evaluated from PBMCs by flow cytometry. Degranulation capacity was evaluated after stimulation with K562 cells in vitro. We found that NK cells were activated in terms of CD69 expression, loss of CD16 and perforin, in all infected patients in comparison with healthy volunteers, independently of the SCV phenotype. Peripheral NK cells in patients with chronic BJI display signs of recent activation and degranulation in vivo in response to CD16-mediated signals, regardless of the type of bacteria involved. This could involve a universal capacity of isolates responsible for chronic BJI to produce undetectable SCVs in vivo, which might be a target of future intervention.

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

    Science.gov (United States)

    2013-08-20

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

  17. SECRETION OF IL-12, IL-27 BY MONONUCLEAR LEUKOCYTES AND EXPRESSION OF THEIR RECEPTORS ON T-LYMPHOCYTES IN CONDITIONS OF DIRECTED CELL INDUCTION IN VITRO IN PULMONARY TUBERCULOSIS

    Directory of Open Access Journals (Sweden)

    I. E. Esimova

    2014-01-01

    Full Text Available The article presents the results of studies concerning secretion of cytokines belonging to interleukin 12 family, i.e., IL-12р70, IL-12р40, IL-12р35 and IL-27 in mononuclear blood leukocytes, as well as expression of their specific receptors on T-lymphocytes from tuberculosis patients under the in vitro conditions of directed (resp., antigen- and cytokine-mediated cell induction. In patients with pulmonary TB, we have registered suppression of both spontaneous and BCG-induced secretion of IL-12р70 and IL- 12р35, along with overproduction of IL-12р40 and IL-27. Furthermore, a decrease in relative contents of СD3+gp130+,CD3+IL12Rβ2+ lymphocytes were revealed in the in vitro IL-12/IL-27 induction tests, as well as increased concentration of T-cells with high expression of the inhibitory molecule WSX-1 (CD3+WSX-1+hi. It was shown that the detectable changes are unidirectional in most cases, being dependent on clinical form of the disease and sensitivity of the etiological agent to anti-tuberculosis drugs.

  18. What Is Chronic Lymphocytic Leukemia?

    Science.gov (United States)

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

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

    Science.gov (United States)

    2013-07-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2014-01-01

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

  1. SHARPIN Regulates Uropod Detachment in Migrating Lymphocytes

    Directory of Open Access Journals (Sweden)

    Jeroen Pouwels

    2013-11-01

    Full Text Available SHARPIN-deficient mice display a multiorgan chronic inflammatory phenotype suggestive of altered leukocyte migration. We therefore studied the role of SHARPIN in lymphocyte adhesion, polarization, and migration. We found that SHARPIN localizes to the trailing edges (uropods of both mouse and human chemokine-activated lymphocytes migrating on intercellular adhesion molecule-1 (ICAM-1, which is one of the major endothelial ligands for migrating leukocytes. SHARPIN-deficient cells adhere better to ICAM-1 and show highly elongated tails when migrating. The increased tail lifetime in SHARPIN-deficient lymphocytes decreases the migration velocity. The adhesion, migration, and uropod defects in SHARPIN-deficient lymphocytes were rescued by reintroducing SHARPIN into the cells. Mechanistically, we show that SHARPIN interacts directly with lymphocyte-function-associated antigen-1 (LFA-1, a leukocyte counterreceptor for ICAM-1, and inhibits the expression of intermediate and high-affinity forms of LFA-1. Thus, SHARPIN controls lymphocyte migration by endogenously maintaining LFA-1 inactive to allow adjustable detachment of the uropods in polarized cells.

  2. Phenotypic and Functional Analysis of Porcine T Lymphocytes

    Institute of Scientific and Technical Information of China (English)

    李华; 陈应华

    2001-01-01

    Porcine and other higher mammals express clusters of differentiation (CD) antigens on the surface of T lymphocytes, such as CD2, CD3, CD4, CD8, etc. However, in porcine, a high percentage of the CD4+ CD8-T lymphocyte subpopulation exist in the peripheral blood and the ratio of the CD4+ and CD8+ T lymphocyte subpopulations is reversed. These differences bring new challenges to better understanding of the phenotype and function of porcine T lymphocytes in antigen recognition and immune response.

  3. Classical and alternative activation and metalloproteinase expression occurs in foam cell macrophages in male and female ApoE null mice in the absence of T- and B-lymphocytes

    Directory of Open Access Journals (Sweden)

    Elaine Mo Hayes

    2014-10-01

    Full Text Available Background: Rupture of advanced atherosclerotic plaques accounts for most life-threatening myocardial infarctions. Classical (M1 and alternative (M2 macrophage activation could promote atherosclerotic plaque progression and rupture by increasing production of proteases, including matrix metalloproteinases (MMPs. Lymphocyte-derived cytokines may be essential for generating M1 and M2 phenotypes in plaques, although this has not been rigorously tested until now.Methods and Results: We validated the expression of M1 markers (iNOS and COX-2 and M2 markers (arginase-1, Ym-1 and CD206 and then measured MMP mRNA levels in mouse macrophages during classical and alternative activation in vitro. We then compared mRNA expression of these genes ex vivo in foam cells from subcutaneous granulomas in fat-fed immune-competent ApoE knockout and immune-compromised ApoE/Rag-1 double knockout mice, which lack all T and B cells. Furthermore, we performed immunohistochemistry in subcutaneous granulomas and in aortic root and brachiocephalic artery atherosclerotic plaques to measure the extent of M1/M2 marker and MMP protein expression in vivo. Classical activation of mouse macrophages with bacterial lipopolysaccharide in vitro increased MMPs-13, -14 and -25 but decreased MMP-19 and TIMP-2 mRNA expressions. Alternative activation with IL-4 increased MMP-19 expression. Foam cells in subcutaneous granulomas expressed all M1/M2 markers and MMPs at ex vivo mRNA and in vivo protein levels, irrespective of Rag-1 genotype. There were also similar percentages of foam cell macrophages carrying M1/M2 markers and MMPs in atherosclerotic plaques from ApoE knockout and ApoE/Rag-1 double knockout mice. Conclusions: Classical and alternative activation leads to distinct MMP expression patterns in mouse macrophages in vitro. M1 and M2 polarization in vivo occurs in the absence of T and B lymphocytes in either granuloma or plaque foam cell macrophages.

  4. Alteration of peripheral blood lymphocyte subsets in acute pancreatitis

    Institute of Scientific and Technical Information of China (English)

    Miroslawa Pietruczuk; Milena I Dabrowska; Urszula Wereszczynska-Siemiatkowska; Andrzej Dabrowski

    2006-01-01

    AIM: To evaluate peripheral blood lymphocyte subsets in patients with acute pancreatitis (AP).METHODS: Twenty patients with mild AP (M-AP) and 15 with severe AP (S-AP) were included in our study. Peripheral blood lymphocytes were examined at d 1-3, 5,10 and 30 by means of flow cytometry.RESULTS: A significant depletion of circulating lymphocytes was found in AP. In the early AP, the magnitude of depletion was similar for T- and B- lymphocytes. In the late course of S-AP, B-lymphocytes were much more depleted than T-lymphocytes. At d 10, strong shift in the CD7+/CD19+ ratio implicating predominance of Tover B-lymphocytes in S-AP was found. Among T-lymphocytes, the significant depletion of the CD4+ population was observed in M-AP and S-AP, while CD8+ cells were in the normal range. Lymphocytes were found to strongly express activation markers: CD69, CD25, CD28,CD38 and CD122. Serum interleukin-2 (IL-2), IL-4, IL-5,IL-10, interferon-γ (IFN-γ) and tumor necrosis factor-α(TNF-α) levels were significantly increased in both forms of AP. The magnitude of elevation of cytokines known to be produced by Th2 was much higher than cytokines produced by Th1 cells.CONCLUSION: AP in humans is characterized by significant reduction of peripheral blood T- and B-lymphocytes.

  5. Acute Lymphocytic Leukemia

    Science.gov (United States)

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

  6. Synthetic consensus HIV-1 DNA induces potent cellular immune responses and synthesis of granzyme B, perforin in HIV infected individuals.

    Science.gov (United States)

    Morrow, Matthew P; Tebas, Pablo; Yan, Jian; Ramirez, Lorenzo; Slager, Anna; Kraynyak, Kim; Diehl, Malissa; Shah, Divya; Khan, Amir; Lee, Jessica; Boyer, Jean; Kim, J Joseph; Sardesai, Niranjan Y; Weiner, David B; Bagarazzi, Mark L

    2015-03-01

    This study evaluated the safety and immunogenicity of PENNVAX-B in 12 HIV infected individuals. PENNVAX-B is a combination of three optimized synthetic plasmids encoding for multiclade HIV Gag and Pol and a consensus CladeB Env delivered by electroporation. HIV infected individuals whose virus was effectively suppressed using highly active antiretroviral therapy (HAART) received PENNVAX-B DNA followed by electroporation with CELLECTRA-5P at study weeks 0, 4, 8, and 16. Local administration site and systemic reactions to PENNVAX-B were recorded after each treatment along with any adverse events. Pain of the treatment procedure was assessed using a Visual Analog Scale. Whole PBMCs were isolated for use in IFN ELISpot and Flow Cytometric assays. PENNVAX-B was generally safe and well tolerated. Overall, the four dose regimen was not associated with any serious adverse events or severe local or systemic reactions. A rise in antigen-specific SFU was detected in the INFγ ELISpot assay in all 12 participants. T cells from 8/12 participants loaded with both granzyme B and perforin in response to HIV antigen, an immune finding characteristic of long-term nonprogressors (LTNPs) and elite controllers (ECs). Thus administration of PENNVAX-B may prove useful adjunctive therapy to ART for treatment and control of HIV infection.

  7. Gamma/delta intraepithelial lymphocytes in the mouse small intestine.

    Science.gov (United States)

    Ogata, Masaki; Itoh, Tsunetoshi

    2016-09-01

    Although many studies of intraepithelial lymphocytes (IELs) have been reported, most of them have focused on αβ-IELs; little attention has been paid to γδ-IELs. The function of γδ-IELs remains largely unclear. In this article, we briefly review a number of reports on γδ-IELs, especially those in the small intestine, along with our recent studies. We found that γδ-IELs are the most abundant (comprising >70 % of the) IELs in the duodenum and the jejunum, implying that it is absolutely necessary to investigate the function(s) of γδ-IELs when attempting to delineate the in vivo defense system of the small intestine. Intraperitoneal injection of anti-CD3 mAb stimulated the γδ-IELs and caused rapid degranulation of them. Granzyme B released from their granules induced DNA fragmentation of duodenal and jejunal epithelial cells (paracrine) and of the IELs themselves (autocrine). However, perforin (Pfn) was not detected, and DNA fragmentation was induced even in Pfn-knockout mice; our system was therefore found to present a novel type of in vivo Pfn-independent DNA fragmentation. We can therefore consider γδ-IELs to be a novel type of large granular lymphocyte without Pfn. Fragmented DNA was repaired in the cells, indicating that DNA fragmentation alone cannot be regarded as an unambiguous marker of cell death or apoptosis. Finally, since the response was so rapid and achieved without the need for accessory cells, it seems that γδ-IELs respond readily to various stimuli, are activated only once, and die 2-3 days after activation in situ without leaving their site. Taken together, these results suggest that γδ-IELs are not involved in the recognition of specific antigen(s) and are not involved in the resulting specific killing or exclusion of the relevant antigen(s).

  8. Role of interferon-γ and cytotoxic T lymphocytes in intraocular tumor rejection.

    Science.gov (United States)

    Ligocki, Ann J; Brown, Joseph R; Niederkorn, Jerry Y

    2016-05-01

    The eye is normally an immunosuppressive environment. This condition is better known as immune privilege and protects the eye from immune-mediated inflammation of tissues that cannot regenerate. However, immune privilege creates a dilemma for the eye when intraocular neoplasms arise. In some cases, immune privilege is suspended, resulting in the immune rejection of intraocular tumors. This study employed a mouse model in which interferon-γ-dependent intraocular tumor rejection occurs. We tested the hypothesis that this rejection requires interferon-γ for the generation and functional capacity of cytotoxic T lymphocyte-mediated rejection of intraocular tumors. Tumors grew progressively in the eyes of interferon-γ knockout mice, even though the mice generated tumor-specific cytotoxic T lymphocyte responses in the periphery. However, interferon-γ knockout mice rejected tumors that were introduced into extraocular sites. Subcutaneous tumor immunization before intraocular challenge led to tumor rejection and preservation of the eye in wild-type mice. By contrast, tumors grew progressively in the eyes of interferon-γ knockout mice despite their ability to generate peripheral tumor-specific cytotoxic T lymphocytes as well as the capacity of CD8(+) T cells to enter the eye as shown by the presence of CD8 and perforin message and CD3(+)CD8(+) leukocytes within the tumor-bearing eye. We found that cytotoxic T lymphocytes generated in wild-type mice and adoptively transferred into interferon-γ knockout mice mediated the rejection of intraocular tumors in interferon-γ knockout hosts. The results indicate that interferon-γ is critical for the initial priming and differentiation of cytotoxic T lymphocytes residing in the periphery to produce the most effect antitumor function within the eye.

  9. Lymphocyte subpopulations in Sheehan's syndrome.

    Science.gov (United States)

    Atmaca, Hulusi; Araslı, Mehmet; Yazıcı, Zihni Acar; Armutçu, Ferah; Tekin, Ishak Özel

    2013-06-01

    The role of autoimmunity in the development of Sheehan's syndrome is obscure. There are a limited number of studies investigating the immunological alterations accompanying Sheehan's Syndrome. Our objective was to evaluate lymphocyte subsets in these patients. We conducted a cross-sectional clinical study. Cytofluorometry was used for the immunophenotyping of peripheral blood leukocytes from patients with Sheehan's syndrome followed up in the endocrine clinic during 2005-2009. Fifteen consecutive patients (mean age 61.6 ± 11.3, range 34-75 years) and 25 healthy controls (mean age 56.7 ± 10.6, range 34-80 years) were included. There was no statistically significant difference between the groups in terms of mean age. The percentages of CD19(+), CD16(+)/56(+), CD8(+)28(-), γδTCR(+), CD8(+); the total lymphocyte counts; and the ratio of CD8(+)28(-)/CD8(+)28(+) were similar (p > 0.05) between patients and controls. Whereas the leucocyte counts (p = 0.003), the percentage of CD3 (+) DR (+) (p Sheehan's syndrome compared to healthy controls. There was a positive correlation between the duration of illness and the percentage of CD3(+)DR(+) (r = 0.53, p = 0.03) expression. Some peripheral lymphocyte cell subsets show marked variation in patients with Sheehan's syndrome in comparison to matched healthy subjects, which may have implications for altered immune regulation in these patients. High CD3 (+) DR (+) expression that correlates with the duration of illness in Sheehan's patients is suggestive of an ongoing inflammation accompanying the slow progression of pituitary dysfunction in Sheehan's syndrome. It is not clear if these cellular alterations contribute to the cause or consequence of pituitary deficiency in Sheehan's syndrome.

  10. Clinical-Grade Human Multipotent Adult Progenitor Cells Block CD8+ Cytotoxic T Lymphocytes.

    Science.gov (United States)

    Plessers, Jeroen; Dekimpe, Emily; Van Woensel, Matthias; Roobrouck, Valerie D; Bullens, Dominique M; Pinxteren, Jef; Verfaillie, Catherine M; Van Gool, Stefaan W

    2016-12-01

    : 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

  11. Effect of Leflunomide on the Abnormal Expression of Lipid Rafts and F-Actin in B Lymphocytes from Patients with Systemic Lupus Erythematosus

    Directory of Open Access Journals (Sweden)

    Guang Fu Dong

    2015-01-01

    Full Text Available Purposes. To investigate the possible changes in B cell subsets and in B cell expression patterns of lipid rafts (LRs and F-actin in patients with SLE and whether leflunomide treatment may have effect on these changes. Methods. The B cell subsets and LRs expression were determined by flow cytometry and confocal microscopy, and F-actin expression was examined by confocal microscopy. Results. CD27+IgD+ B cell subsets were significantly decreased while CD38+CD95+ B cell subsets increased in SLE patients. The LRs levels of B cells were remarkably increased and positively correlated with SLEDAI and anti-dsDNA titer in SLE patients. The expression level of LRs was significantly higher in CD38+ B cells than CD38− B cells and negatively correlated with C3 levels. The increased expression of LRs was associated with reduced expression of F-actin in the B cells from active SLE patients. Furthermore, in vitro treatment of the cells with A771726 reduced the expression level of LRs, attenuated the overaggregation of LRs, and normalized the distribution of F-actin. Conclusions. There were abnormalities in B cell subsets and LRs and F-actin expression of B cell from SLE patients. Modulation of B cell expression of LRs and F-actin by LEF could be a potential therapeutic target for SLE.

  12. Genetic immunization against cervical carcinoma : induction of cytotoxic T lymphocyte activity with a recombinant alphavirus vector expressing human papillomavirus type 16 E6 and E7

    NARCIS (Netherlands)

    Daemen, T; Pries, F; Bungener, L; Kraak, M; Regts, J; Wilschut, J

    2000-01-01

    infection of genital epithelial cells with human papillomavirus (HPV) types 16 and 18 is closely associated with the development of cervical carcinoma. The transforming potential of these high-risk HPVs depends on the expression of the E6 and E7 early viral gene products. Since the expression of E6

  13. Genetic immunization against cervical carcinoma : induction of cytotoxic T lymphocyte activity with a recombinant alphavirus vector expressing human papillomavirus type 16 E6 and E7

    NARCIS (Netherlands)

    Daemen, T; Pries, F; Bungener, L; Kraak, M; Regts, J; Wilschut, J

    2000-01-01

    infection of genital epithelial cells with human papillomavirus (HPV) types 16 and 18 is closely associated with the development of cervical carcinoma. The transforming potential of these high-risk HPVs depends on the expression of the E6 and E7 early viral gene products. Since the expression of E6

  14. Characterization of two heat shock proteins (Hsp70/Hsc70) from grass carp (Ctenopharyngodon idella): evidence for their differential gene expression, protein synthesis and secretion in LPS-challenged peripheral blood lymphocytes.

    Science.gov (United States)

    Zhang, Anying; Zhou, Xiaofei; Wang, Xinyan; Zhou, Hong

    2011-06-01

    Two cDNAs, encoding the stress-inducible 70-kDa heat shock protein (Hsp70) and the constitutively expressed 70-kDa heat shock cognate protein (Hsc70), were isolated from grass carp. The Hsp70 and Hsc70 cDNAs were 2250 bp and 2449 bp in length and contained 1932 bp and 1953 bp open reading frames, respectively. Tissue distribution results showed that Hsp70/Hsc70 was highly expressed in gill, kidney, head kidney and peripheral blood lymphocytes (PBLs). Using grass carp PBLs as a cell model, effects of lipopolysaccharide (LPS) on the mRNA and protein levels of Hsp70/Hsc70 were examined. In this case, LPS increased the mRNA expression of Hsp70 in a time- and dose-dependent manner, but had no effect on Hsc70 mRNA expression. In agreement with this, LPS elevated the intracellular Hsp70 markedly, but not the Hsc70 protein levels in parallel experiments. Furthermore, Hsp70 protein was also detected in culture medium. Moreover, inhibition of LPS on Hsp70 release in a time-dependent manner was observed, indicating that there may be a dynamic balance between Hsp70 stores and Hsp70 release in grass carp PBLs following exposure to LPS. Taken together, these results not only shed new insights into the different regulations of LPS on Hsp70/Hsc70 gene expression, protein synthesis and release, but also provide a basis for further study on the functional role of Hsp70 in fish immune response.

  15. Expression of Fas and Fas ligand in infiltrating lymphocytes in patients with oral lichen planus%口腔扁平苔藓固有层淋巴细胞中Fas及Fas配体的表达变化

    Institute of Scientific and Technical Information of China (English)

    雷蕾; 谭为霞; 周序珑; 郑佩娥

    2010-01-01

    目的 检测Fas及Fas配体(Fas ligand,FasL)在口腔扁平苔藓(oral lichen planus,OLP)固有层淋巴细胞中的蛋白表达,探讨Fas、FasL和活化诱导的细胞死亡(activation-induced cell death,AICD)与OLP发病的关系.方法 采用脱氧核糖核苷酸末端转移酶介导的原位缺口末端标记法检测31例OLP和10例正常口腔黏膜(normal oral mucosa,NOM)中淋巴细胞凋亡情况;分别使用免疫组化法检测组织总淋巴细胞及CD8~+、CD4~+T细胞Fas、FasL的表达.结果 OLP与NOM固有层淋巴细胞凋亡率[分别为(1.9±1.8)%、(11.5±9.0)%]差异有统计学意义(P=0.013).OLP淋巴细胞Fas、FasL表达与NOM组相比明显增强[阳性表达率分别为52%(16/31)、71%(22/31),P值分别为0.005、0.000].OLP中CD8~+与Fas~+细胞双阳性表达率为10%,与NOM组相比无明显升高(P=0.313),而CD8~+与FasL~+细胞双阳性表达率[58%(3/31)]显著升高(P=0.002).CD4~+与Fas~+细胞双阳性表达率为35%(11/31),较NOM组显著升高(P=0.031),其中网纹型的表达明显升高(阳性表达为8/19,P=0.019),但糜烂、萎缩型的表达无显著升高(阳性表达为3/12,P=0.097).CD4~+与FasL~+表达率为16%(5/31),与NOM组相比无明显增强(P=0.182).结论 OLP淋巴细胞凋亡低下;OLP中T细胞亚群Fas、FasL表达不均衡,CD8~+细胞和萎缩、糜烂型OLP中CD4~+细胞可能逃逸AICD,与炎症的持续和进展有关;网纹型OLP中部分CD4~+T细胞可能经历AICD.%Objective To examine the expression of Fas and Fas ligand(FasL)in T lymphocytes of oral lichen planus(OLP)and the effects of Fas,FasL and activation-induced cell death(AICD)on OLP.Methods The oral mucosa samples from patients with OLP and nomal oral mucosa were assessed by in situ terminal deoxynucleotidyl transferase(TdT)-mediated deoxvuridine-5'-triphosphate(dUTP)-biotin nick endlabelling(TUNEL)assay for nucleosomal DNA fragmentation of apoptotic cells in infiltrating lymphocytes.Immunohistochemcial technique was used for detection of

  16. Concomitant heterochromatinisation and down-regulation of gene expression unveils epigenetic silencing of RELB in an aggressive subset of chronic lymphocytic leukemia in males

    Directory of Open Access Journals (Sweden)

    Marteau Jean-Brice

    2010-11-01

    Full Text Available Abstract Background The sensitivity of chronic lymphocytic leukemia (CLL cells to current treatments, both in vitro and in vivo, relies on their ability to activate apoptotic death. CLL cells resistant to DNA damage-induced apoptosis display deregulation of a specific set of genes. Methods Microarray hybridization (Human GeneChip, Affymetrix, immunofluorescent in situ labeling coupled with video-microscopy recording/analyses, chromatin-immunoprecipitation (ChIP, polymerase chain reactions (PCR, real-time quantitative PCR (RT-QPCR and bisulfite genome sequencing were the main methods applied. Statistical analyses were performed by applying GCRMA and SAM analysis (microarray data and Student's t-test or Mann & Whitney's U-test. Results Herein we show that, remarkably, in a resistant male CLL cells the vast majority of genes were down-regulated compared with sensitive cells, whereas this was not the case in cells derived from females. This gene down-regulation was found to be associated with an overall gain of heterochromatin as evidenced by immunofluorescent labeling of heterochromatin protein 1α (HP-1, trimethylated histone 3 lysine 9 (3metH3K9, and 5-methylcytidine (5metC. Notably, 17 genes were found to be commonly deregulated in resistant male and female cell samples. Among these, RELB was identified as a discriminatory candidate gene repressed in the male and upregulated in the female resistant cells. Conclusion The molecular defects in the silencing of RELB involve an increase in H3K9- but not CpG-island methylation in the promoter regions. Increase in acetyl-H3 in resistant female but not male CLL samples as well as a decrease of total cellular level of RelB after an inhibition of histone deacetylase (HDAC by trichostatin A (TSA, further emphasize the role of epigenetic modifications which could discriminate two CLL subsets. Together, these results highlighted the epigenetic RELB silencing as a new marker of the progressive disease in

  17. Expression of B lymphocyte activating factor in decidual and chorionic membranes of women with early spontaneous abortion and its significance%胚胎停育患者蜕膜和绒毛组织中B淋巴细胞刺激因子的表达及意义

    Institute of Scientific and Technical Information of China (English)

    高艳萍; 郝冬梅

    2011-01-01

    目的 探讨胚胎停育患者蜕膜和绒毛组织中B淋巴细胞刺激因子(BAFF)的表达与胚胎停育的关系.方法 随机选择早孕胚胎停育患者30例作为实验组,正常早孕人工流产者30例为对照组.用免疫组化(二步法)法测定蜕膜及绒毛中BAFF的表达情况.结果 BAFF在所有标本中都有表达,但胚胎停育组蜕膜中BAFF的表达均低于对照组(P<0.05),胚胎停育组蜕膜血管内皮中则无表达.结论 BAFF与胚胎停育有一定的关联,可能是导致胚胎停育的原因之一.%Objective To study the correlation between the expression of B lymphocyte activating factor in decidual and chorionic membranes and early spontaneous abortion. Methods Thirty women with early spontaneous abortion served as an experimental group and 30 women who had artificial abortion for normal early pregnancy served as a control group. Expression of B lymphocyte activating factor in decidual and chorionic membranes was detected by immunohistochemistry. Results B lymphocyte activating factor was expressed in all samples of decidual and chorionic membranes. However, its expression level in decidual membrane was lower in experimental group than in control group(P<0.05). B lymphocyte activating factor was not expressed in angioendothelium of experimental group. Conclusion B lymphocyte activating factor is associated with early spontaneous abortion and may be one of the reasons for early spontaneous abortion.

  18. Activation-induced expression of thymic shared antigen-1 on T lymphocytes and its inhibitory role for TCR-mediated IL-2 production.

    Science.gov (United States)

    Kosugi, A; Saitoh, S; Narumiya, S; Miyake, K; Hamaoka, T

    1994-12-01

    We have produced a hamster mAb, PRST1, which reacts with thymic shared Ag-1 (TSA-1), a product of the Ly6 gene family. By cross-blocking experiments, we found that TSA-1 is identical to stem cell Ag-2 (Sca-2). Using PRST1, the changes of TSA-1/Sca-2 expression on mature T cells during the activation process were analyzed. Although freshly isolated T cells did not express detectable TSA-1 on their cell surface, in vitro stimulation of T cells with concanavalin A induced a marked increase of surface TSA-1 expression. The increased expression of TSA-1 on T cells was detected from 12 h after stimulation and was associated with the increase of TSA-1 mRNA. In vivo injection of mice with staphylococcal enterotoxin B (SEB) resulted in the enhanced TSA-1 expression in splenic V beta 8+ T cells. This antigen-specific induction of TSA-1 expression in vivo preceded a detectable increase in numbers of V beta 8- T cells after SEB injection. Functionally, whereas anti-TSA-1 mAb was not mitogenic to T cells, it inhibited anti-CD3-induced IL-2 production by T cell hybridomas. These results indicate that TSA-1/Sca-2 is a unique marker for T cell activation and a signal through this molecule may have a negative feedback role to limit IL-2 production from activated T cells stimulated through the TCR.

  19. Expression profiles of the immune genes CD4, CD8β, IFNγ, IL-4, IL-6 and IL-10 in mitogen-stimulated koala lymphocytes (Phascolarctos cinereus by qRT-PCR

    Directory of Open Access Journals (Sweden)

    Iona E. Maher

    2014-03-01

    Full Text Available Investigation of the immune response of the koala (Phascolarctos cinereus is needed urgently, but has been limited by scarcity of species-specific reagents and methods for this unique and divergent marsupial. Infectious disease is an important threat to wild populations of koalas; the most widespread and important of these is Chlamydial disease, caused by Chlamydia pecorum and Chlamydia pneumoniae. In addition, koala retrovirus (KoRV, which is of 100% prevalence in northern Australia, has been proposed as an important agent of immune suppression that could explain the koala’s susceptibility to disease. The correct balance of T regulatory, T helper 1 (Th1 and Th2 lymphocyte responses are important to an individual’s susceptibility or resistance to chlamydial infection. The ability to study chlamydial or KoRV pathogenesis, effects of environmental stressors on immunity, and the response of koalas to vaccines under development, by examining the koala’s adaptive response to natural infection or in-vitro stimulation, has been limited to date by a paucity of species- specific reagents. In this study we have used cytokine sequences from four marsupial genomes to identify mRNA sequences for key T regulatory, Th1 and Th2 cytokines interleukin 4 (IL-4, interleukin 6 (IL-6, interleukin 10 (IL-10 and interferon gamma (IFNγ along with CD4 and CD8β. The koala sequences used for primer design showed >58% homology with grey short-tailed opossum, >71% with tammar wallaby and 78% with Tasmanian devil amino acid sequences. We report the development of real-time RT-PCR assays to measure the expression of these genes in unstimulated cells and after three common mitogen stimulation protocols (phorbol myristate acetate/ionomycin, phorbol myristate acetate/phytohemagglutinin and concanavalin A. Phorbol myristate acetate/ionomycin was found to be the most effective mitogen to up-regulate the production of IL-4, IL-10 and IFNγ. IL-6 production was not

  20. Cyclophosphamide, Alvocidib, and Rituximab in Treating Patients With High Risk B-Cell Chronic Lymphocytic Leukemia or Small Lymphocytic Lymphoma

    Science.gov (United States)

    2015-11-10

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

  1. Interleukin-21 induces proliferation and modulates receptor expression and effector function in canine natural killer cells.

    Science.gov (United States)

    Shin, Dong-Jun; Lee, Soo-Hyeon; Park, Ji-Yun; Kim, Ju-Sun; Lee, Je-Jung; Suh, Guk-Hyun; Lee, Youn-Kyung; Cho, Duck; Kim, Sang-Ki

    2015-05-15

    Interleukin (IL)-21 is an important modulator of natural killer (NK) cell function. However, little is known about IL-21 function in canine NK cells because the phenotype of these cells remains undefined. In this study, we selectively expanded non-B and non-T large granular NK lymphocytes (CD3(-)CD21(-)CD5(-)CD4(-)TCRαβ(-)TCRγδ(-)) ex vivo from the peripheral blood mononuclear cells (PBMCs) of healthy dogs using a combination of IL-2, IL-15, and IL-21 in the presence of 100 Gy-irradiated K562 cells. We investigated the effects of varying the duration and timing of IL-21 treatment on stimulation of proliferation, expression of NK-related receptors, anti-tumor activity and production of interferon (IFN)-γ. The expanded NK cells in each treatment group became enlarged and highly granular after 21 days in culture. NK cells proliferated rapidly in response to activation by IL-21 for 3 weeks, and IL-21 was able to induce changes in the mRNA expression of NK cell-related receptors and enhance the effector function of NK cells in perforin- and granzyme-B-dependent manners. The duration, frequency and timing of IL-21 stimulation during culture affected the rate of proliferation, patterns of receptor expression, cytokine production, and anti-tumor activity. The optimal conditions for maximizing the IL-21-induced proliferation and effector function of NK cells in the presence of IL-2 and IL-15 were seen in cells treated with IL-21 for the first 7 days of culture but without any further IL-21 stimulation other than an additional 2-day treatment prior to harvesting on day 21. The results of this study suggest that synergistic interactions of IL-21 with IL-2 and IL-15 play an important role in the proliferation, receptor expression, and effector function of canine NK cells. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Treatment with 5-Aza-2'-Deoxycytidine Induces Expression of NY-ESO-1 and Facilitates Cytotoxic T Lymphocyte-Mediated Tumor Cell Killing.

    Science.gov (United States)

    Klar, Agnes S; Gopinadh, Jakka; Kleber, Sascha; Wadle, Andreas; Renner, Christoph

    2015-01-01

    NY-ESO-1 belongs to the cancer/testis antigen (CTA) family and represents an attractive target for cancer immunotherapy. Its expression is induced in a variety of solid tumors via DNA demethylation of the promoter of CpG islands. However, NY-ESO-1 expression is usually very low or absent in some tumors such as breast cancer or multiple myeloma. Therefore, we established an optimized in vitro treatment protocol for up-regulation of NY-ESO-1 expression by tumor cells using the hypomethylating agent 5-aza-2'-deoxycytidine (DAC). We demonstrated de novo induction of NY-ESO-1 in MCF7 breast cancer cells and significantly increased expression in U266 multiple myeloma cells. This effect was time- and dose-dependent with the highest expression of NY-ESO-1 mRNA achieved by the incubation of 10 μM DAC for 72 hours. NY-ESO-1 activation was also confirmed at the protein level as shown by Western blot, flow cytometry, and immunofluorescence staining. The detection and quantification of single NY-ESO-1 peptides presented at the tumor cell surface in the context of HLA-A*0201 molecules revealed an increase of 100% and 50% for MCF7 and U266 cells, respectively. Moreover, the enhanced expression of NY-ESO-1 derived peptides at the cell surface was accompanied by an increased specific lysis of MCF7 and U266 cells by HLA-A*0201/NY-ESO-1(157-165) peptide specific chimeric antigen receptor (CAR) CD8+ T cells. In addition, the killing activity of CAR T cells correlated with the secretion of higher IFN-gamma levels. These results indicate that NY-ESO-1 directed immunotherapy with specific CAR T cells might benefit from concomitant DAC treatment.

  3. Treatment with 5-Aza-2'-Deoxycytidine Induces Expression of NY-ESO-1 and Facilitates Cytotoxic T Lymphocyte-Mediated Tumor Cell Killing.

    Directory of Open Access Journals (Sweden)

    Agnes S Klar

    Full Text Available NY-ESO-1 belongs to the cancer/testis antigen (CTA family and represents an attractive target for cancer immunotherapy. Its expression is induced in a variety of solid tumors via DNA demethylation of the promoter of CpG islands. However, NY-ESO-1 expression is usually very low or absent in some tumors such as breast cancer or multiple myeloma. Therefore, we established an optimized in vitro treatment protocol for up-regulation of NY-ESO-1 expression by tumor cells using the hypomethylating agent 5-aza-2'-deoxycytidine (DAC.We demonstrated de novo induction of NY-ESO-1 in MCF7 breast cancer cells and significantly increased expression in U266 multiple myeloma cells. This effect was time- and dose-dependent with the highest expression of NY-ESO-1 mRNA achieved by the incubation of 10 μM DAC for 72 hours. NY-ESO-1 activation was also confirmed at the protein level as shown by Western blot, flow cytometry, and immunofluorescence staining. The detection and quantification of single NY-ESO-1 peptides presented at the tumor cell surface in the context of HLA-A*0201 molecules revealed an increase of 100% and 50% for MCF7 and U266 cells, respectively. Moreover, the enhanced expression of NY-ESO-1 derived peptides at the cell surface was accompanied by an increased specific lysis of MCF7 and U266 cells by HLA-A*0201/NY-ESO-1(157-165 peptide specific chimeric antigen receptor (CAR CD8+ T cells. In addition, the killing activity of CAR T cells correlated with the secretion of higher IFN-gamma levels.These results indicate that NY-ESO-1 directed immunotherapy with specific CAR T cells might benefit from concomitant DAC treatment.

  4. CRTAM determines the CD4+ cytotoxic T lymphocyte lineage.

    Science.gov (United States)

    Takeuchi, Arata; Badr, Mohamed El Sherif Gadelhaq; Miyauchi, Kosuke; Ishihara, Chitose; Onishi, Reiko; Guo, Zijin; Sasaki, Yoshiteru; Ike, Hiroshi; Takumi, Akiko; Tsuji, Noriko M; Murakami, Yoshinori; Katakai, Tomoya; Kubo, Masato; Saito, Takashi

    2016-01-11

    Naive T cells differentiate into various effector T cells, including CD4(+) helper T cell subsets and CD8(+) cytotoxic T cells (CTL). Although cytotoxic CD4(+) T cells (CD4 +: CTL) also develop from naive T cells, the mechanism of development is elusive. We found that a small fraction of CD4(+) T cells that express class I-restricted T cell-associated molecule (CRTAM) upon activation possesses the characteristics of both CD4(+) and CD8(+) T cells. CRTAM(+) CD4(+) T cells secrete IFN-γ, express CTL-related genes, such as eomesodermin (Eomes), Granzyme B, and perforin, after cultivation, and exhibit cytotoxic function, suggesting that CRTAM(+) T cells are the precursor of CD4(+)CTL. Indeed, ectopic expression of CRTAM in T cells induced the production of IFN-γ, expression of CTL-related genes, and cytotoxic activity. The induction of CD4(+)CTL and IFN-γ production requires CRTAM-mediated intracellular signaling. CRTAM(+) T cells traffic to mucosal tissues and inflammatory sites and developed into CD4(+)CTL, which are involved in mediating protection against infection as well as inducing inflammatory response, depending on the circumstances, through IFN-γ secretion and cytotoxic activity. These results reveal that CRTAM is critical to instruct the differentiation of CD4(+)CTL through the induction of Eomes and CTL-related gene.

  5. [Differential regulation of CCR5 expression on T lymphocytes in healthy donors after mobilization with rhG-CSF and its correlation with aGVHD].

    Science.gov (United States)

    Wang, Meng; Ma, Xiang-Juan; Dong, Yu-Jun; Qiu, Zhi-Xiang; Liu, Wei; Li, Yuan; Wang, Mang-Ju; Sun, Yu-Hua; Ren, Han-Yun

    2013-08-01

    This study was to investigate the differential regulation of CCR5 expression on T cells in healthy donors after mobilization with recombinant human granulocyte colony-stimulating factor (rhG-CSF) and analyze its correlation with acute graft-versus-host disease (aGVHD) so as to understand the possible mechanisms underlying rhG-CSF-induced immune tolerance. Sixty-eight related healthy donor and their corresponding recipient for allogeneic hematopoietic stem cell transplantation (allo-HSCT) were enrolled in this study. The expression of CCR5 on CD4(+) and CD8(+) T cells in the peripheral blood (PB) before and after mobilization were detected by using flow cytometry (FCM) respectively. According to the changes of CCR5 expression on CD4(+) and CD8(+) T cells, the Sixty-two evaluable donors were divided into the downregulated and unchanged/upregulated (non-downregulated) groups, and the incidence of grades II to IV aGVHD in two groups were compared. The results showed that the mean value of CCR5 expression on CD4(+) and CD8(+) T cells in PB was not different significantly after mobilization (P > 0.05). Apparent inconsistency was showed among different individuals. Thirty-four (50%) donors displayed downregulation of CCR5 expression, while 34 (50%) donors manifested unchanged or upregulated CCR5 expression on CD4(+) T cells. CCR5 expression on CD8(+) T cells was downregulated in 42 (61.8%), unchanged or upregulated in 26 (38.3%) donors. The cumulative incidence of grades II to IV aGVHD in the downregulated and non-downregulated groups for CD4(+) T cells were 16.1% and 41.9% (P = 0.032), and recipients with CCR5 downregulation on CD8(+) T cells showed an increased tendency of developing aGVHD (37.8% vs 16.0%, P = 0.065). In conclusion, rhG-CSF mobilization could lead to differential regulation of CCR5 expression on T cells, which might influence the migration of T cells in vivo, decrease T cell trafficking towards GVHD target organs, and thus reduce the incidence of a

  6. Lymphocyte glucocorticoid receptor expression level and hormone-binding properties differ between war trauma-exposed men with and without PTSD

    NARCIS (Netherlands)

    Matic, Gordana; Milutinovic, Danijela Vojnovic; Nestorov, Jelena; Elakovic, Ivana; Jovanovic, Sanja Manitasevic; Perisic, Tatjana; Dunderski, Jadranka; Damjanovic, Svetozar; Knezevic, Goran; Spiric, Zeljko; Vermetten, Eric; Savic, Danka

    2013-01-01

    Objective: Posttraumatic stress disorder (PTSD) has been shown to be associated with altered glucocorticoid receptor (GR) activity. We studied the expression and functional properties of the receptor in peripheral blood mononuclear cells (PBMCs) from non-traumatized healthy individuals (healthy cont

  7. Evaluation of HTLV-1 activity in HAM/TSP patients using proviral load and Tax mRNA expression after In Vitro lymphocyte activation

    Directory of Open Access Journals (Sweden)

    Atefeh Yari

    2014-07-01

    Conclusion: Although proviral load had been addressed as a valuable index for monitoring HTLV-1 infected subjects, the results of this study demonstrated that Tax expression in activated PBMCs along with proviral load assessment in HAM/TSP patients are a more reliable factor for determining the prognosis and monitoring healthy carriers and HAM/TSP patients.

  8. An intrinsic GABAergic system in human lymphocytes.

    Science.gov (United States)

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

    2011-01-01

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

  9. Expresión de L-selectina en linfocitos T y neutrófilos de niños infectados con HIV L-selectin expression on T lymphocytes and neutrophils in HIV infected children

    Directory of Open Access Journals (Sweden)

    Gaddi Eduardo

    2005-04-01

    Full Text Available La capacidad de los leucocitos de abandonar la circulación y migrar hacia los tejidos es un paso crítico de la respuesta inmune. La L-selectina, selectina leucocitaria (CD62L, media la unión de linfocitos a las vénulas endoteliales altas de los ganglios linfáticos periféricos, y también participa en la adhesión de linfocitos, neutrófilos y monocitos al endotelio vascular activado en los sitios de inflamación. En este trabajo se estudiaron los niveles de expresión de L-selectina sobre los linfocitos T y polimorfonucleares neutrófilos en 25 niños HIV(+ sin tratamiento antirretroviral y 25 niños sanos HIV(-, evaluando además su comportamiento en 10 de los pacientes, luego de 6 meses de iniciada la terapéutica específica para el HIV. El número de linfocitos TCD3+, CD4+ y CD8+ que expresan CD62L se encontró significativamente disminuido en los niños HIV(+ con respecto al grupo control. El porcentaje de neutrófilos que expresan CD62L se encontró significativamente disminuido en los pacientes con mayor compromiso inmunológico. Se observó una correlación positiva entre los niveles de LTCD4+ y el porcentaje de neutrófilos que expresan CD62L. Luego de 6 meses de tratamiento antirretroviral no hubo cambios significativos en los niveles de expresión de CD62L sobre LTCD4+ y LTCD8+. La reducción en los niveles de expresión de L-selectina en estos tipos celulares sugiere que durante la infección por HIV las funciones leucocitarias tales como la migración y el asentamiento linfocitario son anormales, contribuyendo al progresivo deterioro inmune.The ability of leukocytes to leave the circulation and migrate into tissues is a critical feature of the immune response. L-selectin (CD62L, the leukocyte selectin, mediates the binding of lymphocytes to high endothelial venules of peripheral lymph nodes and is also involved in lymphocyte, neutrophil and monocyte attachment to vascular endothelium at sites of inflammation. In this study L

  10. Vaccines expressing the innate immune modulator EAT-2 elicit potent effector memory T lymphocyte responses despite pre-existing vaccine immunity.

    Science.gov (United States)

    Aldhamen, Yasser Ali; Seregin, Sergey S; Schuldt, Nathaniel J; Rastall, David P W; Liu, Chyong-Jy J; Godbehere, Sarah; Amalfitano, Andrea

    2012-08-01

    The mixed results from recent vaccine clinical trials targeting HIV-1 justify the need to enhance the potency of HIV-1 vaccine platforms in general. Use of first-generation recombinant adenovirus serotype 5 (rAd5) platforms failed to protect vaccinees from HIV-1 infection. One hypothesis is that the rAd5-based vaccine failed due to the presence of pre-existing Ad5 immunity in many vaccines. We recently confirmed that EAT-2-expressing rAd5 vectors uniquely activate the innate immune system and improve cellular immune responses against rAd5-expressed Ags, inclusive of HIV/Gag. In this study, we report that use of the rAd5-EAT-2 vaccine can also induce potent cellular immune responses to HIV-1 Ags despite the presence of Ad5-specific immunity. Compared to controls expressing a mutant SH2 domain form of EAT-2, Ad5 immune mice vaccinated with an rAd5-wild-type EAT-2 HIV/Gag-specific vaccine formulation significantly facilitated the induction of several arms of the innate immune system. These responses positively correlated with an improved ability of the vaccine to induce stronger effector memory T cell-biased, cellular immune responses to a coexpressed Ag despite pre-existing anti-Ad5 immunity. Moreover, inclusion of EAT-2 in the vaccine mixture improves the generation of polyfunctional cytolytic CD8(+) T cell responses as characterized by enhanced production of IFN-γ, TNF-α, cytotoxic degranulation, and increased in vivo cytolytic activity. These data suggest a new approach whereby inclusion of EAT-2 expression in stringent human vaccination applications can provide a more effective vaccine against HIV-1 specifically in Ad5 immune subjects.

  11. Association of suboptimal health status with psychosocial stress, plasma cortisol and mRNA expression of glucocorticoid receptor α/β in lymphocyte.

    Science.gov (United States)

    Yan, Yu-Xiang; Dong, Jing; Liu, You-Qin; Zhang, Jie; Song, Man-Shu; He, Yan; Wang, Wei

    2015-01-01

    Suboptimal health status (SHS) has become a new public health challenge in China. This study investigated whether high SHS is associated with psychosocial stress, changes in cortisol level and/or glucocorticoid receptor (GR) isoform expression. Three-hundred eighty-six workers employed in three companies in Beijing were recruited. The SHS score was derived from data collection in the SHS questionnaire (SHSQ-25). The short standard version of the Copenhagen Psychosocial Questionnaire (COPSOQ) was used to assess job-related psychosocial stress. The mean value of the five scales of COPSOQ and distribution of plasma cortisol and mRNA expression of GRα/GRβ between the high level of SHS group and the low level of SHS group were compared using a general linear model procedure. Multiple linear regression analysis was used to analyze the effect of psychosocial stress on SHS. We identified three factors that were predictive of SHS, including "demands at work", "interpersonal relations and leadership" and "insecurity at work". Significantly higher levels of plasma cortisol and GRβ/GRα mRNA ratio were observed among the high SHS group. High level of SHS is associated with decreased mRNA expression of GRα. This study confirmed the association between chronic psychosocial stress and SHS, indicating that improving the psychosocial work environment may reduce SHS and then prevent chronic diseases effectively.

  12. Critical role of perforin-dependent CD8+ T cell immunity for rapid protective vaccination in a murine model for human smallpox.

    Directory of Open Access Journals (Sweden)

    Melanie Kremer

    Full Text Available Vaccination is highly effective in preventing various infectious diseases, whereas the constant threat of new emerging pathogens necessitates the development of innovative vaccination principles that also confer rapid protection in a case of emergency. Although increasing evidence points to T cell immunity playing a critical role in vaccination against viral diseases, vaccine efficacy is mostly associated with the induction of antibody responses. Here we analyze the immunological mechanism(s of rapidly protective vaccinia virus immunization using mousepox as surrogate model for human smallpox. We found that fast protection against lethal systemic poxvirus disease solely depended on CD4 and CD8 T cell responses induced by vaccination with highly attenuated modified vaccinia virus Ankara (MVA or conventional vaccinia virus. Of note, CD4 T cells were critically required to allow for MVA induced CD8 T cell expansion and perforin-mediated cytotoxicity was a key mechanism of MVA induced protection. In contrast, selected components of the innate immune system and B cell-mediated responses were fully dispensable for prevention of fatal disease by immunization given two days before challenge. In conclusion, our data clearly demonstrate that perforin-dependent CD8 T cell immunity plays a key role in MVA conferred short term protection against lethal mousepox. Rapid induction of T cell immunity might serve as a new paradigm for treatments that need to fit into a scenario of protective emergency vaccination.

  13. Immunohistochemical expression of B and T-lymphocytes and TGF-β in experimentally transplanted canine venereal tumor Expressão imunoistoquímica de linfócitos T e B e do TGF-β no tumor venéreo transmissível canino experimentalmente transplantado

    Directory of Open Access Journals (Sweden)

    Ana Carolina Trompieri Silveira

    2009-07-01

    Full Text Available The tumor/host relationship may have a determining role in the progression or remission of a tumor. Greater infiltration of leukocytes into tumors has been associated with a better prognosis, although controversy regarding whether these cells have a central role in antitumor immunity still exists. Canine transmissible venereal tumor (TVT is an experimentally transplantable type of tumor that has been used as an experimental model for the tumor/host relationship. The aim of this study was to evaluate the infiltration of T-lymphocytes (CD3, CD4, CD8 and B lymphocytes (CD79-α and the expression of the cytokine TGF-β in TVT, by means of immunohistochemistry (ABC method. The experimental tumors were composed of puppies that developed TVT after transplantation, in the progression (n=8 (Group 1a, latency (n=8 (Group 1b and regression (n=8 (Group 1c phases of the tumor. CD3+ T-lymphocytes predominated in the progression and regression phases, in relation to the latency phase. CD4+ and ¹CD8+ T-lymphocytes were predominant in the progression phase, and with lower expression in the regression phase. The greatest quantities of B-lymphocytes were in the regression phase, with restricted expression in the progression phase. TGF-β was expressed equally in the phases of the transplanted TVT.A relação tumor/hospedeiro pode ter um papel determinante na progressão ou remissão de um tumor. Uma maior infiltração de leucócitos nos tumores tem sido associada a um melhor prognóstico. Entretanto, o papel central dessas células na imunidade antitumoral ainda é controverso. O tumor venéreo transmissível (TVT canino é um tumor transplantável experimentalmente e tem sido utilizado como modelo experimental da relação tumor versus hospedeiro. O objetivo deste estudo foi avaliar a infiltração de linfócitos T (CD3, CD4, CD8, B (CD79-α e a expressão da citocina TGF-β no TVT por meio da imunoistoquímica (método ABC. Os grupos experimentais foram

  14. T lymphocytes from chronic HCV-infected patients are primed for activation-induced apoptosis and express unique pro-apoptotic gene signature.

    Directory of Open Access Journals (Sweden)

    Bin-Bin Zhao

    Full Text Available Although extensive studies have demonstrated the functional impairment of antigen-specific CD4(+ and CD8(+ T-cells during chronic hepatitis C virus (HCV infection, the functional status of global CD4(+ and CD8(+ T-cells remains unclear. In this report, we recruited 42 long-term (~20 years treatment-naïve chronic HCV (CHC patients and 15 healthy donors (HDs to investigate differences in global CD4(+ and CD8(+ T-cells function. We show that CD4(+ and CD8(+ T-cells from CHC patients underwent increased apoptosis after TCR stimulation. Furthermore, IFN-γ, IL-9 and IP-10 were elevated in CHC patients' plasma and promoted activation-induced T-cells death. Global CD4(+ and CD8(+ T-cells also showed unique transcriptional profiles in the expression of apoptosis-related genes. We identified BCL2, PMAIP1, and CASP1 in CD4(+ T-cells and IER3 and BCL2A1 in CD8(+ T-cells from CHC patients as HCV-specific gene signatures. Importantly, the gene expression patterns of CD4(+ and CD8(+ T-cells from CHC patients differ from those in CD4(+ and CD8(+ T-cells from human immunodeficiency virus type 1 (HIV-1 or hepatitis B virus (HBV infected individuals. Our results indicate that chronic HCV infection causes a systemic change in cytokine levels that primes T-cells for activation-induced apoptosis. Furthermore, HCV infection programs unique apoptosis-related gene expression profiles in CD4(+ and CD8(+ T-cells, leading to their enhanced activation-induced apoptosis. These results provide novel insights to the pathogenesis of chronic HCV infection.

  15. Porcine γδ T lymphocytes can be categorized into two functionally and developmentally distinct subsets according to expression of CD2 and level of TCR.

    Science.gov (United States)

    Stepanova, Katerina; Sinkora, Marek

    2013-03-01

    Porcine γδ T cells have two levels of TCRγδ expression. Whereas TCRγδ(med) cells are mostly CD2(+)CD8(-) and CD2(+)CD8(+), TCRγδ(hi) cells are highly enriched for CD2(-)CD8(-). This distribution is independent of bacterial colonization and it is already established in the thymus prior to export of γδ cells to the periphery. Sorting and cultivation experiments revealed that CD2(-)CD8(-) γδ cells are unable to acquire CD2 and CD8, whereas CD2(+) subsets can gain or loose CD8. There is also differential susceptibility for proliferation between CD2(+) and CD2(-) γδ cells. Although CD2(-)CD8(-) almost do not proliferate, proliferation of CD2(+)CD8(-) and CD2(+)CD8(+) is substantial. Population of CD2(-) γδ cells is also absent in CD1(+) immature thymocytes. Additionally, subpopulations of CD2(+) and CD2(-) γδ cells in the thymus differ in expression of auxiliary surface molecules such as CD25, CD45RA/RC, and MHC class II. Moreover, TCRγδ(hi) cells can generate TCRγδ(med) cells but never the opposite. The only exception is the thymus, where a few TCRγδ(med) cells can be induced to TCRγδ(hi) but only under IL-2 influence. The repertoire of TCRδ is polyclonal in all subsets, indicating that there is the same extent of diversification and equal capability of immune responses. Results collectively indicate that CD2 expression determines two lineages of γδ cells that differ in many aspects. Because CD2(-) γδ cells are missing in the blood of humans and mice but are obvious in other members of γδ-high species such as ruminants and birds, our findings support the idea that circulating CD2(-) γδ T cells are a specific lineage.

  16. Krüppel-like factor KLF10 regulates transforming growth factor receptor II expression and TGF-β signaling in CD8+ T lymphocytes.

    Science.gov (United States)

    Papadakis, Konstantinos A; Krempski, James; Reiter, Jesse; Svingen, Phyllis; Xiong, Yuning; Sarmento, Olga F; Huseby, April; Johnson, Aaron J; Lomberk, Gwen A; Urrutia, Raul A; Faubion, William A

    2015-03-01

    KLF10 has recently elicited significant attention as a transcriptional regulator of transforming growth factor-β1 (TGF-β1) signaling in CD4(+) T cells. In the current study, we demonstrate a novel role for KLF10 in the regulation of TGF-β receptor II (TGF-βRII) expression with functional relevance in antiviral immune response. Specifically, we show that KLF10-deficient mice have an increased number of effector/memory CD8(+) T cells, display higher levels of the T helper type 1 cell-associated transcription factor T-bet, and produce more IFN-γ following in vitro stimulation. In addition, KLF10(-/-) CD8(+) T cells show enhanced proliferation in vitro and homeostatic proliferation in vivo. Freshly isolated CD8(+) T cells from the spleen of adult mice express lower levels of surface TGF-βRII (TβRII). Congruently, in vitro activation of KLF10-deficient CD8(+) T cells upregulate TGF-βRII to a lesser extent compared with wild-type (WT) CD8(+) T cells, which results in attenuated Smad2 phosphorylation following TGF-β1 stimulation compared with WT CD8(+) T cells. Moreover, we demonstrate that KLF10 directly binds to the TGF-βRII promoter in T cells, leading to enhanced gene expression. In vivo viral infection with Daniel's strain Theiler's murine encephalomyelitis virus (TMEV) also led to lower expression of TGF-βRII among viral-specific KLF10(-/-) CD8(+) T cells and a higher percentage of IFN-γ-producing CD8(+) T cells in the spleen. Collectively, our data reveal a critical role for KLF10 in the transcriptional activation of TGF-βRII in CD8(+) T cells. Thus, KLF10 regulation of TGF-βRII in this cell subset may likely play a critical role in viral and tumor immune responses for which the integrity of the TGF-β1/TGF-βRII signaling pathway is crucial. Copyright © 2015 the American Physiological Society.

  17. Comparing of Cu/Zn SOD Gene Expression of Lymphocyte Cell and Malondialdehyde Level in Active Men and Women after Physical Training

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    Bakhtiar Tartibian

    2012-07-01

    Full Text Available Background: The purpose of this study is to compare Cu/Zn SOD mRNA and MDA level as a result of a session incremental exercise in active women and men. Materials and Methods: This research is a quasi-experimental study with repeated measurements in which 14 active female and 13 male subjects with age range 22-24 participated voluntarily. Then, blood was taken from brachial vein of the subjects in three stages before and after GXT (Graded exercise test and 3 hours after that and SYBER Green PCR Master mix reagent Kit and Real time-PCR were used to measure Cu/Zn SOD mRNA and spectrophotometer was used to measure MDA level.Results: MDA levels increased significantly in men during the recovery stage and after the exercise (p1=0.012 and p2 =0.014, but it did not increase significantly in active women. Also, MDA difference between the two genders was not reported significant in any of the exercise stages. Cu/Zn SOD gene expression did not increase significantly in either sex.Conclusion: The risk of injury from free radicals is more probable in active men than active women and vigorous physical activity does not significantly increase the Cu/Zn SOD gene expression.

  18. GABA, a natural immunomodulator of T lymphocytes

    DEFF Research Database (Denmark)

    Bjurstöm, Helen; Wang, Junyang; Ericsson, Ida

    2008-01-01

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

  19. The Gene Expression Changes in Collagen-Induced Arthritis Rat Lymphocytes%胶原诱导性关节炎大鼠淋巴细胞基因表达的变化

    Institute of Scientific and Technical Information of China (English)

    刘静; 邸志权; 王晶晶; 胡金芳; 申秀萍; 应森林

    2013-01-01

    Rheumatoid arthritis ( RA) is a chronic debilitating autoimmune disease that results in joint destruction and subsequent loss of function. To better understand its pathogenesis and to facilitate the search for novel RA therapeutics, we studied collagen-induced arthritis rat lymphocyte genes profile expression. Using DNA microarray technique, the lymphocyte genes profile expression of the collagen-induced arthritis rats and the normal rats was compared. Lymphocytes were purified and total RNA was processed for a microarray analysis using Affymetrix Gene Chip technique. Statistical comparison analyses identified differentially expressed genes that distinguished CIA from control rats. Clustering analyses indicated that 822 genes expression of rats with collagen-induced arthritis were different compared with normal rats,including 558 up-regulated genes,and 264 down-regulated genes. Differential genes involved in the function group of immune response,inflammation stimulation,cell activation,carbohydrate binding and the pathway of apoptosis, oxidative stress,eicosanoid synthesis,blood clotting cascade,prostaglandin synthesis regulation,TGF beta signaling pathway and so on. The major overexpressed genes were Ccr2 (chemokine receptor 2) ,Ccrl (chemokine receptor 1) ,Il1b( interleukin 1 beta) ,Vcan (versican) ,IgG-2a(gamma-2a immunoglobulin heavy chain) ,Cyr61(cysteine-rich,angiogenic inducer,61) ,Bmp8a (bone morpho-genetic protein 8a) ,Tgfbl (transforming growth factor,beta 1 ) and RGD1564318 ( similar to immunoglobulin light chain) and the major underexpressed genes were Ifi27l1 (interferon, alpha-inducible protein 27 like) ,Oasla (2'-5' oligoadenylate synthetase 1A) , Glp2(interferon,alpha-inducible protein), Sgkl ( serum/glucocorticoid regulated kinase 1 ), Irf7 ( interferon regulatory factor 7). There are some genes which have been reported associated with RA in these differential expression genes such as Ccr1 ,Ccr2,Il1b, Vcan,Cyr61 ,CD44,Icaml ,TGF-β1, Hgf

  20. Aβ25-35刺激下人淋巴细胞中p53表达水平的变化%p53 expression in human lymphocytes after exposure to Alzheimer's β-amyloid peptide

    Institute of Scientific and Technical Information of China (English)

    谭梦姗; 王树英; 贾建平

    2012-01-01

    Objective To study the possible mechanism of p53 changes, the increased total p53 level and specifically expressed mutant-like p53 in peripheral blood lymphocytes of sporadic Alzheimer's diseases (sAD) patients. Methods Lymphocytes from healthy volunteers were exposed to soluble neuro-toxic-amyloid peptide (Aβ25-35 ) and were evaluated of p53 expression by Western blot analysis. Results Compared with controls,total p53 level was significantly increased (P<0. 01) and mutant-like p53 also expressed over time in low doses of amyloid treatment (10-8mol/l). Meanwhile,although there was a tendency of increased total p53 level as J3 -amyloid concentration increased, no significant differences were found and no difference in mutant p53 expression. Conclusions All these results were consistent with that found in sAD patients and confirmed the important role of soluble Aβ in AD pathogenesis. Thus, this model may play a role in exploring the pathogenesis and medical intervention of AD in the near future.%目的 探讨散发型阿尔茨海默病(sporadic Alzheimer's disease,sAD)患者外周血淋巴细胞中总p53蛋白水平增高,同时出现异常的“突变样”p53表达的可能机制.方法 采用诱导AD早期病理改变的重要物质即淀粉样蛋白Aβ25-35刺激健康人外周血淋巴细胞,通过Western blot检测总p53蛋白及其突变型的表达情况.结果 与对照组对比显示,在低浓度(10-8 mol/L) Aβ25-35作用下,不同时程的淋巴细胞中总p53表达即增高,差异有统计学意义(P<0.01),并可见微量突变型p53的表达;随着Aβ25-35刺激浓度的提高,总p53水平有增高的趋势,但几乎无统计学意义;突变型p53表达无差异.结论 上述结果模拟了sAD患者外周血淋巴细胞中的变化,证实了可溶性A8在AD发病中的重要作用,以及对p53表达的影响.因而,这一模型将有望在探讨AD的发病机制以及筛选防治AD的药物中发挥一定的作用.

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

    Science.gov (United States)

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

    2016-01-01

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

  2. Mitochondrial apoptosis of lymphocyte is induced in type 2 diabetes

    Institute of Scientific and Technical Information of China (English)

    Xu Hui; Chen Yanbo; Li Yanxiang; Xia Fangzhen; Han Bing; Zhang Huixin; Zhai Hualing

    2014-01-01

    Background Lymphocyte function and homeostasis is associated with immune defence to infection.Apoptosis of lymphocytes might be a considerably important component which has an impact on immunity to infections in people with hyperglycemia.The aim of this study was to explore the mitochondrial apoptosis pathway of lymphocyte in diabetic patients.Methods Sixty patients with type 2 diabetes mellitus and fifty healthy volunteers were included in this study.Annexin V and propidiumiodide (Pl) were joined in the isolated lymphocytes and the rate of lymphocyte apoptosis was calculated with flow cytometry.Observation of the lymphocytes was done using transmission electron microscopy; mitochondria had been extracted and then mitochondrial membrane potential (MMP) was detected to assess mitochondrial function; the mRNA level of Bcl-2,cytochrome c (Cyt-C),caspase-9 and caspase-3 were analyzed by real-time reverse transcriptionpolymerase chain reaction (RT-PCR).Results Apoptosis rate of lymphocyte was significantly higher in diabetic group than that in normal control group (P <0.05).Transmission electron microscopy showed lymphocyte shrinkage and breakage,chromatin condensation and less mitochondria; a fall in MMP levels was also evident; Bcl-2 concentration was reduced and the expressions of caspase-9,caspase-3 and Cyt-C were elevated (P <0.05) in diabetic patients.Conclusions The rate of lymphocyte apoptosis was significantly higher in type 2 diabetic patients than that in normal population.Mitochondrial apoptosis pathway may play a very important role in decreasing function of lymphocyte in diabetes.

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

    Science.gov (United States)

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

    1993-06-01

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

  4. 脓毒症患者外周血淋巴细胞PPARγ表达和活性的变化%Changes of PPARγ protein expression and activity in peripheral blood lymphocytes of patients with sepsis

    Institute of Scientific and Technical Information of China (English)

    孙湛; 黄培志; 宋振举; 童朝阳; 朱彪; 姚晨玲

    2009-01-01

    Objective To investigate the changes of PPARγ protein expression and activity of peripheral blood lymphocytes in patients with sepsis and its association with severity and prognosis of sepsis. Method Ac-cording to the guidelines to sepsis set by ACCP/SCCM consensus conference in 2003, 48 patients with sepsis ad-mitted in Emergency and Surgical ICU from December 2007 to March 2008 were enrolled in this perspective study. Sixteen healthy individuals were selected as controls. Patients with metastatic tumors, autoimmune disease, AIDS or under immunosuppressive therapy were excluded. This study was approved by the ethical committee of Zhong-shan Hospital, Fudan University. All patients were divided into mild and severe sepsis groups. Patients were also divided into survivor and non-survivor groups as per 28-day mortality. Peripheral blood lymphocytes were isolated by using Ficoll density gradient centrifugation. PPART protein expression was determined by using Westem Blot-ting. The activity of PPARγ was analyzed by using EMSA. Differences among groups were analyzed by using one-way ANOVA. Results The protein expression and activity of PPARγ were significantly increased in mild sepsis patients (0.56±0.12 and 4.13±0.22, respectively) compared with both healthy controls (0.39±0.07 and 2.42±0.17, respectively) and severe sepsis patients (0.30±0.07 and 1.63±0.12, respectively) (P < 0.05). However, the protein expression and activity of PPARγ were obviously decreased in severe sepsis patients compared with healthy individuals and mild spsis patients (P < 0.05). Survivors from sepsis had significantly higher protein expression and activity (0.54±0.11 and 3.59±0.34, respectively) than non-survivors (0.21±0.08 and 1.94 ±0.25, respectively) (P < 0.05). Conclusions These data suggest that the protein expression and activity of PPARγ in peripheral blood lymphocytes might be valuable biomarkers in assessing the severity and outcome of pa-tients with sepsis.%目的

  5. Monocyte chemoattractant protein-1 secreted by decidual stromal cells inhibits NK cells cytotoxicity by up-regulating expression of SOCS3.

    Directory of Open Access Journals (Sweden)

    Xiaofei Xu

    Full Text Available BACKGROUND: Decidual stromal cells (DSCs are of particular importance due to their pleiotropic functions during pregnancy. Although previous research has demonstrated that DSCs participated in the regulation of immune cells during pregnancy, the crosstalk between DSCs and NK cells has not been fully elucidated. To address this issue, we investigated the effect of DSCs on perforin expression in CD56(+ NK cells and explored the underlying mechanism. METHODOLOGY/PRINCIPAL FINDINGS: Flow cytometry analysis showed perforin production in NK cells was attenuated by DSC media, and it was further suppressed by media from DSCs pretreated with lipopolysaccharide (LPS. However, the expression of granzyme A and apoptosis of NK cells were not influenced by DSC media. ELISA assays to detect cytokine production indicated that monocyte chemoattractant protein-1 (MCP-1 in the supernatant of DSCs conditioned culture significantly increased after LPS stimulation. The inhibitory effect of DSC media on perforin was abolished by the administration of anti-MCP-1 neutralizing antibody. Notably, reduced perforin expression attenuated the cytotoxic potential of CD56(+ NK cells to K562 cells. Moreover, Suppressor of cytokine signaling 3 (SOCS3 expression in NK cells was enhanced by treatment with MCP-1, as measured by RT-PCR and western blot. Interestingly, MCP-1-induced perforin expression was partly abolished by the siRNA induced SOCS3 knockdown. Western blot analysis suggested that both NF-κB and ERK/MAPKs pathway were involved in the LPS-induced upregulation of MCP-1 in DSCs. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that LPS induces upregulation of MCP-1 in DSCs, which may play a critical role in inhibiting the cytotoxicity of NK cells partly by promoting SOCS3 expression. These findings suggest that the crosstalk between DSCs and NK cells may be crucial to maintain pregnancy homeostasis.

  6. Curcumin and Cholecalciferol in Treating Patients With Previously Untreated Stage 0-II Chronic Lymphocytic Leukemia or Small Lymphocytic Lymphoma

    Science.gov (United States)

    2016-10-04

    Contiguous Stage II Small Lymphocytic Lymphoma; Noncontiguous Stage II Small Lymphocytic Lymphoma; Stage 0 Chronic Lymphocytic Leukemia; Stage I Chronic Lymphocytic Leukemia; Stage I Small Lymphocytic Lymphoma; Stage II Chronic Lymphocytic Leukemia

  7. A Truncated form of CD200 (CD200S Expressed on Glioma Cells Prolonged Survival in a Rat Glioma Model by Induction of a Dendritic Cell-Like Phenotype in Tumor-Associated Macrophages

    Directory of Open Access Journals (Sweden)

    Kana Kobayashi

    2016-04-01

    Full Text Available CD200 induces immunosuppression in myeloid cells expressing its receptor CD200R, which may have consequences for tumor immunity. We found that human carcinoma tissues express not only full-length CD200 (CD200L but also its truncated form, CD200S. Although CD200S is reported to antagonize the immunosuppressive actions of CD200L, the role of CD200S in tumor immunity has never been investigated. We established rat C6 glioma cell lines that expressed either CD200L or CD200S; the original C6 cell line did not express CD200 molecules. The cell lines showed no significant differences in growth. Upon transplantation into the neonatal Wistar rat forebrain parenchyma, rats transplanted with C6-CD200S cells survived for a significantly longer period than those transplanted with the original C6 and C6-CD200L cells. The C6-CD200S tumors were smaller than the C6-CD200L or C6-original tumors, and many apoptotic cells were found in the tumor cell aggregates. Tumor-associated macrophages (TAMs in C6-CD200S tumors displayed dendritic cell (DC-like morphology with multiple processes and CD86 expression. Furthermore, CD3+, CD4+ or CD8+ cells were more frequently found in C6-CD200S tumors, and the expression of DC markers, granzyme, and perforin was increased in C6-CD200S tumors. Isolated TAMs from original C6 tumors were co-cultured with C6-CD200S cells and showed increased expression of DC markers. These results suggest that CD200S activates TAMs to become DC-like antigen presenting cells, leading to the activation of CD8+ cytotoxic T lymphocytes, which induce apoptotic elimination of tumor cells. The findings on CD200S action may provide a novel therapeutic modality for the treatment of carcinomas.

  8. Lymphocyte Trafficking to Mucosal Tissues

    DEFF Research Database (Denmark)

    Mikhak, Zamaneh; Agace, William Winston; Luster, Andrew D.

    2015-01-01

    Lymphocytes are the key cells of the adaptive immune system that provide antigen-specific responses tailored to the context of antigen exposure. Through cytokine release and antibody production, lymphocytes orchestrate and amplify the recruitment and function of other immune cells and contribute...... to host defense against invading pathogens and the pathogenesis of many inflammatory diseases. Lymphocyte function is critically dependent on their ability to traffic into the correct anatomic locations at the appropriate times. This process is highly regulated and requires that lymphocytes interact...

  9. Lymphocytic Interstitial Pneumonia.

    Science.gov (United States)

    Panchabhai, Tanmay S; Farver, Carol; Highland, Kristin B

    2016-09-01

    Lymphocytic interstitial pneumonia (LIP) is a rare lung disease on the spectrum of benign pulmonary lymphoproliferative disorders. LIP is frequently associated with connective tissue diseases or infections. Idiopathic LIP is rare; every attempt must be made to diagnose underlying conditions when LIP is diagnosed. Computed tomography of the chest in patients with LIP may reveal ground-glass opacities, centrilobular and subpleural nodules, and randomly distributed thin-walled cysts. Demonstrating polyclonality with immunohistochemistry is the key to differentiating LIP from lymphoma. The 5-year mortality remains between 33% and 50% and is likely to vary based on the underlying disease process.

  10. X-ray irradiation affects Gadd45 mRNA expression in human peripheral blood lymphocytes%X 射线对人周围血淋巴细胞 Gadd45 mRNA 表达影响

    Institute of Scientific and Technical Information of China (English)

    王恰; 李明芳; 杨爱初; 杨宇华; 梁晓阳; 严茂胜

    2015-01-01

    Objective To analyze the relative expression level of Growth arrest and DNA damage gene (Gadd)45 mRNA of human peripheral blood lymphocytes after X-ray irradiation with different doses and time,and to explore the possibility of Gadd45 gene as a biological dosimeter.Methods i)Dose-effect experiment.The healthy human peripheral blood were irradiated by 0.00,0.10,0.25,0.50,1.00,2.00,3.00 and 5.00 Gy X-ray and the lymphocytes were separated for measurement.ii)Time-effect experiment.The healthy human peripheral blood were irradiated by 2.00 Gy X-ray and the lymphocytes were separated and then cultivate for measurement after cultivation time of 0,3,6,12,24 and 48 hours.The real-time fluorescent quantitative polymerase chain reaction method was used to detect Gadd45 mRNA expression level. Results i)The relative expression level of Gadd45 mRNA increased with the increasing dose from 0.00 to 0.50 Gy.The linear regression equation was ^y =1.056 9 +7.132 2 x (determination coefficient was 0.992,P <0.01 ).The relative expression level of Gadd45 mRNA achieved the peak value when the irradiation dose was 0.50 Gy,which was 4.53 times of that with 0.00 Gy dose irradiation.The relative expression level of Gadd45 mRNA gradually decreased in 0.50-5.00 Gy.ii)In the period of 3-12 hours after 2.00 Gy of X-ray irradiation,the relative expression level of Gadd45 mRNA increased with the increasing exposure time.The linear regression equation was ^y =-6.366 0 +4.965 0 x (determination coefficient was 0.932,P <0.05).It achieved the peak value at the time of 12 hours,which was 57.68 times of that with 0-hour time group.During the time of 24-48 hours after irradiation,the relative expression level of Gadd45 mRNA decreased sharply,the 48-hour group was 2.08 times of 0-hour time group.Conclusion X-ray irradiation up-regulated the expression level of Gadd45 mRNA.There was a good dose-effect and time-effect relation in specific dose and time range.Further study is needed to explore if Gadd45

  11. Chronic lymphocytic leukaemia

    Science.gov (United States)

    Kipps, Thomas J.; Stevenson, Freda K.; Wu, Catherine J.; Croce, Carlo M.; Packham, Graham; Wierda, William G.; O’Brien, Susan; Gribben, John; Rai, Kanti

    2017-01-01

    Chronic lymphocytic leukaemia (CLL) is a malignancy of CD5+ B cells that is characterized by the accumulation of small, mature-appearing lymphocytes in the blood, marrow and lymphoid tissues. Signalling via surface immunoglobulin, which constitutes the major part of the B cell receptor, and several genetic alterations play a part in CLL pathogenesis, in addition to interactions between CLL cells and other cell types, such as stromal cells, T cells and nurse-like cells in the lymph nodes. The clinical progression of CLL is heterogeneous and ranges from patients who require treatment soon after diagnosis to others who do not require therapy for many years, if at all. Several factors, including the immunoglobulin heavy-chain variable region gene (IGHV) mutational status, genomic changes, patient age and the presence of comorbidities, should be considered when defining the optimal management strategies, which include chemotherapy, chemoimmunotherapy and/or drugs targeting B cell receptor signalling or inhibitors of apoptosis, such as BCL-2. Research on the biology of CLL has profoundly enhanced our ability to identify patients who are at higher risk for disease progression and our capacity to treat patients with drugs that selectively target distinctive phenotypic or physiological features of CLL. How these and other advances have shaped our current understanding and treatment of patients with CLL is the subject of this Primer. PMID:28102226

  12. TNF/TNFR1 signaling up-regulates CCR5 expression by CD8+ T lymphocytes and promotes heart tissue damage during Trypanosoma cruzi infection: beneficial effects of TNF-α blockade

    Directory of Open Access Journals (Sweden)

    Karina Kroll-Palhares

    2008-06-01

    Full Text Available In Chagas disease, understanding how the immune response controls parasite growth but also leads to heart damage may provide insight into the design of new therapeutic strategies. Tumor necrosis factor-alpha (TNF-α is important for resistance to acute Trypanosoma cruzi infection; however, in patients suffering from chronic T. cruzi infection, plasma TNF-α levels correlate with cardiomyopathy. Recent data suggest that CD8-enriched chagasic myocarditis formation involves CCR1/CCR5-mediated cell migration. Herein, the contribution of TNF-α, especially signaling through the receptor TNFR1/p55, to the pathophysiology of T. cruzi infection was evaluated with a focus on the development of myocarditis and heart dysfunction. Colombian strain-infected C57BL/6 mice had increased frequencies of TNFR1/p55+ and TNF-α+ splenocytes. Although TNFR1-/- mice exhibited reduced myocarditis in the absence of parasite burden, they succumbed to acute infection. Similar to C57BL/6 mice, Benznidazole-treated TNFR1-/- mice survived acute infection. In TNFR1-/- mice, reduced CD8-enriched myocarditis was associated with defective activation of CD44+CD62Llow/- and CCR5+ CD8+ lymphocytes. Also, anti-TNF-α treatment reduced the frequency of CD8+CCR5+ circulating cells and myocarditis, though parasite load was unaltered in infected C3H/HeJ mice. TNFR1-/- and anti-TNF-α-treated infected mice showed regular expression of connexin-43 and reduced fibronectin deposition, respectively. Furthermore, anti-TNF-α treatment resulted in lower levels of CK-MB, a cardiomyocyte lesion marker. Our results suggest that TNF/TNFR1 signaling promotes CD8-enriched myocarditis formation and heart tissue damage, implicating the TNF/TNFR1 signaling pathway as a potential therapeutic target for control of T. cruzi-elicited cardiomyopathy.

  13. TNF/TNFR1 signaling up-regulates CCR5 expression by CD8+ T lymphocytes and promotes heart tissue damage during Trypanosoma cruzi infection: beneficial effects of TNF-alpha blockade.

    Science.gov (United States)

    Kroll-Palhares, Karina; Silvério, Jaline Coutinho; Silva, Andrea Alice da; Michailowsky, Vladimir; Marino, Ana Paula; Silva, Neide Maria; Carvalho, Cristiano Marcelo Espinola; Pinto, Luzia Maria de Oliveira; Gazzinelli, Ricardo Tostes; Lannes-Vieira, Joseli

    2008-06-01

    In Chagas disease, understanding how the immune response controls parasite growth but also leads to heart damage may provide insight into the design of new therapeutic strategies. Tumor necrosis factor-alpha (TNF-alpha) is important for resistance to acute Trypanosoma cruzi infection; however, in patients suffering from chronic T. cruzi infection, plasma TNF-alpha levels correlate with cardiomyopathy. Recent data suggest that CD8-enriched chagasic myocarditis formation involves CCR1/CCR5-mediated cell migration. Herein, the contribution of TNF-alpha, especially signaling through the receptor TNFR1/p55, to the pathophysiology of T. cruzi infection was evaluated with a focus on the development of myocarditis and heart dysfunction. Colombian strain-infected C57BL/6 mice had increased frequencies of TNFR1/p55+ and TNF-alpha+ splenocytes. Although TNFR1-/- mice exhibited reduced myocarditis in the absence of parasite burden, they succumbed to acute infection. Similar to C57BL/6 mice, Benznidazole-treated TNFR1-/- mice survived acute infection. In TNFR1-/- mice, reduced CD8-enriched myocarditis was associated with defective activation of CD44+CD62Llow/- and CCR5+ CD8+ lymphocytes. Also, anti-TNF-alpha treatment reduced the frequency of CD8+CCR5+ circulating cells and myocarditis, though parasite load was unaltered in infected C3H/HeJ mice. TNFR1-/- and anti-TNF-alpha-treated infected mice showed regular expression of connexin-43 and reduced fibronectin deposition, respectively. Furthermore, anti-TNF-alpha treatment resulted in lower levels of CK-MB, a cardiomyocyte lesion marker. Our results suggest that TNF/TNFR1 signaling promotes CD8-enriched myocarditis formation and heart tissue damage, implicating the TNF/TNFR1 signaling pathway as a potential therapeutic target for control of T. cruzi-elicited cardiomyopathy.

  14. Obinutuzumab for previously untreated chronic lymphocytic leukemia.

    Science.gov (United States)

    Abraham, Jame; Stegner, Mark

    2014-04-01

    Obinutuzumab was approved by the Food and Drug Administration in late 2013 for use in combination with chlorambucil for the treatment of patients with previously untreated chronic lymphocytic leukemia (CLL). The approval was based on results of an open-label phase 3 trial that showed improved progression-free survival (PFS) with the combination of obinutuzumab plus chlorambucil compared with chlorambucil alone. Obinutuzumab is a monoclonal antibody that targets CD20 antigen expressed on the surface of pre B- and mature B-lymphocytes. After binding to CD20, obinutuzumab mediates B-cell lysis by engaging immune effector cells, directly activating intracellular death signaling pathways, and activating the complement cascade. Immune effector cell activities include antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis.

  15. Decreased deformability of lymphocytes in chronic lymphocytic leukemia

    Science.gov (United States)

    Zheng, Yi; Wen, Jun; Nguyen, John; Cachia, Mark A.; Wang, Chen; Sun, Yu

    2015-01-01

    This paper reports the first study of stiffness/deformability changes of lymphocytes in chronic lymphocytic leukemia (CLL) patients, demonstrating that at the single cell level, leukemic metastasis progresses are accompanied by biophysical property alterations. A microfluidic device was utilized to electrically measure cell volume and transit time of single lymphocytes from healthy and CLL patients. The results from testing thousands of cells reveal that lymphocytes from CLL patients have higher stiffness (i.e., lower deformability), as compared to lymphocytes in healthy samples, which was also confirmed by AFM indentation tests. This observation is in sharp contrast to the known knowledge on other types of metastatic cells (e.g., breast and lung cancer cells) whose stiffness becomes lower as metastasis progresses.

  16. TNFα and IFNγ but not perforin are critical for CD8 T cell-mediated protection against pulmonary Yersinia pestis infection.

    Science.gov (United States)

    Szaba, Frank M; Kummer, Lawrence W; Duso, Debra K; Koroleva, Ekaterina P; Tumanov, Alexei V; Cooper, Andrea M; Bliska, James B; Smiley, Stephen T; Lin, Jr-Shiuan

    2014-05-01

    Septic pneumonias resulting from bacterial infections of the lung are a leading cause of human death worldwide. Little is known about the capacity of CD8 T cell-mediated immunity to combat these infections and the types of effector functions that may be most effective. Pneumonic plague is an acutely lethal septic pneumonia caused by the Gram-negative bacterium Yersinia pestis. We recently identified a dominant and protective Y. pestis antigen, YopE69-77, recognized by CD8 T cells in C57BL/6 mice. Here, we use gene-deficient mice, Ab-mediated depletion, cell transfers, and bone marrow chimeric mice to investigate the effector functions of YopE69-77-specific CD8 T cells and their relative contributions during pulmonary Y. pestis infection. We demonstrate that YopE69-77-specific CD8 T cells exhibit perforin-dependent cytotoxicity in vivo; however, perforin is dispensable for YopE69-77-mediated protection. In contrast, YopE69-77-mediated protection is severely impaired when production of TNFα and IFNγ by CD8 T cells is simultaneously ablated. Interestingly, TNFα is absolutely required at the time of challenge infection and can be provided by either T cells or non-T cells, whereas IFNγ provided by T cells prior to challenge appears to facilitate the differentiation of optimally protective CD8 T cells. We conclude that cytokine production, not cytotoxicity, is essential for CD8 T cell-mediated control of pulmonary Y. pestis infection and we suggest that assays detecting Ag-specific TNFα production in addition to antibody titers may be useful correlates of vaccine efficacy against plague and other acutely lethal septic bacterial pneumonias.

  17. Expression of T Lymphocyte Subsets in Lesions of Facial Steroid-dependent Dermatitis%面部激素依赖性皮炎皮损中T淋巴细胞亚群的分析

    Institute of Scientific and Technical Information of China (English)

    吴景良; 徐西红; 张理涛

    2013-01-01

    Objective To observe the expression of T lymphocyte subsets in lesions of facial steroid-dependent dermatitis and to explore the pathogenesis.Methods Twenty-five patients with facial steroid-dependent dermatitis and ten patients with facial subacute eczema were diagnosed clinically.Ten healthy skin specimens were taken as controls.The distribution and intensity of expression of T lymphocyte subsets in the specimens were detected by immunohistochemical SP method.Results There was not or little CD4+and CD8+T were detected cell in the superficial dermis of normal skin tissue,but a large number of CD4+and CD8+T cell were expressed at the dermal papilla,superficial dermis in facial subacute eczema and facial steroid-dependent dermatitis.The level of CD8+T cell in facial steroid-dependent dermatitis were higher than that of controls,and there had a significant statistical difference(P<0.017);The level of C D8+T cell in facial subacute eczema were higher than that of controls,and there had a significant statistical difference(P<0.017).The level of CD4+T cell in facial steroid-dependent dermatitis and facial subacute eczema were higher than that of controls,but they hadn't statistically significant difference(P>0.05).Although the CD4+/CD8+ratio in facial steroid-dependent dermatitis increased,compared with controls,there was no statistical difference(P>0.05).Conclusion The change of T lymphocyte subsets in the lesion of facial steroid dependence dermatitis,which maybe participated in immunopathogenesis of facial steroid dependence dermatitis.%目的 探讨T淋巴细胞亚群在面部激素依赖性皮炎发病机制中的作用.方法 应用免疫组织化学SP法检测25例面部激素依赖性皮炎患者皮损、10例面部亚急性湿疹患者皮损、10例外科手术切取的面部外观正常皮肤T淋巴细胞亚群分布特点.结果 正常皮肤组织CD4+,CD8+T细胞在真皮浅层呈阴性或少量表达,面部亚急性湿疹皮损和面部激

  18. Lymphocyte apoptosis: role of uremia and permeability of dialysis membrane.

    Science.gov (United States)

    Soriano, Sagrario; Martín-Malo, Alejandro; Carracedo, Julia; Ramírez, Rafael; Rodríguez, Mariano; Aljama, Pedro

    2005-01-01

    Uremia is associated to host defense mechanism disorders. Lymphocyte apoptosis, which may cause alteration of the immune system, is increased in uremic patients. The aim of the present study was to determine if, in addition to uremia, dialysis membranes with different biocompatibility and permeability have an effect on lymphocyte apoptosis. Cell apoptosis and Fas expression were assessed using flow cytometry in four groups of patients: (1) uremic non-dialyzed (Non-D) patients; (2) hemodialysis (HD) patients on hemophan; (3) low-flux polysulfone, and (4) high-flux polysulfone membrane. Ten healthy volunteers were used as controls. At baseline, lymphocytes from patients on hemophan showed an increase in apoptosis (18.4 +/- 6.9%) as compared with Non-D (7.2 +/- 2.8%; p dialysis patients lymphocyte apoptosis is influenced not only by the biocompatibility but also by the permeability of the dialysis membrane.

  19. Lymphocytes prime activation is required for nicotine-induced calcium waves.

    Science.gov (United States)

    Landais, Emilie; Liautaud-Roger, Francoise; Antonicelli, Frank

    2010-06-01

    Lymphocytes are reported to express nicotinic acetylcholine receptors (nAChR). However, no data are available on the expression of these nAChR on activated lymphocyte relatively to resting lymphocytes. In this study, we examined nAChR subunits expression in PHA-stimulated versus un-stimulated lymphocytes, and four leukemic cell lines. Cell stimulation with nicotine triggered calcium responses only in some experiments conducted with PHA-stimulated lymphocytes. Likewise, only the Jurkat and HL-60 cell lines displayed calcium waves upon nicotine stimulation, whereas the Raji and CCRF-CEM did not. All responding cells displayed an active form of the nicotinic a-7 nAChR. Indeed, use of 2 different sets of primers for the corresponding mRNA showed that expression of the full-length a-7 subunit mRNA was only present in PHA-stimulated lymphocytes for which calcium waves had been evidenced. Microscopy analysis of lymphocytes structure showed a direct relationship between their size, their a-7 nAChR expression, and calcium release upon nicotine stimulation. Then, this relationship suggested that lymphocytes need a prime activation to express the a-7 nAChR, and therefore to release calcium in response to nicotine.

  20. Key role of mitochondria in apoptosis of lymphocytes.

    Science.gov (United States)

    Boichuk, S V; Minnebaev, M M; Mustafin, I G

    2001-12-01

    Changes in the mitochondrial potential, expression of phosphatidylserine, parameters of direct and lateral light scattering, and DNA fragmentation during spontaneous and induced apoptosis in peripheral blood lymphocytes were studied by flow cytofluorometry. Dexamethasone and Ca2+ ionophore A23187 served as inductors of apoptosis. A decrease in the mitochondrial potential is an early sign of spontaneous and induced apoptosis. Phosphatidylserine expression on the outer plasma membrane occurred later and inversely depended on the mitochondrial potential. Our results indicate that the involvement of mitochondria in spontaneous and induced apoptosis accompanied by a decrease in the mitochondrial potential is an early and key event of programmed lymphocyte death. The decrease in the mitochondrial potential of lymphocytes induced degradation of their nuclei (DNA fragmentation) and promoted elimination of apoptotic cells (phosphatidylserine expression).

  1. Obinutuzumab in chronic lymphocytic leukemia.

    Science.gov (United States)

    Dupuis, Jehan

    2015-09-01

    Obinutuzumab is the second next-generation monoclonal anti-CD20 antibody (after ofatumumab) to enter clinical practice in chronic lymphocytic leukemia. Its superiority in association with chlorambucil as compared with chlorambucil alone has led to its approval as a first-line treatment for chronic lymphocytic leukemia, for patients who are not candidates for a more intensive treatment.

  2. Paraflagellar rod protein-specific CD8+ cytotoxic T lymphocytes target Trypanosoma cruzi-infected host cells.

    Science.gov (United States)

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

    2002-08-01

    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.

  3. 登革病毒感染对T细胞和NK细胞表达细胞因子的影响%Effects of Dengue Virus Infection on Expression of Cytokines in T cells and NK Cells

    Institute of Scientific and Technical Information of China (English)

    庞贤武; 田疆; 曾谷城; 刘岩; 方丹云; 晏辉钧; 江丽芳

    2012-01-01

    [目的]本文探讨登革病毒体外感染人外周血单个核细胞后对T细胞和NK细胞表达细胞因子的影响,并初步探讨登革病毒感染引起的免疫效应.[方法]用登革2型病毒(DEN-2)感染健康人外周血单个核细胞(PBMC),流式细胞术检测登革2型病毒后T细胞和NK细胞的表型及细胞因子的表达水平.[结果]DEN-2感染18h后,T细胞明显表达CD69、IFN-y、TNF-α和perforin,NK细胞表达CD69、IFN-γ和perforin;随着感染复数(MOI)增加,表达perforin和IFN-y的T细胞数及表达perforin的NK细胞数上升;随着感染时间的延长,表达perforin和IL-4的T细胞数及表达perforin的NK细胞数逐渐上升,而表达IFN-y的T细胞数及表达IFN-y的NK细胞数逐渐下降;表达perforin的T细胞亚型主要为CD8+T细胞.[结论]发现登革病毒在体外能够诱导T细胞表达CD69、IFN-y、TNF-α、perforin和IL-4,及NK细胞表达CD69、IFN-y和perforin,提示T细胞、NK细胞可能在抗登革病毒感染或免疫损伤中起着重要的作用.%[Objective] To investigate the expression of cytokines in T cells and NK cells from human peripheral blood mononuclear cells in response to dengue virus in vitro, and to explore the immune response after dengue virus infection. [Methods] Flow cytometry was used to detect the phenolype and eytokines of T cells and NK cells from PBMC after DEN-2 infection. [Results] T cells express CD69, IFN-γ, TNF-a, perforin, and NK cells express CD69, IFN-γ, perform 18 hours after DEN-2 infection. The numbers of IFN-7 and perforin expressing T cells and perforin expressing NK cells have a positive correlation with dengue virus dose. The numbers of perforin and IL-4 expressing T cells and perforin expressing NK cells increase, whereas the numbers of IFN-7 expressing T cells and NK cells decrease follow infection times. T cells expressing perforin are mainly CD8+T cells. [Conclusion] With in vitro infection by dengue virus, T cells can express CD69, IFN-7

  4. Differential expression of CD96 surface molecule represents CD8⁺ T cells with dissimilar effector function during HIV-1 infection.

    Directory of Open Access Journals (Sweden)

    Emily M Eriksson

    Full Text Available During HIV-1 infection, immune dysregulation and aberrant lymphocyte functions are well-established characteristics. Cell surface molecules are important for immunological functions and changes in expression can affect lymphocyte effector functions, thereby contributing to pathogenesis and disease progression. In this study we have focused on CD96, a member of the IgG superfamily receptors that have generated increasing recent interest due to their adhesive and co-stimulatory functions in addition to immunoregulatory capacity. CD96 is expressed by both T and NK cells. Although the function of CD96 is not completely elucidated, it has been shown to have adhesive functions and enhance cytotoxicity. Interestingly, CD96 may also have inhibitory functions due to its immunoreceptor tyrosine-based inhibitory motif (ITIM. The clinical significance of CD96 is still comparatively limited although it has been associated with chronic Hepatitis B infection and disease progression. CD96 has not previously been studied in the context of HIV-1 infection, but due to its potential importance in immune regulation and relevance to chronic disease, we examined CD9