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Cytolytic Activity (cytolytic + activity)
Selected AbstractsCD8+ T-cell interaction with HCV replicon cells: Evidence for both cytokine- and cell-mediated antiviral activityHEPATOLOGY, Issue 6 2003Chen Liu The interaction between the host immune response and infected hepatocytes plays a central role in the pathogenesis of hepatitis C virus (HCV). The lack of a suitable animal or in vitro model has hindered our understanding of the host T-cell/HCV interaction. Our aim was to develop an in vitro model to study the mechanisms of HCV-specific T-cell-mediated antiviral and cytolytic function. The HCV replicon was HLA typed and lymphocytes were obtained from an HLA class I-matched subject. CD8+ T cells were expanded with 2 HCV-specific/HLA-restricted peptides for NS3. Lymphocyte preparations were cocultured with HCV replicon (FCA1) and control (Huh7) cells labeled with 51Cr. After a 48-hour incubation, the cells were harvested for RNA extraction. Standard blocking assays were performed in the presence of anti-interferon gamma (IFN-,), anti-tumor necrosis factor , (TNF-,), and anti-FasL. Cytolytic activity was measured by 51Cr release. HCV replicon cells express homozygous HLA-A11 alleles and present HCV nonstructural proteins. HCV-specific expansion of CD8+ cells led to a 10-fold decrease in HCV replication by Northern blot analysis and 21% specific lysis of FCA1 cells (compared with 2% of control Huh7 cells). Twenty percent of this antiviral activity was independent of T-cell binding, suggesting cytokine-mediated antiviral activity. The CD8+ antiviral effect was markedly reduced by blocking either IFN-, or FasL but was unaffected by blocking TNF-,. In conclusion, HCV-specific CD8+ cells inhibit viral RNA replication by cytokine-mediated and direct cytolytic effects. This T-cell/HCV subgenomic replicon system represents a model for the investigation of CD8 cell interaction with HCV-infected hepatocytes. [source] Crystallization and preliminary X-ray analysis of cecropin B from Bombyx moriACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2010Zhongyuan Liu Cecropin B is a 37-residue cationic antimicrobial peptide derived from the haemolymph of Bombyx mori. The precise mechanism by which cecropins exert their antimicrobial and cytolytic activities is not well understood. Crystals of cecropin B were obtained by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant at 289,K. The crystal diffracted to 1.43,Å resolution using X-ray radiation and belonged to the orthorhombic space group P1, with unit-cell parameters a = 15.08, b = 22.75, c = 30.20,Å, , = 96.9, , = 103.1, , = 96.5°. The asymmetric unit contained only one molecule of cecropin B, with a calculated Matthews coefficient of 2.48,Å3,Da,1 and a solvent content of 50.4%. [source] Conformation and lytic activity of eumenine mastoparan: a new antimicrobial peptide from wasp venomCHEMICAL BIOLOGY & DRUG DESIGN, Issue 3 2004M.P. Dos Santos Cabrera Abstract:, Eumenine mastoparan-AF (EMP-AF) is a novel membrane active tetradecapeptide recently isolated from the venom of solitary wasp, Anterhynchium flavomarginatum micado. It was reported previously that EMP-AF peptide presented low cytolytic activities in human erythrocytes and in RBL-2H3 mast cells. In the present work, we observed that this peptide is able to permeate anionic liposomes, and in less extension also the neutral ones. We present evidences showing that the permeation ability is well correlated with the amount of helical conformation assumed by the peptides in these environments. This peptide also showed a broad-spectrum inhibitory activity against Gram-positive and Gram-negative bacteria. The permeability of liposomes and the antibiotic effect showed a significant reduction when C-terminus was deamidated (in acidic form). The removal of the three first amino acid residues from the N-terminus rendered the peptide inactive both in liposomes and in bacteria. The results suggest that the mechanism of action involves a threshold in the accumulation of the peptide at level of cell membrane. [source] Therapy-induced antitumor vaccination by targeting tumor necrosis factor-, to tumor vessels in combination with melphalanEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 12 2007Lorenzo Mortara Abstract Treatment of tumor-bearing mice with mouse (m)TNF-,, targeted to tumor vasculature by the anti-ED-B fibronectin domain antibody L19(scFv) and combined with melphalan, induces a therapeutic immune response. Upon treatment, a highly efficient priming of CD4+ T cells and consequent activation and maturation of CD8+ CTL effectors is generated, as demonstrated by in vivo depletion and adoptive cell transfer experiments. Immunohistochemical analysis of the tumor tissue demonstrated massive infiltration of CD4+ and CD8+ T cells 6,days after treatment and much earlier in the anamnestic response to tumor challenge in cured mice. In fact, the curative treatment with L19mTNF-, and melphalan resulted in long-lasting antitumor immune memory, accompanied by a mixed Th1/Th2-type response and significant in vitro tumor-specific cytolytic activity. Finally, the combined treatment reduced the percentage and absolute number of CD4+CD25+ regulatory T cells in the tumor-draining lymph nodes of mice responding to therapy, and this was associated with the establishment of protective immunity. These findings pave the way for alternative therapeutic strategies based on the targeted delivery of biological and pharmacological cytotoxic compounds that not only kill most of the tumor cells but, more importantly, trigger an effective and long-lasting antitumor adaptive immune response. [source] Detergent-resistant membranes are platforms for actinoporin pore-forming activity on intact cellsFEBS JOURNAL, Issue 4 2006Jorge Alegre-Cebollada Sticholysin II is a pore-forming toxin produced by the sea anemone Stichodactyla helianthus. We studied its cytolytic activity on COS-7 cells. Fluorescence spectroscopy and flow cytometry revealed that the toxin permeabilizes cells to propidium cations in a dose-dependent and time-dependent manner. This permeabilization is impaired by preincubation of cells with cyclodextrin. Isolation of detergent-resistant cellular membranes showed that sticholysin II colocalizes with caveolin-1 in fractions corresponding to raft-like domains. The interaction of sticholysin II with such domains is only lipid dependent as it also occurs in the absence of any other membrane-associated protein. Toxin binding to raft-like lipid vesicles inhibited cell permeabilization. The results suggest that sticholysin II promotes pore formation in COS-7 cells through interaction with membrane domains which behave like cellular rafts. [source] Secretion of the Escherichia coli K-12 SheA hemolysin is independent of its cytolytic activityFEMS MICROBIOLOGY LETTERS, Issue 2 2001Francisco J del Castillo Abstract The Escherichia coli K-12 sheA gene encodes a pore-forming hemolysin that is secreted to the medium by a hitherto unidentified mechanism. To study SheA secretion, we constructed fusions between SheA and the mature form of the periplasmic enzyme ,-lactamase, and performed site-directed mutagenesis on these constructs. The SheA-Bla and Bla-SheA hybrid proteins displayed hemolytic activity and were efficiently exported to the extracellular medium. Our results with mutant hybrid proteins show that secretion of SheA is independent of its cytolytic activity, that secretion is paralleled by a transient leakage of periplasmic contents to the extracellular medium, and that deletion of the 11 C-terminal residues of SheA has no effect on its secretion and cytolytic activity. [source] Fish venom: pharmacological features and biological significanceFISH AND FISHERIES, Issue 2 2009Gisha Sivan Abstract Nearly 1200 species of marine fish are venomous and they account for two-third of the population of venomous vertebrates. Fish venoms are focused as a potential source of pharmacological agents and physiological tools that have evolved to target vital processes in the human body that appear to have more electivity than many drugs. Fish venoms possess cardiovascular, neuromuscular, oedematic and cytolytic activity. Lethal toxins have been isolated and purified, with some having LD50 values comparable to that of snake venoms. Cardiovascular activity seems to be the dominant effect of fish venoms in experimental models. Piscine venom acts both pre- and post-junctionally to produce depolarization of cell membranes. Studies on cytolytic activity of fish venom found that it produces lysis by forming hydrophilic pores in cell membranes which then result in cell lysis. Almost all fish venoms with neuromuscular activity also possesses cytolytic activity, and it is very likely that the two activities are related. Fish venom is known to induce intense and sustained edematogenic response. As piscine venoms have evolved for the same purpose, they show a number of similarities pharmacologically and it seems likely that most of the biological activities of any given toxin can be traced back to its cytolytic activity. A variety of toxins have been isolated from piscine venom. Although there is a complex balance between the components present in the venom of different fish, all of them seem to share similar activity , functionally and pharmacologically as well as structurally. [source] Response of lung ,, T cells to experimental sepsis in miceIMMUNOLOGY, Issue 1 2004Mark Hirsh Summary ,, T cells link innate and adaptive immune systems and may regulate host defence. Their role in systemic inflammation induced by trauma or infection (sepsis) is still obscured. The present study was aimed to investigate functions of lung ,, T cells and their response to experimental sepsis. Mice were subjected to caecal ligation and puncture (CLP) to induce sepsis and acute lung injury (ALI), or to the sham operation. Animals were killed 1, 4, and 7 days postoperatively; lungs were examined by histology, and isolated cells were studied by flow cytometry. Absolute number of ,, T cells progressively increased in lungs during sepsis, and reached a seven-fold increase at day 7 after CLP (3·84 ± 0·41 × 105/lung; P,=,0·0002 versus sham). A cellular dysfunction was revealed one day after CLP, as manifested by low cytolytic activity (22·3 ± 7·1%; P,<,0·05 versus sham), low interferon-, (IFN-,; 8·5 ± 2·5%; P,<,0·05 versus control) and interleukin-10 (IL-10) expression, and high tumour necrosis factor-, expression (19·5 ± 1·7%; P,<,0·05 versus control). The restoration of cytotoxicity, and increase in IFN-, and IL-10 expression was observed at day 7 of CLP-induced sepsis. In summary, our results demonstrate significant progressive accumulation of ,, T cells in lungs during CLP-induced ALI. The temporary functional suppression of lung ,, T cells found early after CLP may influence the outcome of sepsis, possibly being associated with uncontrolled inflammatory lung damage. [source] Differential modulation of CD8, by rat ,, and ,, T cells after activationIMMUNOLOGY, Issue 3 2001Frank Straube Summary Major histocompatibility complex (MHC) class I-restricted ,, T cells express the CD8,, heterodimer, which acts as a MHC class I-specific co-receptor. Rats are so far the only species with frequent expression of the CD8,, by MHC-unrestricted ,, T cells. This study compares CD8,, expression by splenic rat ,, and ,, T cells and reveals a lineage-specific difference in the control of CD8, expression. After activation in vitro, many ,, T cells, but not ,, T cells, persistently down-modulate the expression of CD8,, but not CD8,, at the RNA level. Down-regulation occurred after stimulation with T-cell receptor (TCR)-specific monoclonal antibody (mAb) and interleukin-2 (IL-2) or CD28-mediated costimulation, and after activation with phorbol 12-myristate 13-acetate (PMA) and ionomycin. Functional differences between modulating and non-modulating cells were not found with respect to interferon-, (IFN-,) production and cytolytic activity. The modulation could be indicative for a fundamental difference between ,, and ,, T cells and also limits the use of CD8, as a stable marker of ,, T-cell subsets. Possibly, CD8, modulation provides a mechanism to escape over-stimulation by (auto-)antigens by increasing the threshold of TCR-mediated activation in ,, T cells. [source] Real-time monitoring of the membrane-binding and insertion properties of the cholesterol-dependent cytolysin anthrolysin O from Bacillus anthracis,JOURNAL OF MOLECULAR RECOGNITION, Issue 4 2006Simon Cocklin Abstract Bacillus anthracis has recently been shown to secrete a potently hemolytic/cytolytic protein that has been designated anthrolysin O (ALO). In this work, we initiated a study of this potential anthrax virulence factor in an effort to understand the membrane,binding properties of this protein. Recombinant anthrolysin O (rALO35,512) and two N-terminally truncated versions of ALO (rALO390,512 and rALO403,512) from B. anthracis were overproduced in Escherichia coli and purified to homogeneity. The role of cholesterol in the cytolytic activity of ALO was probed in cellular cholesterol depletion assays using mouse and human macrophage-like lines, and also Drosophila Schneider 2 cells. Challenging the macrophage cells with rALO35,512, but not rALO390,512 or rALO403,512, resulted in cell death by lysis, with this cytolysis being abolished by depletion of the membrane cholesterol. Drosophila cells, which contain ergosterol as their major membrane sterol, were resistant to rALO-mediated cytolysis. In order to determine the molecular mechanism of this resistance, the interaction of rALO with model membranes comprised of POPC alone, or with a variety of structurally similar sterols including ergosterol, was probed using Biacore. Both rALO35,512 and rALO403,512 demonstrated robust binding to model membranes composed of POPC and cholesterol, with amount of protein bound proportional to the cholesterol content. Ergosterol supported greatly reduced binding of both rALO35,512 and rALO403,512, whereas other sterols tested did not support binding. The rALO403,512,membrane interaction demonstrated an equilibrium dissociation constant (KD) in the low nanomolar range, whereas rALO35,512 exhibited complex kinetics likely due to the multiple events involved in pore formation. These results establish the pivotal role of cholesterol in the action of rALO. The biosensor method developed to measure ALO recognition of cholesterol in a membrane environment could be extended to provide a platform for the screening of inhibitors of other membrane-binding proteins and peptides. Copyright© 2006 John Wiley & Sons, Ltd. [source] Chronic Alcohol Consumption Is Associated With an Increased Cytotoxic Profile of Circulating Lymphocytes That May Be Related With the Development of Liver InjuryALCOHOLISM, Issue 5 2010Francisco Javier Laso Background:, Apoptosis has recently emerged as a key component of acute and chronic liver diseases and it could be related to alcoholic liver disease. In the present study, we attempted to analyze the cytotoxic profile of circulating lymphocytes in chronic alcoholic patients grouped according to ethanol intake status and presence of liver disease. Methods:, We investigate the phenotypic and functional behavior of different compartments of peripheral blood (PB) cytotoxic T and natural killer (NK) cells in chronic alcoholic patients without liver disease and active ethanol intake (AWLD group; n = 22), and in subjects with alcohol liver cirrhosis (ALC group; n = 22). Results:, AWLD patients showed an expansion of both CD4+/CD8+ cytotoxic T cells and NK/T cells, in association with an enhanced cytolytic activity against K562 cells and a higher ability to induce in vitro expression of the pro-apoptotic protein APO2.7 in HepG2 cells. Conversely, ethanol intake in ALC patients was associated with decreased NK cell numbers, a reduced cytotoxic activity against K562 cells without significant changes in the expression of APO2.7, and a pro-fibrotic profile of cytokine secretion. Conclusions:, Overall, our results suggest that alcoholic patients display different phenotypical and functional changes in circulating PB cytotoxic lymphocytes according to the presence of alcoholic liver disease, which could be related to the development and progress of liver injury. [source] ,-Endorphin Neuronal Cell Transplant Reduces Corticotropin Releasing Hormone Hyperresponse to Lipopolysaccharide and Eliminates Natural Killer Cell Functional Deficiencies in Fetal Alcohol Exposed RatsALCOHOLISM, Issue 5 2009Nadka I. Boyadjieva Background:, Natural killer (NK) cell dysfunction is associated with hyperresponse of corticotropin releasing hormone (CRH) to immune challenge and with a loss of ,-endorphin (BEP) neurons in fetal alcohol exposed animals. Recently, we established a method to differentiate neural stem cells into BEP neurons using cyclic adenosine monophosphate (cAMP)-elevating agents in cultures. Hence, we determined whether in vitro differentiated BEP neurons could be used for reversing the compromised stress response and immune function in fetal alcohol exposed rats. Methods:, To determine the effect of BEP neuron transplants on NK cell function, we implanted in vitro differentiated BEP neurons into the paraventricular nucleus of pubertal and adult male rats exposed to ethanol or control in utero. The functionality of transplanted BEP neurons was determined by measuring proopiomelanocortin (POMC) gene expression in these cells and their effects on CRH gene expression under basal and after lipopolysaccaride (LPS) challenge. In addition, the effectiveness of BEP neurons in activating NK cell functions is determined by measuring NK cell cytolytic activity and interferon-, (IFN-,) production in the spleen and in the peripheral blood mononuclear cell (PBMC) following cell transplantation. Results:, We showed here that when these in vitro differentiated BEP neurons were transplanted into the hypothalamus, they maintain biological functions by producing POMC and reducing the CRH neuronal response to the LPS challenge. BEP neuronal transplants significantly increased NK cell cytolytic activity in the spleen and in the PBMC and increased plasma levels of IFN-, in control and fetal alcohol exposed rats. Conclusions:, These data further establish the BEP neuronal regulatory role in the control of CRH and NK cell cytolytic function and identify a possible novel therapy to treat stress hyperresponse and immune deficiency in fetal alcohol exposed subjects. [source] Reduction of Perforin, Granzyme B, and Cytokine Interferon , by Ethanol in Male Fischer 344 RatsALCOHOLISM, Issue 4 2003Madhavi Dokur Background: Chronic alcohol consumption can impair the immune system and predispose individuals to an increased risk of cancer and infection. Natural killer (NK) cells are the first line of defense against viral, bacterial, and fungal infections and play an important role in cellular resistance to malignancy and tumor metastasis. We have shown previously that ethanol administration suppresses NK cell cytolytic activity in male Fischer rats. This study analyzed the effects of ethanol on perforin, granzyme B, and the cytokine interferon (IFN)-,, factors that modulate NK cell cytolytic activity, to understand the molecular mechanism involved in ethanol's suppression of NK cell activity. Methods: A group of male Fischer rats was fed an ethanol-containing diet (8.7% v/v), whereas a control group was pair-fed an isocaloric diet. At the end of 2 weeks, animals were decapitated, and spleen tissues were immediately removed and used for analysis of NK cell cytolytic activity, perforin, granzyme B, and IFN-, messenger RNA (mRNA) or protein levels. The mRNA levels of perforin, granzyme B, and IFN-, were evaluated by quantitative real-time polymerase chain reaction, and protein levels of these factors were analyzed by Western blot, enzyme-linked immunosorbent assay, or enzymatic activity assay. Results: Ethanol reduced the NK cell cytolytic activity and decreased the mRNA expression of perforin, granzyme B, and IFN-, in ethanol-fed animals when compared with pair-fed animals. Ethanol also significantly reduced the protein levels of perforin and IFN-, and the enzyme activity of granzyme B in alcohol-fed animals as compared with pair-fed animals. Conclusions: These data suggest that chronic ethanol consumption may suppress NK cell cytolytic activity in male Fischer rats by decreasing the production, activity, or both of granzyme B, perforin, and IFN-,. [source] The role of intrahepatic immune effector cells in inflammatory liver injury and viral control during chronic hepatitis B infectionJOURNAL OF VIRAL HEPATITIS, Issue 3 2003T. J. Tang Summary. Cytotoxic T lymphocytes (CTL) and Kupffer cells play an important role in the immune control of hepatitis B virus (HBV), but may also induce liver injury during infection. We investigated the intrahepatic immune response in liver biopsies of chronic HBV patients in relation to inflammatory liver injury and viral control. Forty-seven liver biopsies from patients with chronic HBV with varying degrees of inflammation (ALT values) were selected. Acute hepatitis and normal liver specimens served as controls. Immune effector cells, cytotoxic effector molecules and cytokine producing cells were quantified after immunohistochemical staining in lobular and portal areas of the biopsies. The intralobular number of CD8+ T-lymphocytes was significantly decreased in biopsies of patients with high ALT (r = ,0.54; P < 0.001). Higher ALT-values were correlated with increased numbers of granzyme+ cells in portal areas (r = 0.65; P < 0.001) and higher numbers of intralobular Fas-L+ cells (r = 0.32; P = 0.05). Fas-L was expressed on Kupffer and lymphoid cells. More intralobular CD8+ T-lymphocytes were found in HBeAg, than in HBeAg+ patients (P = 0.002). But IFN- , and TNF- , producing cells were observed sporadically in chronic HBV patients. Hence, in chronic HBV infection, low viral replication and HBeAg negativity is related to increased presence of intralobular CD8+ T-lymphocytes. Persistence of the virus may be caused by the absence of cells producing anti-viral cytokines in the liver. Inflammatory liver injury during chronic HBV infection is probably not the result of increased numbers of infiltrating CD8+ T-lymphocytes, but of Fas-L expression by Kupffer cells and increased cytolytic activity of cells in portal areas. [source] Mutation in the LPS outer core biosynthesis gene, galU, affects LPS interaction with the RTX toxins ApxI and ApxII and cytolytic activity of Actinobacillus pleuropneumoniae serotype 1MOLECULAR MICROBIOLOGY, Issue 1 2008Mahendrasingh Ramjeet Summary Lipopolysaccharides (LPS) and Apx toxins are major virulence factors of Actinobacillus pleuropneumoniae, a pathogen of the respiratory tract of pigs. Here, we evaluated the effect of LPS core truncation in haemolytic and cytotoxic activities of this microorganism. We previously generated a highly attenuated galU mutant of A. pleuropneumoniae serotype 1 that has an LPS molecule lacking the GalNAc-Gal II-Gal I outer core residues. Our results demonstrate that this mutant exhibits wild-type haemolytic activity but is significantly less cytotoxic to porcine alveolar macrophages. However, no differences were found in gene expression and secretion of the haemolytic and cytotoxic toxins ApxI and ApxII, both secreted by A. pleuropneumoniae serotype 1. This suggests that the outer core truncation mediated by the galU mutation affects the toxins in their cytotoxic activities. Using both ELISA and surface plasmon resonance binding assays, we demonstrate a novel interaction between LPS and the ApxI and ApxII toxins via the core oligosaccharide. Our results indicate that the GalNAc-Gal II-Gal I trisaccharide of the outer core is fundamental to mediating LPS/Apx interactions. The present study suggests that a lack of binding between LPS and ApxI/II affects the cytotoxicity and virulence of A. pleuropneumoniae. [source] Listeriolysin O as cytotoxic component of an immunotoxinPROTEIN SCIENCE, Issue 6 2009Sabine Bergelt Abstract Monoclonal antibodies (mAbs) have been developed over the past years as promising anticancer therapeutics. The conjugation of tumor specific mAbs with cytotoxic molecules has been shown to improve their efficacy dramatically. These bifunctional immunotoxins, consisting of covalently linked antibodies and protein toxins, possess considerable potential in cancer therapy. Many of them are under investigation in clinical trials. As a result of general interest in new toxic components, we describe here the suitability of the bacterial protein Listeriolysin O (LLO) as cytotoxic component of an immunotoxin. Unique characteristics of LLO, such as its acidic pH optimum and the possibility to regulate the cytolytic activity by cysteine-oxidation, make LLO an interesting toxophore. Oxidized LLO shows a substantially decreased cytolytic activity when compared with the reduced protein as analyzed by hemolysis. Both oxidized and reduced LLO exhibit a cell-type-unspecific toxicity in cell culture with a significantly higher toxicity of reduced LLO. For cell-type-specific targeting of LLO to tumor cells, LLO was coupled to the dsFv fragment of the monoclonal antibody B3, which recognizes the tumor-antigen Lewis Y. The coupling of LLO to dsFv-B3 was performed via cysteine-containing polyionic fusion peptides that act as a specific heterodimerization motif. The novel immunotoxin B3-LLO could be shown to specifically eliminate antigen positive MCF7 cells with an EC50 value of 2.3 nM, whereas antigen negative cell lines were 80- to 250-fold less sensitive towards B3-LLO. [source] Enhancement of Immunogenicity of Jeg3 Cells by Ectopic Expression of HLA-A*0201 and CD80AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 3 2003Serpil Koc Problem: The choriocarcinoma cell line Jeg3 suppresses immunity in vitro by secretion of soluble factors like leukemia inhibitory factor suppressing leukocyte activation. The cells lack expression of classical human leukocyte antigen (HLA)-A and -B alleles but express some HLA-C, and non-classical HLA-G and -E. Upon binding to killing inhibitory receptor on natural killer (NK) cells, HLA-G prevents activation of cytolytic activity. We investigated whether Jeg3 cells are capable of immune stimulation after complementation with classical HLA and T cell costimulatory signal CD80. Method of study: Jeg3 cells were transduced to express HLA-A*0201 and/or CD80. Parental Jeg3 or transfectants Jeg3-A2, Jeg3-CD80 or Jeg3-CD80-A2 were used to stimulate allogeneic resting and activated peripheral blood lymphocytes (PBL). The different cell lines were loaded with a HLA-A2-restricted Epstein-Barr virus (EBV) recall antigen peptide epitope and antigen presenting ability was examined. T cell lines specific for Jeg3 and transfectants were generated from HLA-A2 matched and nonmatched donors and compared for expansion, phenotypes and cytolytic activity. Results: While all Jeg3 cell lines induced only marginal proliferation of resting T cells, phytohemagglutinin (PHA)-activated T cells were stimulated by CD80 or CD80-A2 expressing Jeg3. Only the transfectant Jeg3-CD80-A2 was capable of specific T cell stimulation by EBV recall antigen presentation. T cell lines of HLA-A2 non-matched donors stimulated with the Jeg3 transfectants showed significant expansion only when HLA-A2 and the costimulus CD80 were present. T cells from HLA-A2 positive donors did not expand significantly or differentially. No NK cells grew under any condition. In Jeg3-CD80-A2 stimulated T cells lines CD8+ cells expanded preferentially. These T cells exerted cytolytic activity toward all Jeg3 cell lines. Conclusion: Our data suggest that, in spite of immunosuppressive mechanisms, proliferative and cytolytic T cell responses are induced by Jeg3 cells when classical HLA- and/or costimulatory signals are present on the cells. [source] Enhancement of immunogenicity of JEG-3 Cells by ectopic expression of HLA-A*0201 and CD80AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 3 2002Serpil Koc The chorioncarcinoma cell line JEG-3 escapes immunity by secretion of leukocyte inhibitory factor suppressing leukocyte proliferation. The cells lack expression of classical HLA alleles but express nonclassical HLA-G, which binds to killer inhibitory receptor of natural killer cells, preventing cytolysis. We investigated whether JEG-3 cells are capable of immune stimulation after introduction of classical HLA and T-cell costimulatory signals. JEG-3 cells were transduced with vectors for HLA-A*0201 and/or CD80. Parental JEG-3, or JEG-3/A2, JEG-3/CD80, or JEG-3/A2/CD80 were used to stimulate allogeneic T cells. While parental JEG-3 cells induced only marginal proliferation of resting T cells, HLA-A2 or CD80 expressing JEG-3 induced enhanced proliferation. Double transfectants were most efficient. This difference was more obvious when T cells were preactivated by PHA. T cell lines restimulated with JEG-3 transfectants were characterized for expansion, phenotypes, and cytolytic activity. HLA-A2 matched and nonmatched donors were compared. T cells stimulated with JEG-3/A2 or JEG-3/CD80 were cytolytic towards parental JEG-3 cells. Again double positive JEG-3/A2/CD80 induced highest cytolytic activity, most obvious in HLA-nonmatched donors suggesting alloreactivity to HLA. Our data suggest that, in spite of immunosuppressive mechanisms, proliferative and cytolytic T cell responses are induced by JEG-3 cells when classical HLA and/or costimulatory signals are present on the cells. [source] Rapamycin and MPA, But Not CsA, Impair Human NK Cell Cytotoxicity Due to Differential Effects on NK Cell PhenotypeAMERICAN JOURNAL OF TRANSPLANTATION, Issue 9 2010D. N. Eissens Cyclosporin A (CsA), rapamycin (Rapa) and mycophenolic acid (MPA) are frequently used for GVHD prophylaxis and treatment after allogeneic stem cell transplantation (SCT). As NK cells have received great interest for immunotherapeutic applications in SCT, we analyzed the effects of these drugs on human cytokine-stimulated NK cells in vitro. Growth-kinetics of CsA-treated cultures were marginally affected, whereas MPA and Rapa severely prevented the outgrowth of CD56bright NK cells. Single-cell analysis of NK cell receptors using 10-color flow cytometry, revealed that CsA-treated NK cells gained a similar expression profile as cytokine-stimulated control NK cells, mostly representing NKG2A+KIR,NCR+ cells. In contrast, MPA and Rapa inhibited the acquisition of NKG2A and NCR expression and NK cells maintained an overall NKG2A,KIR+NCR+/, phenotype. This was reflected in the cytolytic activity, as MPA- and Rapa-treated NK cells, in contrast to CsA-treated NK cells, lost their cytotoxicity against K562 target cells. Upon target encounter, IFN-, production was not only impaired by MPA and Rapa, but also by CsA. Overall, these results demonstrate that CsA, MPA and Rapa each have distinct effects on NK cell phenotype and function, which may have important implications for NK cell function in vivo after transplantation. [source] Immune hierarchy among HIV-1 CD8+ T cell epitopes delivered by dendritic cells depends on MHC-I binding irrespective of mode of loading and immunization in HLA-A*0201 miceAPMIS, Issue 11 2009HENRIK N. KLOVERPRIS Recent human immunodeficiency virus type 1 (HIV-1) vaccination strategies aim at targeting a broad range of cytotoxic T lymphocyte (CTL) epitopes from different HIV-1 proteins by immunization with multiple CTL epitopes simultaneously. However, this may establish an immune hierarchical response, where the immune system responds to only a small number of the epitopes administered. To evaluate the feasibility of such vaccine strategies, we used the human leukocyte antigen (HLA)-A*0201 transgenic (tg) HHD murine in vivo model and immunized with dendritic cells pulsed with seven HIV-1-derived HLA-A*0201 binding CTL epitopes. The seven peptides were simultaneously presented on the same dendritic cell (DC) or on separate DCs before immunization to one or different lymphoid compartments. Data from this study showed that the T-cell response, as measured by cytolytic activity and ,-interferon (IFN-,)-producing CD8+ T cells, mainly focused on two of seven administered epitopes. The magnitude of individual T-cell responses induced by immunization with multiple peptides correlated with their individual immunogenicity that depended on major histocompatibility class I binding and was not influenced by mode of loading or mode of immunization. These findings may have implications for the design of vaccines based on DCs when using multiple epitopes simultaneously. [source] Presence of CD4+CD8+ double-positive T cells with very high interleukin-4 production potential in lesional skin of patients with systemic sclerosisARTHRITIS & RHEUMATISM, Issue 10 2007Yann Parel Objective Fibrotic skin changes in systemic sclerosis (SSc) are preceded by the appearance of an inflammatory infiltrate rich in T cells. Since no direct comparison with T cells in normal skin has been performed previously, this study was undertaken to functionally characterize T cells in the skin of patients with early active SSc and in normal skin. Methods We characterized coreceptor expression, T cell receptor (TCR) usage, cytokine production, and helper and cytolytic activity of T cell lines and clones established from skin biopsy specimens from 6 SSc patients and 4 healthy individuals. Immunofluorescence analysis of skin biopsy and peripheral blood samples was performed to confirm the presence of specific subsets in vivo. Results A distinct subset expressing both CD4 and CD8,/, coreceptors at high levels (double-positive [DP]) was present in T cell lines from SSc and normal skin. DP T cells actively transcribed both accessory molecules, exerted clonally distributed cytolytic and helper activity, and expressed TCR clonotypes distinct from those in CD4+ or CD8+ single-positive (SP) T cells. In SSc skin, DP T cells produced very high levels of interleukin-4 (IL-4) compared with CD4+ SP T cells. Furthermore, DP T cells were directly identified in SSc skin, thus providing evidence that they are a distinct subset in vivo. Conclusion The present findings show that T cells with the unusual CD4+CD8+ DP phenotype are present in the skin. Their very high level of IL-4 production in early active SSc may contribute to enhanced extracellular matrix deposition by fibroblasts. [source] Effect of millimeter waves on natural killer cell activationBIOELECTROMAGNETICS, Issue 1 2005V.R. Makar Abstract Millimeter wave therapy (MMWT) is being widely used for the treatment of many diseases in Russia and other East European countries. MMWT has been reported to reduce the toxic effects of chemotherapy on the immune system. The present study was undertaken to investigate whether millimeter waves (MMWs) can modulate the effect of cyclophosphamide (CPA), an anticancer drug, on natural killer (NK) cell activity. NK cells play an important role in the antitumor response. MMWs were produced with a Russian-made YAV-1 generator. The device produced modulated 42.2,±,0.2 GHz radiation through a 10,×,20 mm rectangular output horn. Mice, restrained in plastic tubes, were irradiated on the nasal area. Peak SAR at the skin surface and peak incident power density were measured as 622,±,100 W/kg and 31,±,5 mW/cm2, respectively. The maximum temperature elevation, measured at the end of 30 min, was 1 °C. The animals, restrained in plastic tubes, were irradiated on the nasal area. CPA injection (100 mg/kg) was given intraperitoneally on the second day of 3-days exposure to MMWs. All the irradiation procedures were performed in a blinded manner. NK cell activation and cytotoxicity were measured after 2, 5, and 7 days following CPA injection. Flow cytometry of NK cells showed that CPA treatment caused a marked enhancement in NK cell activation. The level of CD69 expression, which represents a functional triggering molecule on activated NK cells, was increased in the CPA group at all the time points tested as compared to untreated mice. However, the most enhancement in CD69 expression was observed on day 7. A significant increase in TNF-, level was also observed on day 7 following CPA administration. On the other hand, CPA caused a suppression of the cytolytic activity of NK cells. MMW irradiation of the CPA treated groups resulted in further enhancement of CD69 expression on NK cells, as well as in production of TNF-,. Furthermore, MMW irradiation restored CPA induced suppression of the cytolytic activity of NK cells. Our results show that MMW irradiation at 42.2 GHz can up-regulate NK cell functions. Bioelectromagnetics 26:10,19, 2005. © 2004 Wiley-Liss, Inc. [source] Carbon-ion beam treatment induces systemic antitumor immunity against murine squamous cell carcinomaCANCER, Issue 15 2010Akinao Matsunaga MD Abstract BACKGROUND: Carbon-ion beam (CIB) treatment is a powerful tool for controlling primary tumors in the clinical setting. However, to date, few clinical or experimental studies have investigated the effects of CIB treatment on tumor recurrence and antitumor immunity. METHODS: A multiple challenge test was performed using syngenic and nude mouse models of a poorly immunogenic squamous cell carcinoma cell line (SCCVII) after CIB treatment at a clinically available dose (77 kiloelectron volts [keV]/,m) to primary tumors. To further examine changes in antitumor immunity in this model, the authors used dendritic cell (DC)-based immunotherapy. RESULTS: In a syngenic model, CIB treatment itself resulted not only in efficient elimination of the primary tumor but also in a dramatic reduction of tumor formation after secondary tumor challenge at a contralateral site (P < .0001). Conversely, CIB treatment eliminated neither the primary nor the secondary tumor in nude mice. This antitumor effect produced by CIB treatment was enhanced significantly by combining it with DC immunotherapy (P = .0007). Combined CIB and DC treatment induced more intense cytolytic activity than CIB in a chromium-release assay. The third challenge tests, which included challenge with a third-party tumor cell line (FM3A) and effector depletion, revealed that the antitumor effects were the results of tumor-specific, long-lasting antitumor immunity through CD8-positive T lymphocytes. CONCLUSIONS: To the authors' knowledge, this is the first demonstration of strong antitumor immunity induced by CIB treatment in a dermal tumor, and this effect was enhanced by combining it with DC-based immunotherapy. The authors concluded that this combination warrants further investigation as a promising modality for the prevention of tumor recurrence. Cancer 2010. © 2010 American Cancer Society. [source] | ||||||||||||||||