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Alan M Krensky - One of the best experts on this subject based on the ideXlab platform.
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15 kDa Granulysin Causes Differentiation of Monocytes to Dendritic Cells but Lacks Cytotoxic Activity
Journal of immunology (Baltimore Md. : 1950), 2012Co-Authors: Carol Clayberger, Michael W Finn, Tianhong Wang, Reena V. Saini, Christine Wilson, Valarie A. Barr, Marianna Sabatino, Luciano Castiello, David F. Stroncek, Alan M KrenskyAbstract:Granulysin is expressed as two isoforms by human cytotoxic cells: a single mRNA gives rise to 15 kDa Granulysin, a portion of which is cleaved to a 9 kDa protein. Studies with recombinant 9 kDa Granulysin have demonstrated its cytolytic and proinflammatory properties, but much less is known about the biologic function of the 15 kDa isoform. In this study, we show that the subcellular localization and functions of 9 and 15 kDa Granulysin are largely distinct. Nine kilodalton Granulysin is confined to cytolytic granules that are directionally released following target cell recognition. In contrast, 15 kDa Granulysin is located in distinct granules that lack perforin and granzyme B and that are released by activated cytolytic cells. Although recombinant 9 kDa Granulysin is cytolytic against a variety of tumors and microbes, recombinant 15 kDa Granulysin is not. The 15 kDa isoform is a potent inducer of monocytic differentiation to dendritic cells, but the 9 kDa isoform is not. In vivo, mice expressing Granulysin show markedly improved antitumor responses, with increased numbers of activated dendritic cells and cytokine-producing T cells. Thus, the distinct functions of Granulysin isoforms have major implications for diagnosis and potential new therapies for human disease.
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Granulysin Delivered by Cytotoxic Cells Damages Endoplasmic Reticulum and Activates Caspase-7 in Target Cells
Journal of immunology (Baltimore Md. : 1950), 2011Co-Authors: Reena V. Saini, Michael W Finn, Alan M Krensky, Tianhong Wang, Christine Wilson, Carol ClaybergerAbstract:Granulysin is a human cytolytic molecule present in cytotoxic granules with perforin and granzymes. Recombinant 9-kDa Granulysin kills a variety of microbes, including bacteria, yeast, fungi, and parasites, and induces apoptosis in tumor cells by causing intracellular calcium overload, mitochondrial damage, and activation of downstream caspases. Reasoning that Granulysin delivered by cytotoxic cells may work in concert with other molecules, we crossed Granulysin transgenic (GNLY+/−) mice onto perforin (perf)- or granzyme B (gzmb)-deficient mice to examine Granulysin-mediated killing in a more physiologic whole-cell system. Splenocytes from these animals were activated in vitro with IL-15 to generate cytolytic T cells and NK cells. Cytotoxic cells expressing Granulysin require perforin, but not granzyme B, to cause apoptosis of targets. Whereas granzyme B induces mitochondrial damage and activates caspases-3 and -9 in targets, cytotoxic cell-delivered Granulysin induces endoplasmic reticulum stress and activates caspase-7 with no effect on mitochondria or caspases-3 and -9. In addition, recombinant Granulysin and cell-delivered Granulysin activate distinct apoptotic pathways in target cells. These findings suggest that cytotoxic cells have evolved multiple nonredundant cell death pathways, enabling host defense to counteract escape mechanisms employed by pathogens or tumor cells.
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expression and purification of 15 kda Granulysin utilizing an insect cell secretion system
Protein Expression and Purification, 2011Co-Authors: Michael W Finn, Carol Clayberger, Alan M KrenskyAbstract:Granulysin is an antimicrobial and proinflammatory protein expressed in activated human T cells and natural killer cells. A single mRNA produces the 15 kDa isoform which is then cleaved at the amino and carboxy termini to produce the 9 kDa isoform. Recombinant 9 kDa Granulysin has been studied in detail but little is known about the function of the 15 kDa isoform, and no protocol has been published describing expression and purification of this form. Two commercially available preparations of the recombinant 15 kDa Granulysin contain tags that may affect function. Here we describe for the first time a method to produce 15 kDa Granulysin as a secreted protein from insect cells. The 15 kDa Granulysin is purified using a HiTrap Heparin column and a Resource S column. A typical a yield of purified 15 kDa Granulysin is 0.6 mg/L of insect cell supernatant.
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induction of Granulysin and perforin cytolytic mediator expression in 10 week old infants vaccinated with bcg at birth
Clinical & Developmental Immunology, 2011Co-Authors: Patricia L Semple, Alan M Krensky, Marcia Watkins, Virginia Davids, Willem A Hanekom, Gilla Kaplan, Stanley R RessAbstract:Background. While vaccination at birth with Mycobacterium bovis Bacilli Calmette-Guerin (BCG) protects against severe childhood tuberculosis, there is no consensus as to which components of the BCG-induced immune response mediate this protection. However, Granulysin and perforin, found in the granules of cytotoxic T lymphocytes and Natural Killer (NK) cells, can kill intracellular mycobacteria and are implicated in protection against Mycobacterium tuberculosis. Methods. We compared the cellular expression of Granulysin and perforin cytolytic molecules in cord blood and peripheral blood from 10-week-old infants vaccinated at birth with either Japanese or Danish BCG, administered either intradermally or percutaneously. Results. In cord blood, only CD56+ NK cells expressed Granulysin and perforin constitutively. These cytolytic mediators were upregulated in CD4+ and CD8+ cord blood cells by ex vivo stimulation with BCG but not with PPD. Following BCG vaccination of neonates, both BCG and PPD induced increased expression of Granulysin and perforin by CD4+ and CD8+ T cells. There was no difference in expression of cytolytic molecules according to vaccination route or strain. Conclusions. Constitutive expression of perforin and Granulysin by cord blood NK-cells likely provides innate immunity, while BCG vaccination-induced expression of these cytolytic mediators may contribute towards protection of the neonate against tuberculosis.
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Granulysin production and anticryptococcal activity is dependent upon a far upstream enhancer that binds stat5 in human peripheral blood cd4 t cells
Journal of Immunology, 2010Co-Authors: Junji Xing, Alan M Krensky, Christopher H. Mody, Shuai Wang, Chunfu ZhengAbstract:Previous studies have demonstrated that STAT5 is critical for expression of Granulysin and antimicrobial activity. Because the signaling pathway and the resultant microbicidal activity are defective in HIV-infected patients, the mechanism by which STAT5 leads to Granulysin expression is of great interest. In the current study, IL-2–stimulated CRL-2105 CD4+ T cells expressed Granulysin and killed Cryptococcus neoformans similar to primary CD4+ T cells. The enhancer activity of the upstream element of the Granulysin promoter was analyzed in primary CD4+ T cells and CRL-2105 T cells with a luciferase reporter assay, and a STAT5 binding site, 18,302 to 18,177 bp upstream of the transcription start site, was identified as an enhancer. Additionally, the enhancer functioned in the context of heterologous SV40 promoter irrespective of its transcriptional orientation. Chromatin immunoprecipitation and EMSAs demonstrated that the enhancer element bound STAT5 both in vivo and in vitro, and mutation of the STAT5 binding site abrogated its enhancer activity. Furthermore, overexpression of a dominant negative STAT5a abolished the enhancer activity of the STAT5 binding site and abrogated the anticryptococcal activity of IL-2–stimulated primary CD4+ T cells. Taken together, these data provide details about the complex regulation leading to Granulysin expression and anticryptococcal activity in primary CD4+ T cells.
Carol Clayberger - One of the best experts on this subject based on the ideXlab platform.
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15 kDa Granulysin Causes Differentiation of Monocytes to Dendritic Cells but Lacks Cytotoxic Activity
Journal of immunology (Baltimore Md. : 1950), 2012Co-Authors: Carol Clayberger, Michael W Finn, Tianhong Wang, Reena V. Saini, Christine Wilson, Valarie A. Barr, Marianna Sabatino, Luciano Castiello, David F. Stroncek, Alan M KrenskyAbstract:Granulysin is expressed as two isoforms by human cytotoxic cells: a single mRNA gives rise to 15 kDa Granulysin, a portion of which is cleaved to a 9 kDa protein. Studies with recombinant 9 kDa Granulysin have demonstrated its cytolytic and proinflammatory properties, but much less is known about the biologic function of the 15 kDa isoform. In this study, we show that the subcellular localization and functions of 9 and 15 kDa Granulysin are largely distinct. Nine kilodalton Granulysin is confined to cytolytic granules that are directionally released following target cell recognition. In contrast, 15 kDa Granulysin is located in distinct granules that lack perforin and granzyme B and that are released by activated cytolytic cells. Although recombinant 9 kDa Granulysin is cytolytic against a variety of tumors and microbes, recombinant 15 kDa Granulysin is not. The 15 kDa isoform is a potent inducer of monocytic differentiation to dendritic cells, but the 9 kDa isoform is not. In vivo, mice expressing Granulysin show markedly improved antitumor responses, with increased numbers of activated dendritic cells and cytokine-producing T cells. Thus, the distinct functions of Granulysin isoforms have major implications for diagnosis and potential new therapies for human disease.
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Granulysin Delivered by Cytotoxic Cells Damages Endoplasmic Reticulum and Activates Caspase-7 in Target Cells
Journal of immunology (Baltimore Md. : 1950), 2011Co-Authors: Reena V. Saini, Michael W Finn, Alan M Krensky, Tianhong Wang, Christine Wilson, Carol ClaybergerAbstract:Granulysin is a human cytolytic molecule present in cytotoxic granules with perforin and granzymes. Recombinant 9-kDa Granulysin kills a variety of microbes, including bacteria, yeast, fungi, and parasites, and induces apoptosis in tumor cells by causing intracellular calcium overload, mitochondrial damage, and activation of downstream caspases. Reasoning that Granulysin delivered by cytotoxic cells may work in concert with other molecules, we crossed Granulysin transgenic (GNLY+/−) mice onto perforin (perf)- or granzyme B (gzmb)-deficient mice to examine Granulysin-mediated killing in a more physiologic whole-cell system. Splenocytes from these animals were activated in vitro with IL-15 to generate cytolytic T cells and NK cells. Cytotoxic cells expressing Granulysin require perforin, but not granzyme B, to cause apoptosis of targets. Whereas granzyme B induces mitochondrial damage and activates caspases-3 and -9 in targets, cytotoxic cell-delivered Granulysin induces endoplasmic reticulum stress and activates caspase-7 with no effect on mitochondria or caspases-3 and -9. In addition, recombinant Granulysin and cell-delivered Granulysin activate distinct apoptotic pathways in target cells. These findings suggest that cytotoxic cells have evolved multiple nonredundant cell death pathways, enabling host defense to counteract escape mechanisms employed by pathogens or tumor cells.
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expression and purification of 15 kda Granulysin utilizing an insect cell secretion system
Protein Expression and Purification, 2011Co-Authors: Michael W Finn, Carol Clayberger, Alan M KrenskyAbstract:Granulysin is an antimicrobial and proinflammatory protein expressed in activated human T cells and natural killer cells. A single mRNA produces the 15 kDa isoform which is then cleaved at the amino and carboxy termini to produce the 9 kDa isoform. Recombinant 9 kDa Granulysin has been studied in detail but little is known about the function of the 15 kDa isoform, and no protocol has been published describing expression and purification of this form. Two commercially available preparations of the recombinant 15 kDa Granulysin contain tags that may affect function. Here we describe for the first time a method to produce 15 kDa Granulysin as a secreted protein from insect cells. The 15 kDa Granulysin is purified using a HiTrap Heparin column and a Resource S column. A typical a yield of purified 15 kDa Granulysin is 0.6 mg/L of insect cell supernatant.
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Biology and clinical relevance of Granulysin
Tissue antigens, 2009Co-Authors: Alan M Krensky, Carol ClaybergerAbstract:Granulysin is a cytolytic and proinflammatory molecule first identified by a screen for genes expressed 'late' (3-5 days) after activation of human peripheral blood mononuclear cells. Granulysin is present in cytolytic granules of cytotoxic T lymphocytes and natural killer cells. Granulysin is made in a 15-kDa form that is cleaved into a 9-kDa form at both the amino and the carboxy termini. The 15-kDa form is constitutively secreted, and its function remains poorly understood. The 9-kDa form is released by receptor-mediated granule exocytosis. Nine kiloDalton Granulysin is broadly cytolytic against tumors and microbes, including gram-positive and gram-negative bacteria, fungi/yeast and parasites. It kills the causative agents of both tuberculosis and malaria. Granulysin is also a chemoattractant for T lymphocytes, monocytes and other inflammatory cells and activates the expression of a number of cytokines, including regulated upon activation T cell expressed and secreted (RANTES), monocyte chemoattractant protein (MCP)-1, MCP-3, macrophage inflammatory protein (MIP)-1 alpha, interleukin (IL)-10, IL-1, IL-6 and interferon (IFN)-alpha. Granulysin is implicated in a myriad of diseases including infection, cancer, transplantation, autoimmunity, skin and reproductive maladies. Small synthetic forms of Granulysin are being developed as novel antibiotics. Studies of the full-length forms may give rise to new diagnostics and therapeutics for use in a wide variety of diseases.
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In vitro and in vivo antimicrobial activity of Granulysin-derived peptides against Vibrio cholerae
The Journal of antimicrobial chemotherapy, 2008Co-Authors: Ana Paula Galvão Da Silva, Alan M Krensky, Shu Chen Lyu, Donovan Unks, Renata Zbozien-pacamaj, Xi Chen, Carol ClaybergerAbstract:Received 20 September 2007; returned 26 November 2007; revised 14 January 2008; accepted 24 January 2008 Objectives: To determine the antibacterial activity of synthetic peptides derived from the cationic antimicrobial peptide Granulysin against Vibrio cholerae. Methods: The antibacterial activity of Granulysin-derived peptides was assessed in vitro by microtitre and cfu assays. Toxicity against human peripheral blood mononuclear cells (PBMCs) was measured by propidium iodide uptake and haemolysis by measuring the levels of haemoglobin released after incubation of red blood cells (RBCs) with Granulysin peptides. The ability of Granulysin peptides to control bacterial growth in vivo was tested by the treatment of suckling mice infected with V. cholerae with Granulysin peptides, administered by gavage 1 h after infection and determining the number of bacteria in the small and large intestines 24 h after infection. Results: All peptides tested inhibited V. cholerae growth in vitro, and they were more effective against stationary phase cells. Two peptides, G12.21 and G14.15, effectively controlled bacterial growth in vivo. The peptides did not lyse RBCs and, with the exception of two peptides, exhibited very little toxicity against human PBMCs. Conclusions: These results suggest that Granulysin-derived peptides are candidates for the development of new agents for the treatment of V. cholerae infection.
Shigeru Saito - One of the best experts on this subject based on the ideXlab platform.
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Increased prevalence of peripheral blood Granulysin-producing cytotoxic T lymphocytes in preeclampsia
Journal of Reproductive Immunology, 2011Co-Authors: Attila Molvarec, Arihiro Shiozaki, Mika Ito, Gergely Toldi, Balázs Stenczer, András Szarka, Akitoshi Nakashima, Barna Vásárhelyi, János Rigó, Shigeru SaitoAbstract:Abstract Preeclampsia (PE) is a severe complication of pregnancy characterized by an excessive maternal systemic inflammatory response with activation of both the innate and adaptive arms of the immune system. Granulysin is a cytolytic and pro-inflammatory molecule expressed by activated human cytotoxic T lymphocytes and natural killer (NK) cells. Recent data show that serum Granulysin levels are elevated in preeclampsia. The purpose of this study was to determine whether the proportion of peripheral blood cytotoxic T lymphocytes and NK cells that express intracellular Granulysin is altered in PE. Twenty-two preeclamptic patients and 29 healthy pregnant women were involved in this case-control study. Intracellular Granulysin expression of lymphocytes was determined with flow cytometric examination. In healthy pregnant women, the majority of NK cells and a small fraction of cytotoxic T cells expressed Granulysin in their cytoplasma (median (25–75 percentile): 53.5 (45.6–68.0)% and 13.8 (8.5–23.1)%, respectively). In PE, the percentage of Granulysin-positive cytotoxic T lymphocytes was markedly increased, while the proportion of Granulysin-producing NK cells was unchanged as compared to healthy pregnant women (for cytotoxic T cells: 34.1 (19.3–45.6)%, p p >0.05). Maternal age of healthy pregnant women showed a significant inverse correlation with the frequency of Granulysin-expressing NK cells (Spearman R =−0.44, p R =0.43, p
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Granulysin Produced by Uterine Natural Killer Cells Induces Apoptosis of Extravillous Trophoblasts in Spontaneous Abortion
The American journal of pathology, 2008Co-Authors: Akitoshi Nakashima, Arihiro Shiozaki, Mika Ito, Kazuyuki Ogawa, Kinya Nagata, Yasushi Takamori, Subaru Myojo, Mikiko Tatematsu, Masatoshi Sakai, Shigeru SaitoAbstract:Immune changes are known to occur in recurrent spontaneous abortion, but it is unclear whether either maternal natural killer (NK) cells or T cells attack fetus-derived trophoblasts. To clarify the immunological causes of spontaneous abortion, we examined the relationship between cytotoxic granule proteins in decidual lymphocytes, such as Granulysin, granzyme B, and perforin, and the induction of apoptosis in extravillous trophoblasts (EVTs). The number of Granulysin-positive CD56bright NK cells increased significantly in the decidua basalis during spontaneous abortion compared with normal pregnancy; however, granzyme B- and perforin-positive cells did not change. Interestingly, the expression of Granulysin was also detected in the nuclei of EVTs in spontaneous abortion samples. When IL-2-stimulated CD56bright NK cells were cocultured with EVT cells (HTR-8/SV40neo), Granulysin was found initially in the cytoplasm and then accumulated in the nuclei of the HTR-8/SV40neo cells. Furthermore, transfected cells expressing a GFP-Granulysin fusion protein induced apoptosis in HTR-8/SV40neo cells independently of caspases. Our results suggest that Granulysin-positive uterine NK cells attack EVTs; subsequently, the uNK-derived Granulysin actively accumulates in the nuclei of EVTs, causing the death of EVTs due to apoptosis. These data support a new apoptosis pathway for trophoblasts via uNK-derived Granulysin, suggesting that Granulysin is involved in spontaneous abortion.
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Plasma Granulysin concentrations and preeclampsia risk.
Clinical biochemistry, 2006Co-Authors: Chunfang Qiu, Shigeru Saito, Kazuyuki Ogawa, Kinya Nagata, Masatoshi Sakai, Michelle A. WilliamsAbstract:Abstract Objective: Epidemiological, clinical and histological data suggest intriguing similarities between preeclampsia and graft–host-rejection. Granulysin, a novel biomarker of overall cellular immunity, is secreted by natural killer cells and cytotoxic T lymphocytes, which are associated with graft–host-rejection. Plasma Granulysin was elevated in Japanese preeclamptic women. Design and Methods: 50 preeclampsia cases and 50 normotensive controls (USA) were studied. Plasma Granulysin at delivery was determined using enzyme immunoassay. Logistic regression procedures were used to estimate odds ratios (OR) and 95% confidence intervals (CI). Results: Granulysin were elevated in preeclampsia cases compared with controls (3.01 ± 0.18 vs. 2.22 ± 0.14 ng/mL, p Conclusions: These data offer further evidence of a predominant Th1 immune status associated with preeclampsia. Prospective studies are needed to determine whether Granulysin is elevated early in pregnancy.
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Serum Granulysin is a marker for Th1 type immunity in pre-eclampsia
Clinical and experimental immunology, 2004Co-Authors: Masatoshi Sakai, K. Ogawa, K. Nagata, Arihiro Shiozaki, Satoshi Yoneda, Yasushi Sasaki, Shigeru SaitoAbstract:Recent studies suggest that pre-eclampsia is associated with a Th1 predominant state and may be considered a failure of tolerance. Granulysin is a cytotoxic granule protein of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). Recently, we developed an enzyme-linked immunosorbent assay (ELISA) system for detecting serum Granulysin, and reported that serum Granulysin is a useful marker to evaluate the cell-mediated immunity. In this study, we show that the serum levels of Granulysin were significantly elevated in pre-eclamptic patients compared with those in normal pregnancy subjects. In addition, the serum Granulysin levels in pre-eclamptic patients were well associated with mean blood pressure, percentage of peripheral blood Th1 cells and Th1/Th2 ratios. The present results suggest that the serum Granulysin levels would be a useful and novel serum marker to evaluate the Th1/Th2 balance, especially Th1 type immunity in pre-eclampsia.
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The placental bed of patients with preeclampsia has increased density of Granulysin positive immunocytes
American Journal of Obstetrics and Gynecology, 2004Co-Authors: Yeon Mee Kim, Shigeru Saito, Mi Ran Kim, Gi Jin Kim, Jimmy Espinoza, Ricardo Gomez, Chong Jai Kim, Bo Hyun Yoon, Moshe Mazor, Roberto RomeroAbstract:70 71 THE PLACENTAL BED OF PATIENTS WITH PREECLAMPSIA HAS INCREASED DENSITY OF Granulysin POSITIVE IMMUNOCYTES YEON MEE KIM, MI RAN KIM, GI JIN KIM, JIMMY ESPINOZA, RICARDO GOMEZ, CHONG JAI KIM, BO HYUN YOON, MOSHE MAZOR, SHIGERU SAITO, ROBERTO ROMERO, Wayne State University School of Medicine, Department of Pathology, Detroit, Michigan, Perinatology Research Branch, NICHD, NIH, DHHS, Bethesda, Maryland, CEDIP, Sotero del Rio Hospital, Puente Alto, Chile, Chile, Seoul National University College of Medicine, Seoul, Korea, South Korea, Soroka University Medical Center, Beer Sheva, Korea, Israel, Toyama Medical and Pharmaceutical University, Department of Obstetrics and Gynecology, Toyama, Japan, Japan OBJECTIVE: Granulysin, a novel cytolytic molecule of natural killer cells and cytotoxic T lymphocytes, induces cell-mediated apoptosis in mammalian cells and has antimicrobial properties. Granulysin mRNA expression is increased during acute allograft rejection and the serum Granulysin level is increased in preeclampsia. This has been interpreted as supporting evidence of an abnormal allogeneic response biased toward a Th-1 type response (Clin Exp Immunol 2004; 136:114). This study was conducted to characterize the expression of Granulysin in the fetomaternal interface (placental bed) of women with and without preeclampsia. STUDY DESIGN: Placental bed biopsies were obtained from patients with: (1) preterm delivery and intact membranes (PTD) without chorioamnionitis (n = 15); and (2) preterm severe pre-eclampsia (n = 15). Patients were matched for gestational age. Granulysin and CD3 (T cell marker) expressions were determined in the placental bed using double immunohistochemistry. Image analysis to determine the number of Granulysin positive cells was conducted in 10 microscopic fields (!400). Non-parametric statistics were employed for analysis. RESULTS: (1) Granulysin was present in scattered mononuclear cells and polymorphonuclear leukocytes in the decidua basalis, the myometrium and vascular wall in the placental bed. (2) The median number of Granulysin positive cells was significantly higher in patients with preeclampsia than in patients without preeclampsia (P ! .0001). (3) The median number of Granulysin/ CD3+ T lymphocytes was significantly lower in patients with preeclampsia than in those without preeclampsia (P = .0001). CONCLUSION: The number of Granulysin positive immunocytes is increased in the placental bed of women with preeclampsia. These results suggest the SMFM Abstracts S31
Kazuyuki Ogawa - One of the best experts on this subject based on the ideXlab platform.
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Granulysin Produced by Uterine Natural Killer Cells Induces Apoptosis of Extravillous Trophoblasts in Spontaneous Abortion
The American journal of pathology, 2008Co-Authors: Akitoshi Nakashima, Arihiro Shiozaki, Mika Ito, Kazuyuki Ogawa, Kinya Nagata, Yasushi Takamori, Subaru Myojo, Mikiko Tatematsu, Masatoshi Sakai, Shigeru SaitoAbstract:Immune changes are known to occur in recurrent spontaneous abortion, but it is unclear whether either maternal natural killer (NK) cells or T cells attack fetus-derived trophoblasts. To clarify the immunological causes of spontaneous abortion, we examined the relationship between cytotoxic granule proteins in decidual lymphocytes, such as Granulysin, granzyme B, and perforin, and the induction of apoptosis in extravillous trophoblasts (EVTs). The number of Granulysin-positive CD56bright NK cells increased significantly in the decidua basalis during spontaneous abortion compared with normal pregnancy; however, granzyme B- and perforin-positive cells did not change. Interestingly, the expression of Granulysin was also detected in the nuclei of EVTs in spontaneous abortion samples. When IL-2-stimulated CD56bright NK cells were cocultured with EVT cells (HTR-8/SV40neo), Granulysin was found initially in the cytoplasm and then accumulated in the nuclei of the HTR-8/SV40neo cells. Furthermore, transfected cells expressing a GFP-Granulysin fusion protein induced apoptosis in HTR-8/SV40neo cells independently of caspases. Our results suggest that Granulysin-positive uterine NK cells attack EVTs; subsequently, the uNK-derived Granulysin actively accumulates in the nuclei of EVTs, causing the death of EVTs due to apoptosis. These data support a new apoptosis pathway for trophoblasts via uNK-derived Granulysin, suggesting that Granulysin is involved in spontaneous abortion.
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Reduction in resting plasma Granulysin as a marker of increased training load.
Exercise immunology review, 2007Co-Authors: Cecilia M. Shing, Xiumin Zhang, Kazuyuki Ogawa, Ryoichi Nagatomi, Jonathan M. Peake, Katsuhiko Suzuki, David G. Jenkins, Jeff S. CoombesAbstract:Granulysin is a cytolytic granule protein released by natural killer cells and activated cytotoxic T lymphocytes. The influence of exercise training on circulating Granulysin concentration is unknown, as is the relationship between Granulysin concentration, natural killer cell number and natural killer cell cytotoxicity. We examined changes in plasma Granulysin concentration, natural killer cell number and cytotoxicity following acute exercise and different training loads. Fifteen highly trained male cyclists completed a baseline 40-km cycle time trial (TT401) followed by five weeks of normal training and a repeat time trial (TT402). The cyclists then completed four days of high intensity training followed by another time trial (TT403) on day five. Following one final week of normal training cyclists completed another time trial (TT404). Fasting venous blood was collected before and after each time trial to determine Granulysin concentration, natural killer cell number and natural killer cell cytotoxicity. Granulysin concentration increased significantly after each time trial (P 0.05). Exercise did not significantly alter natural killer cell cytotoxicity on a per cell basis, and there were no significant differences between the four time trials. In conclusion, plasma Granulysin concentration increases following moderate duration, strenuous exercise and is decreased in response to a short-term period of intensified training.
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exhaustive exercise induces differential changes in serum Granulysin and circulating number of natural killer cells
Tohoku Journal of Experimental Medicine, 2006Co-Authors: Xiumin Zhang, Kaori Matsuo, Arta Farmawati, Yohei Higashi, Kazuyuki Ogawa, Kinya Nagata, Ryoichi NagatomiAbstract:The circulating number of natural killer (NK) cells largely changes after an acute bout of physical exercise. Granulysin is a cytolytic granule protein with a broad range of antimicrobial and tumoricidal activities produced and released by human NK cells and cytolytic T lymphocytes. Since NK cells constitutively produce Granulysin, most serum Granulysin in healthy humans is derived from NK cells. Serum graulysin levels in the healthy humans may therefore reflect the size of whole-body NK cell population in the body. The aim of this study was to determine the effect of an acute bout of exhaustive exercise on serum Granulysin in comparison with the circulating number of NK cells. Six healthy, young male volunteers participated in the study. Each subject underwent both exhaustive exercise and resting sessions in a random order with at least a seven-day interval. Subjects were asked to run to exhaustion on a treadmill with an incremental graded protocol. Blood samples were collected before, immediately after, and 1 hr, 3 hr, 6 hr, 12 hr and 24 hr after exercise. Serum Granulysin levels were measured by enzyme-linked immunosorbent assay (ELISA). NK cells were determined by flow cytometry. Exhaustive exercise induced a 4.8-fold increase in peripheral blood NK cells, but no significant change in serum Granulysin. Our results support the hypothesis that exhaustive exercise-induced changes in the circulating number of NK cells represent a redistribution of lymphocytes, rather than the change in the size of whole-body NK cell population.
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Plasma Granulysin concentrations and preeclampsia risk.
Clinical biochemistry, 2006Co-Authors: Chunfang Qiu, Shigeru Saito, Kazuyuki Ogawa, Kinya Nagata, Masatoshi Sakai, Michelle A. WilliamsAbstract:Abstract Objective: Epidemiological, clinical and histological data suggest intriguing similarities between preeclampsia and graft–host-rejection. Granulysin, a novel biomarker of overall cellular immunity, is secreted by natural killer cells and cytotoxic T lymphocytes, which are associated with graft–host-rejection. Plasma Granulysin was elevated in Japanese preeclamptic women. Design and Methods: 50 preeclampsia cases and 50 normotensive controls (USA) were studied. Plasma Granulysin at delivery was determined using enzyme immunoassay. Logistic regression procedures were used to estimate odds ratios (OR) and 95% confidence intervals (CI). Results: Granulysin were elevated in preeclampsia cases compared with controls (3.01 ± 0.18 vs. 2.22 ± 0.14 ng/mL, p Conclusions: These data offer further evidence of a predominant Th1 immune status associated with preeclampsia. Prospective studies are needed to determine whether Granulysin is elevated early in pregnancy.
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Analysis of serum Granulysin in patients with hematopoietic stem-cell transplantation: Its usefulness as a marker of graft-versus-host reaction
American journal of hematology, 2006Co-Authors: Masayuki Nagasawa, Kazuyuki Ogawa, Kinya Nagata, Takeshi Isoda, Sukeyuki Itoh, Michiko Kajiwara, Tomohiro Morio, Norio Shimizu, Masataka Nakamura, Shuki MizutaniAbstract:Granulysin is a newly identified CTL/NK cell-related cytotoxic protein, which is secreted in both constitutive and Ca-dependent manner. To evaluate its significance in stem-cell transplantation (SCT), serum Granulysin was measured by newly established ELISA method in 26 patients undergoing SCT (21 allogeneic and 5 autologous). In the allogeneic SCT, Granulysin was transiently increased in 3 weeks, which was not observed in autologous SCT. In acute GVHD, serum Granulysin was markedly increased and correlated with the severity of GVHD. Elevation of Granulysin was not necessarily associated with increase of sIL2R or IFN-γ. In vitro, allospecific T cells released Granulysin in an allo-specific manner, and it was correlated with allo-specific cytotoxic activity. These results indicate that increased release of Granulysin presents alloreactivity and serum Granulysin is useful as a marker of GVHD in SCT. Am. J. Hematol. 81:340–348, 2006. © 2006 Wiley-Liss, Inc.
Daniel Rukavina - One of the best experts on this subject based on the ideXlab platform.
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Possible role of Granulysin in pathogenesis of osteoarthritis.
Medical hypotheses, 2015Co-Authors: Tatjana Kehler, Gordana Laškarin, Marin Dominovic, Drazen Massari, Viktor Peršić, Ivan Rosovic, Josip Laginja, Daniel RukavinaAbstract:Increased presence of immune mediator and cytotoxic/apoptotic molecule Granulysin was noticed in different tissues during pathological processes with the domination of Th1 over Th2 mediated immunity. Beside Granulysin expression in T and NKT cells, activated NK cells are thought to be the major source of chemotactic 15 kDa and cytotoxic 9 kDa Granulysin in vivo. As NK cells are the principal joint's tissue-infiltrating lymphocyte subset, we hypothesized that Granulysin mediated human cell death (apoptosis) could be responsible for the relatively silent damage of the joint's tissue without clinically notable signs of systemic inflammation in the patients with osteoarthritis (OA). The analyzes of the presence and frequency of Granulysin expressing lymphocytes at protein and gene levels in peripheral blood and synovial samples and/or the samples of joint's tissue after the joint replacement therapy in patients with OA could give the initial insight to evaluate our hypothesis. It would be of the particular interest to differentiate the expression of 9 kDa and 15 kDa Granulysin forms in the effector cells, since only the shorter form exhibits cytotoxic properties. The measurement of Granulysin mediated early apoptosis in human NK sensitive K562 cells could be suitable in vitro model for evaluating Granulysin activity. Furthermore, disturbed balance of pro-inflammatory and anti-inflammatory cytokines in OA patients, could influence the level of the Granulysin expression. Having in mind that the Granulysin and its regulation is still unknown in the pathogenesis of OA, it could be worth to explore this important pro-inflammatory, cytotoxic/apoptotic mediator.
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Granulysin expression and colocalization with perforin in decidual and peripheral blood lymphocytes of the first trimester pregnancy
Journal of Reproductive Immunology, 2014Co-Authors: Marin Dominovic, Gordana Laškarin, Danijela Veljkovic Vujaklija, Lana Glavan Gacanin, Daniel RukavinaAbstract:Shorter 9 kDa Granulysin isoform often colocalizes with perforin in the same granules of cytotoxic NK and T cells. Granulysin rapidly enter into the target cells via perforin pores inducing apoptotic death. The longer, 15 kDa form does not colocalize with perforin in the same granules and it exerts regulatory functions after secretion by activated cells. We aimed to analyze the expression of Granulysin by different antibodies within the early pregnancy decidual lymphocytes (DL) and peripheral blood lymphocytes (PBL) in vitro and colocalization of Granulysin and perforin. MATERIAL AND METHODS: DL were isolated by enzymatic digestion and gradient density centrifugation. PBL were obtained by gradient density centrifugation. Intracellular Granulysin was labelled by flow cytometry, using rabbit polyclonal anti-Granulysin antibodies (provided by Prof. Clayberger, Chicago, USA) and RF10 anti-Granulysin monoclonal antibody - mAb (MBL, Japan) in CD3 and CD56 labelled lymphocytes. Granulysin and perforin were visualized by immunofluorescence, and analyzed by confocal microscopy using ImageJ 1.44p quantification software. RESULTS: There is a difference between colocalization of Granulysin and perforin in DL and PBL, with much more colocalization in PBL. Upon the cell activation the colocalization pattern of DL is much more similar to the one observed in PBL. Significant difference Granulysin labelling by polyclonal and monoclonal RF10 antibodies in decidual NK cells is observed as well. In the decidual NK cells there is significant difference in the Granulysin labelling by polyclonal and monoclonal RF10 antibodies, which can be interpreted as a difference in the granuylsin isoform expression. CONCLUSION: Investigation of the dynamics of colocalization following the cell activation showed significant increase of colocalization in DL, pointing to the "functional maturation" of the cytolytic potential of these cells.
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Cell Death Mechanisms at the Maternal-Fetal Interface: Insights into the Role of Granulysin
Clinical & developmental immunology, 2011Co-Authors: Danijela Veljkovic Vujaklija, Marin Dominovic, Sonja Sucic, Tamara Gulic, Daniel RukavinaAbstract:During mammal pregnancy, a sensitive balance between hormones, cytokines, humoral factors, and local cellular interactions must be established. Cytotoxic cells infiltrating the decidua are heavily equipped with cytolytic molecules, in particular perforin and Granulysin. Granulysin is especially abundant in NK cells which are able to spontaneously secrete high quantities of Granulysin. Besides being a potent bactericidal and tumoricidal molecule, Granulysin is also found to be a chemoattractant and a proinflammatory molecule. The precise role(s) of Granulysin at the maternal-fetal interface has not been elucidated yet. It is possible that it behaves as a double-edged sword simultaneously acting as an immunomodulatory and a host defense molecule protecting both the mother and the fetus from a wide spectrum of pathogens, and on the other hand, in case of an NK cell activation, acting as an effector molecule causing the apoptosis of semiallograft trophoblast cells and consequently leading to various pregnancy disorders or pregnancy loss.
