Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.79 (granzyme B)
 3,301 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The human CSP-B/CGL-1 gene is the homologue of the mouse granzyme B/CCPI gene and encodes a cytotoxic T-lymphocyte-specific serine protease. We have used regulatory sequences upstream from the CSP-B gene to drive human growth hormone gene expression in transgenic mice. Eleven founder mice were screened for transgene expression in activated T-cells. Expression was detected in 10 mice; levels of expression were integration site-dependent. The transgene was not expressed in resting lymphocytes but could be activated by treatment with concanavalin A or interleukin-2, indicating that CSP-B regulatory sequences are responsive to signals originating at either the T-cell receptor or the interleukin-2 receptor. Transgene expression was detected at the whole organ level only in lymph nodes and small intestine, where endogenous mouse CCPI mRNA was also present. The time course of transgene activation in T-lymphocytes was similar to that of the mouse CCPI gene. No differences in levels of expression of the transgene were observed in activated lymphocyte populations that had been depleted of either CD4+ or CD8+ cells; in contrast, the mouse CCPI gene was expressed primarily in CD8+ cells. Six CD4+ T-cell clones with Th0, Th1, or Th2 phenotypes were generated from a transgenic animal. All clones expressed moderate to high levels of the transgene, but only three clones expressed mouse CCPI, indicating that the transgene is disregulated in CD4+ T-cell subsets. The CSP-B regulatory unit represents a novel reagent for targeting gene expression to activated T-lymphocytes.
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PMID:The 5'-flanking region of the human CGL-1/granzyme B gene targets expression of a reporter gene to activated T-lymphocytes in transgenic mice. 176 44

Lysis of target cells by cytolytic T lymphocytes (CTL) is associated with the exocytosis of cytoplasmic granules. Purified granules from CTL cell lines contain a pore-forming protein (perforin), tree serine esterases, granzyme A (60,000 MW), granzyme B (29,000 MW), and granzyme C (27,000 MW). We have compared the kinetics of appearance of cytolytic activity with that of perforin and granzyme A activity during activation of lymphocytes from normal animals with leukoagglutinin (LA) and recombinant interleukin-2 (rIL-2). Unstimulated lymph node cells do not express any of these activities, which appear between Day 3 and Day 4 of stimulation and increase rapidly to reach a pronounced peak on Day 6. On Day 7 all the activities are considerably lower, even though the cells still proliferate exponentially. There is a good correlation between the kinetics of appearance of all of these activities. Using antisera against perforin and against granzyme C, one can detect positive cytoplasmic granules in a small fraction of cells on Day 3; by Day 5, 80-90% of the cells are stained. This proportion decreases again on Day 7.
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PMID:Appearance of granule-associated molecules during activation of cytolytic T-lymphocyte precursors by defined stimuli. 326 Feb 14

Cultured natural killer (NK) cells derived from CD3- CD56+ high-density small lymphocytes (HDLs) exhibit similar morphology and high levels of non-major histocompatibility complex-restricted (NK) cytotoxicity equivalent to those of cultured NK cells from CD3- CD56+ low-density large granular lymphocytes (LGLs). To examine the similarities and differences between NK cells from HDLs and NK cells from LGLs, we investigated the expression of three distinct members of the granule serine protease (granzyme) family within cultured CD3- CD56+ LGLs and HDLs. CD3- subpopulations of nonadherent peripheral blood mononuclear cells, LGLs (density < 1.063 g/ml), and HDLs (density > 1.063 g/ml) were stimulated to proliferate in culture. The cultured cells from each population were entirely CD3- CD56+ and were indistinguishable in terms of their increased granularity and size once activated. All cultured CD3- CD56+ LGLs and HDLs displayed cytolytic activity against K562 and immunoglobulin-coated P815. Western analysis detected perforin in both cultured LGL and HDL populations. Cultured HDLs and LGLs both expressed BLT-esterase activity and human granzyme A mRNA. Granzyme B mRNA and protein and Asp-ase activity were detected in unstimulated and cultured LGLs and cultured HDLs. By contrast, unstimulated HDLs did not express significant levels of granzyme B. High levels of Hu-Met-1 granzyme mRNA and Met-ase activity were detected only in cultured LGLs. Thus, despite the development of large granular morphology during proliferation, interleukin-2 cultured CD3- CD56+ HDLs display a different pattern of granzyme expression from CD3- CD56+ LGLs. These data also further suggest an unusually restricted expression of the Hu-Met-1 granzyme in LGLs.
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PMID:Distinct granzyme expression in human CD3- CD56+ large granular- and CD3- CD56+ small high density-lymphocytes displaying non-MHC-restricted cytolytic activity. 753 Feb 84

The human serine proteases granzymes A and B are expressed in cytoplasmic granules of activated cytotoxic T lymphocytes and natural killer cells. Recombinant granzyme A and granzyme B proteins were produced in bacteria, purified and then used to raise specific mouse monoclonal antibodies. Seven monoclonal antibodies (mAb) were raised against granzyme A, which all recognized the same or overlapping epitopes. They reacted specifically in an immunoblot of interleukin-2 (IL-2) stimulated PBMNC with a disulfide-linked homodimer of 43 kDa consisting of 28 kDa subunits. Seven mAb against granzyme B were obtained, which could be divided into two groups, each recognizing a different epitope. On an immunoblot, all mAb reacted with a monomer of 33 kDa protein. By immunohistochemistry, these mAb could be used to detect granzymes A and B expression in activated CTL and NK cells. The availability of these mAb may facilitate studies on the role of human cytotoxic cells in various immune reactions and may contribute to a better understanding of the role of granzymes A and B in the cytotoxic response in vivo.
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PMID:Production and characterization of monoclonal antibodies raised against recombinant human granzymes A and B and showing cross reactions with the natural proteins. 768 40

In order to better understand the immunopathology of acute complications of lung transplantation we have analysed the different parameters of cytotoxic cell and macrophage activation during the course of pulmonary allograft rejection and cytomegalovirus pneumonia. In transplanted patients presenting with an acute pulmonary allograft rejection, a cytomegalovirus pneumonia or no complication (control group), we have studied, first serum markers of immune activation: interleukin-2 soluble receptor (IL-2sR), neopterin, IL-6, TNF soluble receptors (TNF-sR55 and TNF-sR75). Secondly the intrapulmonary compartmentalisation of allogenic and antiviral responses were evaluated by studying bronchoalveolar lavage fluid (BAL). The level of IL-6 was measured in BAL supernatants and the gene expression of two cytokines (IL-1 beta and IL-6) and two markers of activated cytotoxic cells (granzyme B and perforin) were studied by in situ hybridisation on the alveolar cells. Acute pulmonary allograft rejection was characterised by the paucity of systemic stigmata of immune activation and by the intrapulmonary compartmentalisation of the inflammatory response principally expressed by an increase in alveolar concentration of IL-6, TNF-sR55 and TNF-sR75, and an increased expression of the IL-1 beta gene. Cytomegalovirus pneumonia is accompanied by an intense local and systemic inflammatory activity as evidenced by the serum level of IL-2sR, neopterin, TNF-sR55 and TNF-sR75, the alveolar concentration of IL-6, TNF-sR55 and TNF-sR75, and the expression of monokine (IL-1 beta, IL-6) and of cytotoxic mediator (granzyme b, perforin) genes by BAL cells. These mediators could participate in the elaboration of an acute or chronic inflammatory response which would be potentially deleterious for the graft.
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PMID:[Immunopathology of cytomegalovirus pneumonia and allograft rejection in lung transplantation. Group of Pulmonary Transplantation of the University Paris-Sud]. 783 5

One mechanism by which cytotoxic T lymphocytes and natural killer cells inflict target cell death depends upon secreting the contents of their specialized cytoplasmic granules, containing a pore-forming protein, perforin, and a family of homologous serine proteases ("granzymes") with various enzyme activities. We used a granzyme B-specific mouse anti-human monoclonal antibody 2C5 and Western blotting to demonstrate that nuclear extracts of human interleukin-2-stimulated peripheral blood mononuclear cells, the human NK leukemia cell line YT, and the rat NK leukemia cell line RNK-16 contain abundant granzyme B. In interleukin-2-activated peripheral blood mononuclear cells, more than 50% of the total cellular granzyme B was present in the nuclear lysate. Nuclear granzyme B had an apparent molecular mass of approximately 32 kDa in human cells and approximately 30 kDa in RNK-16 and was eluted from immobilized heparin at the same NaCl concentration as granzyme B from cytoplasmic granules. Granzyme B that was affinity-purified with 2C5 from the nuclei of YT or human LAK cells was capable of efficiently cleaving synthetic peptide thiobenzyl ester substrates with the same specificity (peptide cleavage after aspartic acid) as granule-localized granzyme B. By contrast perforin, which colocalizes with granzymes in cytotoxic granules, was not detectable in nuclear lysates. Granzyme B was also demonstrated to be present in the nucleus and cytoplasmic granules of YT by immunohistochemical staining with monospecific anti-granzyme B antisera. Other protease activities (tryptase and peptide cleavage after methionine) were also readily detectable in nuclear and cytoplasmic lysates of YT, RNK-16, and LAK cells, as determined by the cleavage of the synthetic substrates N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT) and Boc-Ala-Ala-Met-S-benzyl, except that BLT-esterase activity was absent from the nucleus of YT. The localization of serine proteases in the nucleus was restricted to lymphocytes with cytotoxic capacity, as non-cytotoxic cell lines expressed high levels of peptide cleavage after methionine and tryptase activities in their cytoplasm, but possessed no nuclear serine protease activity. Furthermore, non-cytotoxic monkey kidney COS-7 cells transfected with an SV40-driven expression plasmid incorporating full-length human granzyme B cDNA contained abundant cytoplasmic granzyme B, but demonstrated minimal nuclear granzyme B accumulation. We conclude that serine proteases of NK cells are not restricted to cytolytic granules and, further, that their capacity to access the nucleus may have implications for the role of these enzymes in eliciting target cell death.
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PMID:Granule serine proteases are normal nuclear constituents of natural killer cells. 803 81

The frequency of peripheral blood cells expressing the perforin gene or the granzyme B gene was evaluated by in situ hybridization in nine patients suffering from metastatic melanoma and treated with recombinant interleukin-2 (rIL-2). A spontaneous expression of both genes was detected in five to seven patients. rIL-2 administration increased the frequency of positive cells in all patients (P < 0.03 for each gene), the highest frequency being reached in the patients who already expressed these genes prior to rIL-2 treatment (P < 0.02). Expressions of the granzyme B gene and of the perforin gene were strongly correlated before IL-2 treatment and they were similarly affected by rIL-2 administration. In contrast, their modification under treatment did not correlate with that of CD56+ cell counts, of natural killer activity and of sCD8 release. This indicates that perforin and granzyme B gene expressions are markers of cytotoxic cell activation independent of those previously described, and that they should be further evaluated in patients with malignancies to delineate their potential value in predicting clinical outcome.
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PMID:Increased expression of perforin and granzyme B genes in patients with metastatic melanoma treated with recombinant interleukin-2. 804 27

Staphylococcal enterotoxin superantigens (SAg) bind class II major histocompatibility complex (MHC) molecules on antigen-presenting cells (APC) and upon cell-to-cell contact stimulate proliferation of T cells expressing appropriate V beta gene products. In addition, SAg can also deliver negative signals to Ag-specific T cells resulting in a state of unresponsiveness or a loss of viability. The present study examines the functional consequences of a direct interaction of SAg with alloAg-specific class II MHC+ CD4+ T cell lines (TCL). Our results demonstrate that SAg induce programmed death (apoptosis) in a majority of Ag-specific CD4+ T cells accompanied by genomic DNA fragmentation. SAg binding to Ag-specific TCL resulted in a rapid mobilization of intracellular free calcium ([Ca2+]i) and transcription of a number of cytokine genes including interleukin-2(IL-2), IL-4, interferon-gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granzyme B indicating the activation of primed T cells. Both SAg-induced cytokine gene expression as well as subsequent death were significantly inhibited by a tyrosine kinase inhibitor herbimycin A and also by cyclosporin A. SAg-induced death of primed T cells was also inhibited by monoclonal antibodies (mAb) directed at the CD11a/CD18 molecule but not those reactive with other T cell surface molecules such as CD2, CD7, CD28, CD29 or CD49d. None of these mAb, including anti-CD11a/CD18, had any effect on SAg-induced expression of IL-2 and IL-4 genes or SAg-induced [Ca2+]i response. Addition of cytokines such as IL-1 alpha, IL-2, IL-4, IL-6, GM-CSF, IFN-gamma, tumor necrosis factor (TNF-alpha, or TNF-beta), or neutralizing Ab to these cytokines had no effect on SAg-induced death of Ag-specific TCL. The T cells which survived the death-inducing effects of SAg showed down-regulation of the CD3/T cell receptor and up-regulation of CD2 and HLA-DR expression, and upon re-exposure to the same SAg upregulated expression of mRNA for IL-2 and IFN-gamma. Presentation of SAg by B7+ ICAM-1+ LFA-3+ DR+ professional APC was also able to induce the death of Ag-specific TCL. Together these results suggest that the activation with SAg causes programmed death of Ag-specific TCL cells via a mechanism that requires late participation of the CD11a/CD18 molecule.
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PMID:Activation with superantigens induces programmed death in antigen-primed CD4+ class II+ major histocompatibility complex T lymphocytes via a CD11a/CD18-dependent mechanism. 810 Jul 73

Using a polymerase chain reaction strategy we identified a serine proteinase inhibitor (serpin) in human bone marrow that is related to the cellular serpin proteinase inhibitor 6 (PI-6) and the viral serpin cytokine response modifier A (CrmA). This serpin, proteinase inhibitor 9 (PI-9), has an unusual reactive center P1(Glu)-P1'(Cys), which suggests that it inhibits serine proteinases that cleave after acidic residues. The only known serine proteinase with this specificity is granzyme B, a granule cytotoxin produced by cytotoxic lymphocytes. To test the interaction of PI-9 with granzyme B we prepared recombinant hexa-histidine tagged PI-9 in a yeast expression system. Addition of the recombinant protein to native granzyme B resulted in an SDS-resistant complex typical of serpin-serine proteinase interactions. Further analysis showed that complex formation followed bimolecular kinetics with a second order rate constant of 1.7 +/- 0.3 x 10(6) M-1 s-1, which is in the range for a physiologically significant serpin-proteinase interaction. Recombinant PI-9 also completely abrogated granzyme B and perforin-mediated cytotoxicity in vitro. Examination of PI-9 mRNA distribution demonstrated that it is expressed in immune tissue, primarily in lymphocytes. The highest levels of PI-9 mRNA and protein were observed in natural killer cell leukemia cell lines and in interleukin-2 stimulated peripheral blood mononuclear cells, which also produce granzyme B. Like PI-6, PI-9 was shown to be a cytosolic protein that is not secreted. Fractionation of natural killer cells and stimulated peripheral blood mononuclear cells demonstrated that PI-9 is in a separate subcellular compartment to granzyme B. These results suggest that PI-9 serves to inactivate misdirected granzyme B following cytotoxic cell degranulation. This may explain why cytotoxic cells are not damaged by their own granzyme B during destruction of abnormal cells.
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PMID:A cytosolic granzyme B inhibitor related to the viral apoptotic regulator cytokine response modifier A is present in cytotoxic lymphocytes. 891 Mar 77

Pentoxifylline (PTX), a methylxanthine derivative, is known to inhibit the production of the TH1 cytokines interleukin-2, tumour necrosis factor-alpha and interferon-gamma. Because these cytokines play an important role in promoting the development of cell-mediated immunity, we hypothesized that PTX would also interfere with the generation of cytotoxic effector cells in response to an immunological stimulus. In this study we used a mouse model system to investigate the effect of PTX on the induction of non-specific killer lymphocytes by anti-CD3 monoclonal antibody. Anti-CD3-induced T-cell proliferation, and the generation of anti-CD3-activated killer (AK) cells was inhibited in a dose-dependent fashion by PTX (25-100 micrograms/ml). The inhibitory effect of PTX could not be attributed to a defect in the recognition/adhesion phase of cytolysis because AK cells generated in the presence of PTX conjugated normally with P815 tumour target cells. However, AK cell expression of the cytoplasmic granule-associated cytolytic effector molecules granzyme B and perforin was markedly reduced when AK cells were induced in the presence of PTX. In eontrast, PTX had no effect on AK cell expression of Fas ligand, a cell-surface cytolytic effector molecule which is involved in granule-independent cytotoxicity. PTX thus has a profound inhibitory effect in vitro on the induction of granule-dependent cytolytic effector mechanisms in a mouse model system.
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PMID:Pentoxifylline inhibits granzyme B and perforin expression following T-lymphocyte activation by anti-CD3 antibody. 908 6


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