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Biomedical subjects

A M Krensky

Publications and source records attributed to A M Krensky.

At least 19 recordsLinked to original sources
PubMed2000

Proliferating cell nuclear antigen as the cell cycle sensor for an HLA-derived peptide blocking T cell proliferation.

Synthetic peptides corresponding to structural regions of HLA molecules are novel immunosuppressive agents. A peptide corresponding to residues 65-79 of the alpha-chain of HLA-DQA03011 (DQ65-79) blocks cell cycle progression from early G1 to the G1 restriction point, which inhibits cyclin-dependent kinase-2 activity and phosphorylation of the retinoblastoma protein. A yeast two-hybrid screen identified proliferating cell nuclear Ag (PCNA) as a cellular ligand for this peptide, whose interaction with PCNA was further confirmed by in vitro biochemistry. Electron microscopy demonstrates that the DQ65-79 peptide enters the cell and colocalizes with PCNA in the T cell nucleus in vivo. Binding of the DQ65-79 peptide to PCNA did not block polymerase delta (pol delta)-dependent DNA replication in vitro. These findings support a key role for PCNA as a sensor of cell cycle progression and reveal an unanticipated function for conserved regions of HLA molecules.

Cell Cycle↗
PubMed2000

Interleukin-1beta induction of the chemokine RANTES promoter in the human astrocytoma line CH235 requires both constitutive and inducible transcription factors.

The chemokine RANTES is an important mediator of inflammatory processes. In this report, we describe the DNA sequence and transcription factor requirements for interleukin-1beta (IL-1beta) induction of the RANTES promoter in the human astrocytoma line CH235. RANTES promoter sequences between -278 and +55 are sufficient for IL-1beta-inducibility. In vitro DNA binding assays demonstrate constitutive binding of Sp1, HMG, Ets domain, and bZIP family members to their cognate sites in the RANTES promoter, whereas NF-kappaB and IRF-1 bind in an IL-1beta-inducible manner. IL-1beta-inducibility of the RANTES promoter requires both constitutive and inducible transcription factors. The formation of a higher order nucleoprotein complex, or 'enhanceosome', may be critical for IL-1beta induction of the RANTES promoter.

Astrocytoma↗
PubMed2000

Self-recognition of CD1 by gamma/delta T cells: implications for innate immunity.

The specificity of immunoglobulins and alpha/beta T cell receptors (TCRs) provides a framework for the molecular basis of antigen recognition. Yet, evolution has preserved a separate lineage of gamma/delta antigen receptors that share characteristics of both immunoglobulins and alpha/beta TCRs but whose antigens remain poorly understood. We now show that T cells of the major tissue gamma/delta T cell subset recognize nonpolymorphic CD1c molecules. These T cells proliferated in response to CD1+ presenter cells, lysed CD1c+ targets, and released T helper type 1 (Th1) cytokines. The CD1c-reactive gamma/delta T cells were cytotoxic and used both perforin- and Fas-mediated cytotoxicity. Moreover, they produced granulysin, an important antimicrobial protein. Recognition of CD1c was TCR mediated, as recognition was transferred by transfection of the gamma/delta TCR. Importantly, all CD1c-reactive gamma/delta T cells express V delta 1 TCRs, the TCR expressed by most tissue gamma/delta T cells. Recognition by this tissue pool of gamma/delta T cells provides the human immune system with the capacity to respond rapidly to nonpolymorphic molecules on professional antigen presenting cells (APCs) in the absence of foreign antigens that may activate or eliminate the APCs. The presence of bactericidal granulysin suggests these cells may directly mediate host defense even before foreign antigen-specific T cells have differentiated.

Amino Acid Sequence↗
PubMed2000

Granulysin: a novel antimicrobial peptide of cytolytic T lymphocytes and natural killer cells.

Granulysin is a novel antimicrobial protein produced by human cytolytic T lymphocytes and natural killer cells. It is active against a broad range of microbes, including Gram-positive and Gram-negative bacteria, fungi, and parasites. The fact that it kills Mycobacterium tuberculosis is particularly important, since the current vaccine (Bacille Calmette-Guerin, BCG) is of limited efficacy and antibiotic resistance is increasing. Although functionally related to other antibacterial peptides, defensins and magainins, granulysin is structurally distinct. Like porcine NK lysin and amoebapores made by Entamoeba histolytica, granulysin is related to saposins, small lipid-associated proteins present in the central nervous system. The identification of this novel molecule indicates a broader and perhaps more significant role for T lymphocytes in both innate and acquired antimicrobial defenses.

Amino Acid Sequence↗
PubMed2000

A human class II MHC-derived peptide antagonizes phosphatidylinositol 3-kinase to block IL-2 signaling.

MHC molecules bind antigenic peptides and present them to T cells. There is a growing body of evidence that MHC molecules also serve other functions. We and others have described synthetic peptides derived from regions of MHC molecules that inhibit T-cell proliferation or cytotoxicity in an allele-nonspecific manner that is independent of interaction with the T-cell receptor. In this report, we describe the mechanism of action of a synthetic MHC class II-derived peptide that blocks T-cell activation induced by IL-2. Both this peptide, corresponding to residues 65-79 of DQA*03011 (DQ 65-79), and rapamycin inhibit p70 S6 kinase activity, but only DQ 65-79 blocks Akt kinase activity, placing the effects of DQ 65-79 upstream of mTOR, a PI kinase family member. DQ 65-79, but not rapamycin, inhibits phosphatidylinositol 3-kinase (PI 3-kinase) activity in vitro. The peptide is taken up by cells, as demonstrated by confocal microscopy. These findings indicate that DQ 65-79 acts as an antagonist with PI 3-kinase, repressing downstream signaling events and inhibiting proliferation. Understanding the mechanism of action of immunomodulatory peptides may provide new insights into T-cell activation and allow the development of novel immunosuppressive agents.

Amino Acid Sequence↗
PubMed1999

Biosynthesis of granulysin, a novel cytolytic molecule.

Granulysin is a newly described lytic molecule expressed by CTL and NK cells. Three mRNA (519, 520, and 522) and two protein products of 15 and 9 kDa are encoded by the granulysin gene. Stable transfectants overexpressing the predominate 520 mRNA were generated to determine the protein products originating from the translation of this message. A transfectant of the NK cell tumor YT overexpressed both 15 and 9 kDa proteins while a transfectant of the T cell tumor HuT78 produced mainly 15 kDa granulysin. Thus the 520 mRNA is sufficient for production of both 15 and 9 kDa granulysin. 9 kDa granulysin accumulated via post-translational processing of 15 kDa protein and was present intracellularly but not in the cell culture supernatant, indicating specific retention of the 9 kDa protein. An inhibitor of granule acidification, concanamycin A, blocked the processing of 15 kDa granulysin to the 9 kDa form. A deduced structural difference between the two forms of the protein and a decrease in lytic activity of 9 kDa granulysin at granule pH suggest two mechanisms by which a granulysin expressing cell is protected from autolysis during the biosynthesis of this potentially harmful molecule.

Anti-Bacterial Agents↗
PubMed1999

RFLAT-1: a new zinc finger transcription factor that activates RANTES gene expression in T lymphocytes.

RANTES (Regulated upon Activation, Normal T cell Expressed and Secreted) is a chemoattractant cytokine (chemokine) important in the generation of inflammatory infiltrate and human immunodeficiency virus entry into immune cells. RANTES is expressed late (3-5 days) after activation in T lymphocytes. Using expression cloning, we identified the first "late" T lymphocyte associated transcription factor and named it "RANTES Factor of Late Activated T Lymphocytes-1" (RFLAT-1). RFLAT-1 is a novel, phosphorylated, zinc finger transcription factor that is expressed in T cells 3 days after activation, coincident with RANTES expression. While Rel proteins play the dominant role in RANTES gene expression in fibroblasts, RFLAT-1 is a strong transactivator for RANTES in T cells.

Adult↗
PubMed1999

HLA-derived peptides as novel immunomodulatory therapeutics.

A growing body of experimental evidence demonstrates that synthetic peptides corresponding to linear sequences of MHC (HLA in humans) proteins have immunomodulatory effects in vitro and in vivo in animal models and in humans. Although the original concept was that these peptides inhibited antigen recognition at the MHC-T cell receptor interface via physical blockade, it is now clear that the mechanisms responsible for the myriad of functional effects are more complex. Recent findings show that some peptides affect signal transduction and cell cycle progression. Fragments of MHC molecules can dampen or downregulate immune responses via a variety of mechanisms. Some soluble MHC molecules or synthetic peptides are capable of inducing and maintaining immunologic tolerance in animals. This information suggests that synthetic peptides themselves or drugs mimicking their effects may represent a new class of immunotherapeutics.

Animals↗
PubMed1998

An antimicrobial activity of cytolytic T cells mediated by granulysin.

Cytolytic T lymphocytes (CTLs) kill intracellular pathogens by a granule-dependent mechanism. Granulysin, a protein found in granules of CTLs, reduced the viability of a broad spectrum of pathogenic bacteria, fungi, and parasites in vitro. Granulysin directly killed extracellular Mycobacterium tuberculosis, altering the membrane integrity of the bacillus, and, in combination with perforin, decreased the viability of intracellular M. tuberculosis. The ability of CTLs to kill intracellular M. tuberculosis was dependent on the presence of granulysin in cytotoxic granules, defining a mechanism by which T cells directly contribute to immunity against intracellular pathogens.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1998

Granulysin-induced apoptosis. I. Involvement of at least two distinct pathways.

Granulysin is a newly described cytolytic molecule released by CTL and NK cells via granule-mediated exocytosis. It shares homology with saposin-like proteins, including NK-lysin and amoebapores, and has been implicated in the lysis of tumor cells and microbes. In the present study we show that recombinant granulysin alone induces apoptosis of Jurkat cells. This apoptosis is associated with a sixfold increase in the ceramide/sphingomyelin ratio, implicating the activation of sphingomyelinases. Granulysin- and ceramide-induced apoptosis are similar in that they both are only minimally inhibited by the more selective cysteine protease p32 (caspase 3)-like caspase inhibitor N-acetyl-Asp-Glu-Val-Asp aldehyde, while they are significantly inhibited by the more general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk). Nevertheless, while Z-VAD-fmk almost completely inhibits ceramide-induced apoptosis, a Z-VAD-fmk-resistant component was observed using granulysin. Granulysin also causes apoptosis in cells depleted of sphingomyelin by prolonged treatment with the ceramide synthase inhibitor fumonisin B1. These data indicate that granulysin induces target cell death by both ceramide- and caspase-dependent and -independent pathways.

Amino Acid Chloromethyl Ketones↗
PubMed1998

Inhibition of cell cycle progression by a synthetic peptide corresponding to residues 65-79 of an HLA class II sequence: functional similarities but mechanistic differences with the immunosuppressive drug rapamycin.

A synthetic peptide corresponding to a region of the alpha1 alpha-helix of DQA03011 (DQ 65-79) inhibits the proliferation of human PBL and T cells in an allele-nonspecific manner. It blocks proliferation stimulated by anti-CD3 mAb, PHA-P, and alloantigen, but not by PMA and ionomycin. Substitution of each amino acid with serine shows that residues 66, 68, 69, 71-73, and 75-79 are critical for function. Inhibition of proliferation is long lasting and is not reversible with exogenous IL-2. The peptide can be added 24 to 48 h after stimulation and still block proliferation. The DQ 65-79 peptide does not affect expression of IL-2 or IL-2R; however, IL-2-stimulated proliferation is inhibited. Cell cycle progression is blocked at the G1/S transition, and the activity of cdk2 (cyclin-dependent kinase 2) kinase is impaired by the continued presence of p27. Although these results suggest a mechanism similar to that of rapamycin, the peptide inhibition is not reversed with FK-506, which indicates a distinct mechanism.

Amino Acid Sequence↗
PubMed1998

Chemokines, lymphocytes and viruses: what goes around, comes around.

Chemokines are believed to be the long-sought soluble mediators of selective lymphocyte recruitment. As most selectin-integrin interactions are nonselective, it is thought that the discrimination seen during lymphocyte infiltration into tissues is brought about by the actions of distinct chemokines. Developments over the past year have demonstrated the expanding roles of these factors in lymphocyte chemoattraction, normal trafficking, and viral immunity.

Animals↗
PubMed1998

Pediatric Transplant Grand Rounds. Pediatric kidney transplantation at Stanford.

(1) We believe that we have achieved excellent graft survival with pediatric kidney transplantation because: (a) we have had no technical losses; (b) there have been no primary immunologic losses within 4 years following transplantation; (c) we have avoided ATN. (2) Our cyclosporine dosage has been greater than the average dosage reported by the NAPRTCS, and we believe that this has led to: (a) a low incidence of rejection episodes; (b) because of this, good 1 and 2 year average serum creatinine levels. (3) In the management of adult-sized kidneys in infants and small children I have discussed: (a) the rationale and strategy to prevent vascular thrombosis, ATN and primary non-function; (b) the importance of optimizing intravascular volume, as well as renal and aortic blood flow. (4) With regard to the management of congenital urologic abnormalities I have discussed: (a) the strategy to avoid unnecessary surgery and to avoid scar around the aorta and the vena cava, particularly in infants and small children; (b) my philosophy regarding the abnormal bladder and the successful use of the small defunctionalized urinary bladder. We believe that these have been the primary ingredients to the success we have seen. I also harken back and continue to practice the adage advanced by my former mentor Dr. Fred Belzer that 'no kidney is better than a bad kidney!' And this could not be more true than in pediatric kidney transplantation, where graft failure enhanced by suboptimal graft quality may potentially both cripple the child and shorten his/her life.

Adult↗
PubMed1997

Processing, subcellular localization, and function of 519 (granulysin), a human late T cell activation molecule with homology to small, lytic, granule proteins.

CTL and NK cells share a common cytolytic mechanism that involves regulated exocytosis of lytic molecules stored within cytoplasmic granules. Here we describe the processing, subcellular localization, and function of a T and NK cell-specific granule protein that shares homology with small, lytic granule-associated molecules. The gene coding for this protein, 519, is expressed late after T cell activation. Antisera raised against a 519/glutathione-S-transferase fusion protein and a series of peptides derived from the 519 protein sequence permitted the identification of two small CTL protein products of 15 and 9 kDa that are exocytosed after stimulation through the TCR. The 9-kDa product is a processed form of 519 and differs from the 15-kDa product in both its amino and carboxyl terminus. While both 519 proteins are found in cytoplasmic granules, the 9-kDa form is also present in dense, highly cytolytic granules. Functional studies indicate that this protein is lytic against tumor cell targets. The cell type- and stage-specific expression pattern of 519 along with its subcellular localization are reminiscent of molecules that play a vital role in granule-mediated cytolysis by CTL and NK cells. Its lytic activity suggests the involvement of 519 in CTL effector function.

Amino Acid Sequence↗
PubMed1997

Granulysin, a new human cytolytic granule-associated protein with possible involvement in cell-mediated cytotoxicity.

A primary process by which cytotoxic T lymphocytes (CTL) and natural killer (NK) cells lyse target cells involves the regulated exocytosis of granules present in the cytoplasm of the effector. These granules contain proteins, such as perforin and the granzymes, that play a direct role in the killing process. The localization of a human T and NK cell-specific protein, granulysin (formerly 519), to cytolytic granules suggests that additional mechanisms may be involved in granule-mediated cytolysis. This protein shares homology with small, granule-associated molecules and is a member of a larger family of proteins known as saposin-like proteins (SAPLIP). SAPLIP share common structural features allowing for association with lipids while retaining the ability to mediate a variety of different functions. Expression of granulysin is induced late after T-cell activation, similar to perforin and the granzymes. Two prominent protein products of 15 and 9kDa were identified in CTL. The 9kDa form localizes to dense, highly cytolytic granules and contains the SAPLIP homology domain. A recombinant granulysin protein, corresponding to the 9kDa form, is cytolytic against tumor cell targets.

Amino Acid Sequence↗