Biomedical subjects

C Clayberger

Publications and source records attributed to C Clayberger.

At least 19 recordsLinked to original sources

PubMed2001

A distinct pathway of cell-mediated apoptosis initiated by granulysin.

Granulysin is an antimicrobial and tumoricidal molecule expressed in granules of CTL and NK cells. In this study, we show that granulysin damages cell membranes based upon negative charge, disrupts the transmembrane potential (Deltapsi) in mitochondria, and causes release of cytochrome c. Granulysin-induced apoptosis is blocked in cells overexpressing Bcl-2. Despite the release of cytochrome c, procaspase 9 is not processed. Nevertheless, activation of caspase 3 is observed in granulysin-treated cells, suggesting that granulysin activates a novel pathway of CTL- and NK cell-mediated death distinct from granzyme- and death receptor-induced apoptosis.

Antigens, Differentiation, T-Lymphocyte↗

PubMed2001

Granulysin expression is a marker for acute rejection and steroid resistance in human renal transplantation.

Differentiating etiologies of transplant dysfunction without biopsy and optimizing therapy for acute rejection by predicting steroid resistance will reduce patient morbidity. Granulysin is a cytolytic molecule released by CTL and NK cells and coexpressed with effectors of acute allograft rejection, like perforin and granzymes. Granulysin mRNA and protein expression were studied in peripheral blood lymphocytes (PBL; n = 61 total, n = 10 with intercurrent infections) and biopsy tissue from adult and children renal transplant recipients (n = 97) by competitive quantitative-reverse transcriptase-PCR (QC-RT-PCR) and immunohistochemistry. Differences in cell phenotypes were studied in steroid sensitive and resistant acute rejection biopsies. Granulysin was studied in phytohemagglutinin (PHA) stimulated cell lines (donor PBL and CD45RO(+) T cells) by FACS, Western blotting, and RT-PCR after pretreating with cyclosporine A (CSA), azathioprine, mycophenolic acid, and steroids. Granulysin mRNA was significantly increased in patient PBL and transplant biopsies during acute rejection (p < 0.0001) and infection (p < 0.001). Rejecting biopsies alone (n = 53) had mononuclear cell granulysin staining. Steroid resistant biopsies (n = 25) had denser granulysin staining (>2 cells/high power field) and CD45RO(+) lymphocytes, when compared with steroid sensitive (n = 28) rejecting tissue. Granulysin levels were unchanged after azathioprine and mycophenolic acid treatment, decreased after treating activated PBL with steroids and cyclosporine A (CSA), and paradoxically, increased (p < 0.05) after treating CD45RO(+) CTL with CSA. Elevated PBL granulysin is a peripheral marker for acute rejection and infection and dense granulysin staining a tissue marker for steroid resistance. Memory CTL abound in steroid resistant grafts and may have a markedly different response to CSA immunotherapy, suggesting a possible mechanism for steroid resistance.

Acute Disease↗

PubMed2001

Granulysin blocks replication of varicella-zoster virus and triggers apoptosis of infected cells.

Granulysin, a lytic protein present in cytolytic granules of human natural killer and cytotoxic T cells, entered cells infected with varicella-zoster virus (VZV). Exposure to granulysin accelerated death of infected cells as assessed by apoptosis markers. The functional domain of granulysin that mediated its antiviral effects was amino acid 23-51; this domain also mediates the additional antitumor cell effects of granulysin. Because granulysin is a product of natural killer cells and T lymphocytes, it is possible that its antiviral activity may act as a mediator of innate and adaptive immune mechanisms.

Amino Acid Chloromethyl Ketones↗

PubMed2001

Granulysin: a novel antimicrobial.

Granulysin is a novel lytic molecule produced by human cytolytic T-lymphocytes (CTLs) and natural killer (NK) cells. It is active against a broad range of microbes, including Gram-positive and -negative bacteria, parasites and Mycobacterium tuberculosis. It is functionally related to other antibacterial peptides, like defensins and magainins, but is structurally distinct. It has structural similarity to porcine NK-lysin and to amoebapores made by Entamoeba histolytica. Synthetic peptides derived from granulysin have differential activity against eukaryotic cells and bacteria. Selective bactericidal peptides may have therapeutic roles as novel antibiotics.

Amino Acid Sequence↗

PubMed2000

Granulysin, a T cell product, kills bacteria by altering membrane permeability.

Granulysin, a protein located in the acidic granules of human NK cells and cytotoxic T cells, has antimicrobial activity against a broad spectrum of microbial pathogens. A predicted model generated from the nuclear magnetic resonance structure of a related protein, NK lysin, suggested that granulysin contains a four alpha helical bundle motif, with the alpha helices enriched for positively charged amino acids, including arginine and lysine residues. Denaturation of the polypeptide reduced the alpha helical content from 49 to 18% resulted in complete inhibition of antimicrobial activity. Chemical modification of the arginine, but not the lysine, residues also blocked the antimicrobial activity and interfered with the ability of granulysin to adhere to Escherichia coli and Mycobacterium tuberculosis. Granulysin increased the permeability of bacterial membranes, as judged by its ability to allow access of cytosolic ss-galactosidase to its impermeant substrate. By electron microscopy, granulysin triggered fluid accumulation in the periplasm of M. tuberculosis, consistent with osmotic perturbation. These data suggest that the ability of granulysin to kill microbial pathogens is dependent on direct interaction with the microbial cell wall and/or membrane, leading to increased permeability and lysis.

Amino Acid Sequence↗

PubMed2000

A non-peptide functional antagonist of the CCR1 chemokine receptor is effective in rat heart transplant rejection.

Chemokines like RANTES appear to play a role in organ transplant rejection. Because RANTES is a potent agonist for the chemokine receptor CCR1, we examined whether the CCR1 receptor antagonist BX471 is efficacious in a rat heterotopic heart transplant rejection model. Treatment of animals with BX471 and a subtherapeutic dose of cyclosporin (2.5 mg/kg), which is by itself ineffective in prolonging transplant rejection, is much more efficacious in prolonging transplantation rejection than animals treated with either cyclosporin or BX471 alone. We have examined the mechanism of action of the CCR1 antagonist in in vitro flow assays over microvascular endothelium and have discovered that the antagonist blocks the firm adhesion of monocytes triggered by RANTES on inflamed endothelium. Together, these data demonstrate a significant role for CCR1 in allograft rejection.

Animals↗

PubMed2000

An immunosuppressive and anti-inflammatory HLA class I-derived peptide binds vascular cell adhesion molecule-1.

BACKGROUND: A synthetic peptide corresponding to residues 75-84 of HLA-B2702 modulates immune responses in rodents and humans both in vitro and in vivo. METHODS: We used a yeast two-hybrid screening, an in vitro biochemical method, and an in vivo animal model. RESULTS: Two cellular receptors for this novel immunomodulatory peptide were identified using a yeast two-hybrid screen: immunoglobulin binding protein (BiP), a member of the heat shock protein 70 family, and vascular cell adhesion molecule (VCAM)-1. Identification of BiP as a ligand for this peptide confirms earlier biochemical findings, while the interaction with VCAM-1 suggests an alternative mechanism of action. Binding to the B2702 peptide but not to closely related variants was confirmed by ligand Western blot analysis and correlated with immunomodulatory activity of each peptide. In mice, an ovalbumin-induced allergic pulmonary response was blocked by in vivo administration of either the B2702 peptide or anti-VLA-4 antibody. CONCLUSIONS: We propose that the immunomodulatory effect of the B2702 peptide is caused, in part, by binding to VCAM-1, which then prevents the normal interaction of VCAM-1 with VLA-4.

Amino Acid Sequence↗

PubMed2000

Bactericidal and tumoricidal activities of synthetic peptides derived from granulysin.

Granulysin, a 9-kDa protein localized to human CTL and NK cell granules, is cytolytic against tumor cells and microbes. Molecular modeling predicts that granulysin is composed of five alpha-helices separated by short loop regions. In this report, synthetic peptides corresponding to the linear granulysin sequence were characterized for lytic activity. Peptides corresponding to the central region of granulysin lyse bacteria, human cells, and synthetic liposomes, while peptides corresponding to the amino or carboxyl regions are not lytic. Peptides corresponding to either helix 2 or helix 3 lyse bacteria, while lysis of human cells and liposomes is dependent on the helix 3 sequence. Peptides in which positively charged arginine residues are substituted with neutral glutamine exhibit reduced lysis of all three targets. While reduction of recombinant 9-kDa granulysin increases lysis of Jurkat cells, reduction of cysteine-containing granulysin peptides decreases lysis of Jurkat cells. In contrast, lysis of bacteria by recombinant granulysin or by cysteine-containing granulysin peptides is unaffected by reducing conditions. Jurkat cells transfected with either CrmA or Bcl-2 are protected from lysis by recombinant granulysin or the peptides. Differential activity of granulysin peptides against tumor cells and bacteria may be exploited to develop specific antibiotics without toxicity for mammalian cells.

Adjuvants, Immunologic↗

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

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

A novel method for measuring CTL and NK cell-mediated cytotoxicity using annexin V and two-color flow cytometry.

An assay based on two-color flow cytometry has been developed to measure CTL and NK cell-mediated cytotoxicity. After effector/target cells are incubated together, CTL or NK populations are stained with an effector cell specific PE-conjugated mAb. Subsequently, annexin V-FITC binds to cells expressing phosphatidylserine (an early marker of apoptosis) on the cell surface. Target cells are gated upon as PE-negative and quantified with respect to their annexin V positivity. The shift from annexin Vneg to annexin Vhi is a discrete event such that all target cells fall within discernible populations with respect to annexin V. There is a strong correlation between cytotoxicity measured with our assay and a standard 51Cr release assay (r2 = 0.989). The PE/annexin V assay shows increased sensitivity at early timepoints after target/effector cell mixing. In addition, this method allows for analysis of target cells at the single cell level. Therefore, we have described a promising new technique to measure in vitro cell-mediated cytotoxicity. It avoids the potential difficulties of working with radioactive isotopes, and offers increased sensitivity and versatility.

Annexin A5↗

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↗

PubMed1997

HLA-derived peptides as novel immunosuppressives.

Over the past decade the use of synthetic peptides corresponding to linear sequences of HLA molecules has progressed from a concept to a reality. These peptides are currently being evaluated in clinical trials. In animal models these peptides, given over 1 week with cyclosporin alone, induced long-term immunological tolerance (Figure 3). They may similarly induce tolerance in humans. The next major hurdle for such tolerogenic drugs, however, is to prove efficacy in clinical circumstances. Many of the drugs used to treat transplant patients today (steroids, cyclosporin, azathioprine, mycophenolate mofetil) may actually inhibit the 'active' processes of induction and maintenance of immunological tolerance. Demonstration that new drugs induce tolerance will require efficacy studies in other immune-mediated diseases in which monotherapy is feasible (such as psoriasis), before further advances can be made in the induction of transplant tolerance. In addition, rapid assays of rejection must be developed in order to reverse tolerance induction failures without graft damage or loss. Lastly, it will require heroic physicians, surgeons, and patients to make immunological tolerance a reproducible clinical reality.

Graft Survival↗

PubMed1997

Interactions of HLA-B*4801 with peptide and CD8.

Functional properties of the B*4801 allotype were investigated using HLA class I-deficient 221 cells transfected with B*4801 cDNA. From pool sequence analysis of endogenously bound peptides, B*4801 was shown to select for nonamer peptides having glutamine or lysine at position 2 and leucine at the carboxyl-terminus. In an in vitro cell-cell binding assay, B*4801 binds CD8 alpha homodimers weakly due to the presence of a threonine residue at position 245 in the alpha 3 domain. A mutant B*4801 molecule in which alanine replaces threonine 245, binds CD8 alpha homodimers at levels comparable to those of other HLA class I allotypes. Despite the low affinity of B*4801 for CD8 alpha, alloreactive T-cells that recognize B*4801 molecules expressed by the 221 transfectant are inhibited by anti-CD8 monoclonal antibodies. Analysis of 25 B*48-expressing individuals from various populations showed threonine 245 was encoded by every B*48 allele.

Alleles↗

PubMed1996

Tumor-specific, cytotoxic T-lymphocyte response after idiotype vaccination for B-cell, non-Hodgkin's lymphoma.

Patients with non-Hodgkin's B-cell lymphoma who received an antitumor vaccine of idiotypic ig protein showed humoral and proliferative immune responses. Because immunity to some antigens, including tumor antigens and human pathogenic viruses, may be better correlated with the cytolytic cellular immune response, we evaluated 16 non-Hodgkin's lymphoma patients immunized with autologous idiotypic ig molecules for changes in tumor-specific cytotoxic T-lymphocyte precursor (CTLp) frequency using limiting dilution analysis. Eleven patients had a significant increase in tumor-specific CTLp. Eight of these 11 patients remain without evidence of disease or with stable minimal disease. In contrast, all five patients who did not have a significant change in tumor-specific CTLp have developed progressive disease. Patient vaccination with tumor associated protein antigens can increase tumor-specific CTLp frequencies. The correlation of increased tumor specific CTLp with freedom from progression is significant at P = .002. This study indicates that measurement of CTLp frequencies are relevant to the clinical evaluation of human tumor vaccines and suggests that cell-mediated cytolytic immune responses may be an important determinant of vaccine efficacy.

Adult↗

PubMed1996

HLA-derived peptides which inhibit T cell function bind to members of the heat-shock protein 70 family.

Synthetic peptides corresponding to sequences of HLA class I molecules have inhibitory effects on T cell function. The peptides investigated in this study have sequences corresponding to the relatively conserved region of the alpha 1 helix of HLA class I molecules that overlaps the "public epitope" Bw4/Bw6. These HLA-derived peptides exhibit inhibitory effects on T lymphocytes and have beneficial effects on the survival of allogenic organ transplants in mice and rats. Peptides corresponding to the Bw4a epitope appear most potent as they inhibit the differentiation of T cell precursors into mature cytotoxic T lymphocytes (CTL) and target cell lysis by established CTL lines and clones. To elucidate the mechanism through which these peptides mediate their inhibitory effect on T lymphocytes, peptide binding proteins were isolated from T cell lysates. We show that the inhibitory Bw4a peptide binds two members of the heat-shock protein (HSP) 70 family, constitutively expressed HSC70 and heat-inducible HSP70. Peptide binding to HSC/HSP70 is sequence specific and follows the rules defined by the HSC70 binding motif. Most intriguing, however, is the strict correlation of peptide binding to HSC/HSP70 and the functional effects such that only inhibitory peptides bind to HSC70 and HSP70 whereas noninhibitory peptides do not bind. This correlation suggests that small molecular weight HLA-derived peptides may modulate T cell responses by directly interacting with HSPs. In contrast to numerous reports of HSP70 expression at the surface of antigen-presenting cells and some tumor cells, we find no evidence that HSC/HSP70 are expressed at the surface of the affected T cells. Therefore, we believe that the peptides' immunodulatory effects are not mediated through a signaling event initiated by interaction of peptide with surface HSP, but favor a model similar to the action of other immunomodulatory compounds, FK506 and cyclosporin A, with a role for HSC/HSP70 similar to that for immunophilins, FKBPs and CyP40.

Amino Acid Sequence↗

PubMed1996

Human CD4+ and CD8+ T lymphocytes are both cytotoxic to Toxoplasma gondii-infected cells.

Studies to determine if Toxoplasma gondii-specific human T cells lyse parasite-infected cells have yielded conflicting results. Furthermore, attempts to obtain human cytotoxic CD8+ T lymphocytes have been difficult because of the lack of a reproducible system for their generation. By using paraformaldehyde-fixed, T. gondii-infected peripheral blood mononuclear cells as antigen-presenting cells, we developed a method whereby T. gondii-specific T-cell lines can be reproducibly generated. Six T. gondii-specific T-cell lines were generated from an individual chronically infected with T. gondii. Cytofluorometric analysis of these lines revealed > 99% CD3+, 85 to 95% CD3+ alpha beta T-cell-receptor-positive (TCR+), 5 to 9% CD3+ gamma delta TCR+, 50 to 70% CD4+, and 20 to 40% CD8+ cells when cells were examined during the first 3 weeks of stimulation and >99% CD3+, >99% CD3+ alpha beta TCR+, < 1% CD3+ gamma delta TCR+, 20 to 40% CD4+, and 60 to 80% CD8+ cells when cells were examined between 5 and 11 weeks. Both CD4+ and CD8+ T cells had remarkable cytotoxic activity against T. gondii-infected target cells (30 to 50% specific Cr release at an effector-to-target ratio of 30:1) but not against uninfected target cells ( < 10% at an effector-to-target ratio of 30:1). Cytotoxic activity by the whole T-cell lines was not T. gondii strain specific. Whole T-cell lines were cytotoxic for target cells infected with the C56 and ME49 strains and the RH strain (which was used to infect peripheral blood mononuclear cells). T. gondii-specific T-cell lines displayed the predominant expression of V beta 7 TCR. The CDR3 regions of the V beta 7 TCRs of these T-cell lines showed a striking degree of sequence identity (oligoclonality). T-cell lines obtained by the method reporter here can be used to characterize functional activity of T-lymphocyte subsets in humans infected with T. gondii.

Amino Acid Sequence↗

PubMed1996

Structure of HLA molecules and immunosuppressive effects of HLA derived peptides.

Elucidation of the structure of MHC molecules has provided profound new insights into their function in antigen presentation. In addition, structural studies have implicated certain regions of MHC molecules in specific functions. Although much of MHC biology has concentrated on the extensive polymorphism among these molecules, there is also evolutionary pressure to maintain the relatively monomorphic portions of these molecules. Drs. Krensky and Clayberger have found that synthetic peptides corresponding to linear sequences of HLA molecules have immunomodulatory effects both in vitro and in vivo. In this paper, they review the structure of HLA molecules and their studies of HLA derived peptides as novel immunotherapeutics. Members of the heat shock protein 70 family are implicated in the HLA derived peptide immunosuppressive pathway.

Alleles↗