Biomedical subjects
Publications and source records attributed to A J Barrett.
Explore the source record for details and available documents.
We previously showed that a peptide (PR1) derived from the primary granule enzyme proteinase 3 induced peptide specific cytotoxic T lymphocytes (CTL) in a normal HLA-A2.1+ individual. These CTL showed HLA-restricted cytotoxicity to myeloid leukemias (which overexpress proteinase 3). To further investigate their antileukemic potential, we studied the ability of PR1-specific CTL, derived from two HLA-A2.1+ normal individuals, to inhibit colony-forming unit granulocyte-macrophage (CFU-GM) from normal and leukemic individuals. CTL from 20 day PR1 peptide-pulsed lymphocyte cultures showed 89% to 98% HLA-A2.1-restricted colony inhibition of chronic myeloid leukemia targets. Colony formation in normal HLA-A2.1+ bone marrow or HLA-A2.1- CML cells was not inhibited. Sequencing of the exon encoding PR1 showed that colony inhibition was not caused by polymorphic differences in proteinase 3 between effectors and targets. Analysis by flow cytometry showed that proteinase 3 was overexpressed in the leukemia targets compared with normal marrow targets (median channel fluorescence 1,399 v 298, P = .009). These results show that PR1-specific allogeneic T cells preferentially inhibit leukemic CFU-GM based on overexpression of proteinase 3, and that proteinase 3-specific CTL could be used for leukemia-specific adoptive immunotherapy.
Fanconi Anemia (FA) is a rare genetic disorder characterized by progress pancytopenia, congenial abnormalities, and a predisposition to malignancy. Therapy is currently limited to allogeneic marrow transplantation; patients lacking a suitable donor usually die from aplasia or acute leukemia. Recently, mutation in a novel gene named FACC (Fanconi anemia C-complementing) has been identified as causing one type of FA. FACC mutations, which introduce splicing errors or stop codons, have been identified in approximately 15% of FA patients. We have recently been successful in functional complementation of four FA cell lines using retroviral vectors to transfer a copy of the normal FACC gene. We also analyzed the ability of our viral vectors to functionally correct hematopoietic progenitor cells from a patient bearing a splice donor mutation. As for the lymphoid cell lines, these CD34-enriched cells were extremely sensitive to MMC. After infection of these progenitor cells with viral vectors bearing normal FACC, the progenitors gave rise to increased numbers of colonies both in the absence and presence of up to 5nM MMC, whereas control cells were completely destroyed by 1nM MMC. In summary, we have demonstrated that: (1) retroviral vectors can be engineered to transfer a normal FACC gene to FA(C) lymphoid cell lines and primary hematopoietic cells; (2) introduction of a normal FACC gene into CD34+ progenitors markedly enhances their growth in the absence and presence of MMC. This study is designed to determine whether hematopoietic progenitors transduced with the normal FACC gene can be reinfused safely into FA(C) patients. CD34+ cells obtained from G-CSF mobilized peripheral blood will be transduced ex vivo over a 72-hour period in the presence of IL-3, IL-6, and Stem Cell Factor with the FACC retroviral vector. These transduced cells will be reinfused into FA(C) patients. Patients will be monitored for toxicities as well as evidence of successful gene transfer and expression. The procedure will be repeated up to a total of 4 times with each treatment 2-4 months apart. Theoretically, these rescued stem cells should have a selective growth advantage within the hypoplastic FA marrow environment in vivo.
Membrane glutamate carboxypeptidase (mGCP) hydrolyses pteroylpoly-gamma-glutamates, methotrexate tri-gamma-glutamate and N-acetyl-aspartyl-alpha-glutamate. The enzyme is thought to be required for intestinal uptake of folate, for the resistance of some tumours to methotrexate, and for the metabolism of N-acetyl-aspartyl-glutamate, an abundant neuropeptide. It has recently been reported that mGCP is a protein also known as prostate-specific membrane antigen, homologous with transferrin receptor. This allows us to predict the domain structure of mGCP. Moreover, we have been able to assign the catalytic domain of mGCP to peptidase family M28, which contains cocatalytic zinc metallopeptidases. On the basis of the known structure of an aminopeptidase in family M28, we predict that Asp377, Asp387, Glu425, Asp453 and His553 are ligands of two atoms of zinc bound in the catalytic site of mGCP, and suggest that the aminopeptidases of Vibrio and Streptomyces can serve as valuable models in the design of inhibitors for this medically important enzyme.
BACKGROUND: Hepatitis-associated aplastic anemia is a variant of aplastic anemia in which aplastic anemia follows an acute attack of hepatitis. The aplastic anemia, however, is often fatal if untreated. To characterize the illness, investigate the role of hepatitis viruses, and assess the response to immunosuppressive treatment, we studied patients with the syndrome who were referred to the National Institutes of Health (NIH). METHODS: Standard hematologic and biochemical tests and measurements of bone marrow cellularity were used to monitor the patients' response to treatment. Serum was assayed for antibodies and antigens related to hepatitis A, B, and C viruses and for the RNA of hepatitis C and GB virus C by the polymerase chain reaction. All patients were treated with antithymocyte globulin and cyclosporine. RESULTS: Ten patients with hepatitis-associated aplastic anemia were referred to the NIH between 1990 and 1996; all had the typical features of this syndrome. There was evidence of activated CD8 T lymphocytes in the blood. Serologic tests for hepatitis A, B, and C viruses were negative; RNA of hepatitis C virus was undetectable in all patients, but RNA of GB virus C was detected in three patients. Seven of the patients responded to intensive immunosuppressive treatment; the three who did not respond all died within one year of treatment, two from complications of stem-cell or marrow transplantation. CONCLUSIONS: The hepatitis of the hepatitis-associated aplastic anemia does not appear to be caused by any of the known hepatitis viruses. We recommend immunosuppressive treatment for patients who do not have an HLA-matched related donor available for bone marrow transplantation. Several features of the syndrome suggest that it is mediated by immunopathologic mechanism.
Legumain is a cysteine endopeptidase that shows strict specificity for hydrolysis of asparaginyl bonds. The enzyme belongs to peptidase family C13, and is thus unrelated to the better known cysteine peptidases of the papain family, C1 (Rawlings, N. D., and Barrett, A. J. (1994) Methods Enzymol. 244, 461-486). To date, legumain has been described only from plants and a blood fluke, Schistosoma mansoni. We now show that legumain is present in mammals. We have cloned and sequenced human legumain and part of pig legumain. We have also purified legumain to homogeneity (2200-fold, 8% yield) from pig kidney. The mammalian sequences are clearly homologous with legumains from non-mammalian species. Pig legumain is a glycoprotein of about 34 kDa, decreasing to 31 kDa on deglycosylation. It is an asparaginyl endopeptidase, hydrolyzing Z-Ala-Ala-Asn-7-(4-methyl)coumarylamide and benzoyl-Asn-p-nitroanilide. Maximal activity is seen at pH 5.8 under normal assay conditions, and the enzyme is irreversibly denatured at pH 7 and above. Mammalian legumain is a cysteine endopeptidase, inhibited by iodoacetamide and maleimides, but unaffected by compound E64 (trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane). It is inhibited by ovocystatin (cystatin from chicken egg white) and human cystatin C with Ki values < 5 nM. We discuss the significance of the discovery of a cysteine endopeptidase of a new family and distinctive specificity in man and other mammals.
Interferon-alpha (IFN-alpha) is an established treatment for chronic myelogenous leukemia (CML) in chronic phase, but the mechanism of its antileukemic activity is not clear. One possible mechanism of action might include the induction of apoptosis, and especially Fas-mediated cell killing may play an important role in the elimination of malignant cells. We investigated Fas receptor (Fas-R) expression and the consequences of Fas-R triggering in CML patients. Using two-color flow cytometry, we found a significantly higher number of Fas-R-expressing CD34+ cells in the bone marrow (BM) of CML patients compared with normal subjects. We have previously shown that IFN-gamma induces Fas-R expression on CD34+ cells; in this study, we investigated whether IFN-alpha induces Fas-R expression on CML progenitor cells. Dose-dependent induction of Fas-R expression was observed after IFN-alpha stimulation of CD34+ cells from CML BM. In methylcellulose culture, IFN-alpha alone at a therapeutic concentration showed only marginal antiproliferative effects on both normal and CML BM progenitors. In contrast, a Fas-R agonist, the anti-CD95 monoclonal antibody CH11, inhibited colony formation from normal progenitors, and the inhibition was even stronger on CML progenitors. When CML BM cells were cultured in the presence of IFN-alpha, Fas-R-mediated inhibition of colony growth was potentiated in a dose-dependent fashion, consistent with IFN-alpha induction of Fas-R expression. This functional effect did not require the presence of accessory cells, since similar results were obtained with purified CD34+ cells. In suspension cultures, we demonstrated that suppression of CML hematopoiesis by IFN-alpha and Fas-R agonist was exerted through Fas-R-mediated induction of apoptosis. Our findings suggest that the Fas-R/Fas-ligand system might be involved in the immunologic regulation of CML progenitor growth and that its effect can be amplified by IFN-alpha.
It is now clear that the graft-versus-leukemia (GVL) effect which accompanies allogeneic bone marrow transplantation for hematological malignancies is a powerful therapeutic weapon which, if harnessed, could improve our ability to treat refractory malignant disorders. Advances in the understanding of the alloimmune response now provide a clearer picture of the mechanisms involved in the GVL reaction: the CD4+ T cell plays a central role in the orchestration of leukemia cell killing. The immunogenicity of the leukemia is also a major factor determining the effectiveness of the GVL response. The characterization of antigens restricted to leukemia and hematopoietic tissues should make it eventually possible to produce specific and powerful antileukemic alloresponses in donor lymphocytes by adoptive immunotherapy or by vaccines.
There is good evidence that T lymphocytes play an important role in the graft-versus-leukemia (GVL) effect following allogeneic bone marrow transplantation (BMT) for hematologic malignancies. However, the role of natural killer (NK) cells in GVL is less clear. To further investigate a possible association of NK cells with GVL we studied 15 patients undergoing BMT for chronic myeloid leukemia (CML), correlating T-cell (CD4+ and CD8+) and NK-cell (CD16+ 56+) recovery with relapse and graft-versus-host disease (GVHD). Patients were studied on three occasions up to 9 months after BMT, for lymphocyte surface phenotype and for spontaneous and IL-2-stimulated (LAK cell) cytotoxic function. Circulating CD8+ and NK but not CD4+ cell numbers were significantly lower in five patients who relapsed compared with those remaining in remission after BMT (mean 0.03 vs 0.32 x 10(9)/l, p = 0.002 for CD8+ cells: mean 0.03 vs 0.11 x 10(9)/l, p = 0.002 for NK cells). There was no correlation of CD4+. CD8+, or NK cell numbers and development of grade-II or more acute GVHD. Spontaneous NK cytotoxic function rose to within the normal range in the first month after BMT. LAK function remained low during the study period. These results link NK cell recovery more closely with a GVL than with a GVH effect.
The high relapse rate after T-cell depleted bone marrow transplantation (BMT) for chronic myeloid leukaemia (CML) and the ability of donor lymphocyte transfusions to induce stable remission in patients relapsing after BMT has emphasized the importance of alloreacting donor T-lymphocytes in the graft-versus-leukaemia (GVL) effect in this disease. The mechanisms underlying the GVL response and its relationship with graft-versus-host disease are becoming better defined. There is accumulating evidence that the CD4+ T-cell plays a central role in the GVL response. The prominent role of GVL in the cure of CML after BMT may be associated with the fact that CML cells are strongly immunogenic. New transplant strategies are being devised to separate GVL from graft-versus-host reactions. Future developments focus on the identification of leukaemia-specific antigens and the amplification of T-cell responses against them.
New understanding of the alloresponse following bone marrow transplantation supports the possibility that the graft-versus-host disease (GVHD) response can be separated from a favorable graft-versus-leukemia (GVL) effect. We used chronic myeloid leukemia (CML) cells to generate 122 recipient-reactive T cell clones from a closely HLA-matched sibling responder. Clones were tested for their proliferative response to stimulator CML cells or PHA-transformed (non-leukemic) lymphoblasts. Of 78 clones tested, 32 recognized both leukemia cells and PHA blasts, 19 only CML and four only PHA blasts. The remainder were non-specific responders. This functional specificity corresponded to distinct patterns of T cell receptor (TCR) V beta usage: clones recognizing CML cells preferentially used V beta 5, V beta 6/7 while clones recognizing both CML and PHA blasts or only PHA blasts preferentially used V beta 3 and V beta 8. It may therefore be possible to identify in vitro-generated myeloid leukemia-restricted donor T cells by their pattern of V beta usage. TCR V beta antibodies could thus be used to select and expand leukemia-restricted donor T cells for transfusion after BMT to specifically enhance the GVL response.
We studied patients relapsing with myeloid leukemias following allogeneic bone marrow transplantation (BMT) for evidence of immune escape by clonal evolution of the leukemia. Relapsed cells from four out of five patients had a reduced ability to stimulate proliferation of lymphocytes from an HLA-mismatched responder. There was decreased susceptibility to lysis by CTL in three and reduced susceptibility to NK-mediated lysis in one. Relapsed leukemias had marked alterations in expression of critical surface molecules involved in immune responsiveness. Three had decreased expression of MHC class I and II, with no change or increase in CD54 (ICAM-1) or CD80 (B7.1). None of these responded to treatment with donor lymphocytes. Three patients showed no change, or increased expression of MHC with no change or decrease in ICAM-1 or B7.1. Two achieved remission - one in response to donor lymphocytes and one following withdrawal of cyclosporine. In one patient transplanted with myelodysplastic syndrome in transformation, interferon-gamma upregulated expression of MHC molecules in relapsed cells and increased their stimulatory capacity and target susceptibility to unmatched responder lymphocytes. These results suggest that immune escape through clonal evolution of the leukemia is a common occurrence in patients who relapse with myelogenous leukemias after BMT.
Twenty-five transfusion-dependent myelodysplastic syndrome (MDS) patients (with < 20% blasts) were treated in a phase II study with antithymocyte globulin (ATG) at 40 mg/kg/d for four doses and then followed with blood counts every 2 weeks and clinic visits every 3 months, for a median of 14 months (range 1-38 months). 11 (44%) patients responded and became transfusion-independent after ATG, including three complete responses, six partial responses, and two minimal responses. Responses were observed in 9/14 patients (64%) with refractory anaemia (RA) and 2/6 patients (33%) with refractory anaemia with excess blasts (RAEB). Median response duration was 10 months (range 3-38 months). The Kaplan-Meier estimate of overall survival was 84% at 38 months, with one early death due to pneumonia and two deaths from disease progression to leukaemia. Side-effects consisted mainly of mild serum sickness in all patients. A single course of ATG restored haemopoiesis in some patients with MDS and was well tolerated.
The N-terminal amino-acid sequence of pig dipeptidyl-peptidase II (EC 3.4.14.2; DPP II) recently published (Huang, K., Takagaki, M., Kani, K. and Ohkubo, I. (1996) Biochim. Biophys. Acta 1290, 149-156) proves that the enzyme is homologous with lysosomal Pro-X carboxypeptidase (EC 3.4.16.2), and belongs to peptidase family S28 in clan SC. This is consistent with a number of biochemical similarities between these two prolyl bond-cleaving serine peptidases. DPP II is not related to granzymes, as was suggested by Huang et al.
Proteinase 3 is present in high concentration in the primary granules of acute and chronic myeloid leukemia blasts, and may represent a potential T-cell target antigen. We screened proteinase 3 against the binding motif of HLA-A2.1. Based on its high predicted binding, a 9-mer peptide, "PR-1," was synthesized and tested for binding to HLA-A2.1 using the T2 cell line. PR-1 at 100 micrograms/mL significantly increased expression of HLA-A2.1, with median channel of fluorescence increasing from 22 to 294. Binding half-life was determined to be 1,460 minutes by I125-labeled beta 2-microglobulin incorporation. HLA-A2.1+ peripheral blood mononuclear cells from a normal donor were used to generate a T-cell line specific for PR-1. The line demonstrated 85% PR-1-specific lysis at an E:T ratio of 50:1, compared with 20% lysis without PR-1, using T2 cells as targets. It also showed 79% specific lysis to fresh chronic myelogenous leukemia blasts, 54% to fresh acute myelogenous leukemia blasts, and only background lysis (< 20%) to HLA-A2.1+ normal allogeneic marrow cells. The amount of lysis of HLA-A2.1+ myeloid cells was proportional to cytoplasmic proteinase 3 expression. Thus, HLA-A2.1-restricted cytotoxic T cells, raised against a peptide contained in proteinase 3, preferentially lysed fresh human leukemic cells.
Explore the source record for details and available documents.
The natural history of PRV is characterized by a prolonged period of myeloproliferation chiefly affecting the red cell series. It can be controlled by venesection and cytotoxic agents. In this case report the patient declined further 'standard' chemotherapy to control his disease and the use of relatively low doses of IFN-a was successful in our patient in controlling myeloproliferation and stabilizing the disease over a prolonged period. These results support the use of IFN-a, not only as a means of myelosuppression in PRV but also as an agent with the potential to prevent disease progression.
Existing evidence supports that CD4+ T lymphocytes play a role in the graft-versus-leukaemia (GVL) reaction after allogeneic bone marrow transplantation (BMT) for chronic myeloid leukaemia (CML), not only as initiators of the immune response but also as effectors of GVL. In BMT between HLA-identical pairs this CD4-mediated GVL would require CML cells to process and present antigens through MHC class II molecules. To investigate whether CML cells are capable of processing and presenting antigens, and suitable targets for CD4+ T-cell-mediated cytotoxicity, we generated HLA-DR1-restricted CD4+ cytotoxic T-cell clones that specifically recognized tuberculous purified protein derivative (PPD). We have shown that CML cells and B lymphoblastoid cell line (B-LCL) cells but not PHA-blasts from patients with CML processed exogenous antigen, PPD, and induced proliferative and cytotoxic CD4+ T-cell responses. Antigen presentation was blocked by antibodies to HLA-DR but not to MHC class I and by treatment with chloroquine and brefeldin. This indicates that CML cells use a classic MHC class II antigen processing pathway to present PPD antigens to CD4+ T cells. Cytotoxicity to CML was shown by antibody blocking studies to be mediated mainly through fas antigen. These findings indicate that donor CD4+ T cells alone are sufficient to mediate GVL effects following allogeneic BMT for CML.