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Biomedical subjects

A J Barrett

Publications and source records attributed to A J Barrett.

At least 109 records · Page 6Linked to original sources
PubMed1996

Interaction of natural killer cells with MHC class II: reversal of HLA-DR1-mediated protection of K562 transfectant from natural killer cell-mediated cytolysis by brefeldin-A.

Major histocompatibility complex (MHC) class I antigens on tumour cell surfaces have been shown to modulate target susceptibility to natural killer (NK) cell-mediated lysis in some, although not all, systems investigated. MHC class II expression may also affect NK cell function, but the mechanism by which MHC class II antigen regulates NK cell activity has not been fully examined. In this study we induced HLA-DR1 expression by gene transfection into the classic NK-sensitive K562 cell line to study the interaction of NK cells with MHC class II molecules and the effect of brefeldin-A (BFA), an endogenous antigen-processing pathway blocker, on NK-target cell interaction. We demonstrated that the expression of HLA-DR1 on the cell surface reduced K562 cell susceptibility to NK lysis by peripheral blood monuclear cells and a NK cell line. The effect was demonstrable in prolonged (8 hr) cytotoxicity assays and was blocked by pretreatment of target cells with anti-HLA-DR antibody. Treatment of K562 DR transfectant with BFA abrogated the resistance of K562 transfectant to NK-mediated cytolysis. These findings indicate that HLA class II molecules regulate NK cell function and target recognition, and suggest that endogenous peptides presented through MHC molecules are responsible for regulating NK cytolysis.

Brefeldin A↗
PubMed1996

Helper and cytotoxic lymphocyte responses to chronic myeloid leukaemia: implications for adoptive immunotherapy with T cells.

We studied 19 patients with chronic myeloid leukaemia (CML) to characterize T-cell autologous responses to leukaemia. Third party stimulated alloresponses in mixed lymphocyte reactions were normal in all patients. Using the helper T-lymphocyte precursor frequency (HTLPf) assay we demonstrated a low frequency of T helper cells recognizing autologous leukaemia cells from CML blood (1/850 000) and marrow (1/965 000). However, similar frequencies to autologous bone marrow and lymphoid cells were also found in normal individuals. In 11 patients studied, HLA-matched siblings had a higher HTLPf to leukaemia than the patient's autologous response (P < 0.004). Alloresponse in mixed lymphocyte reactions (MLR), and autologous HTLPf to leukaemia, were comparable at all stages of disease progression and time from diagnosis, and independent of treatment given. In order to generate autologous cytotoxic lymphocyte responses to CML, lymphocytes were stimulated with CML cells. Cultures were fed again with CML cells and examined for cytotoxicity after 21 d. Strong lymphokine-activated killer (LAK) cytotoxicity was found against K562 and Daudi cell lines, and to Epstein-Barr virus-transformed allogeneic and autologous lymphoblastoid cell lines. Autologous leukaemia cells were lysed to a lesser extent in only 3/13 patients tested. The findings indicate that immune reactivity in CML is normal but suggest that CML cells are relatively resistant to lysis by cytotoxic T cells. The results do not support the existence of a leukaemia-specific T-cell response in CML.

Adult↗
PubMed1996

Specific depletion of alloreactivity against haplotype mismatched related individuals by a recombinant immunotoxin: a new approach to graft-versus-host disease prophylaxis in haploidentical bone marrow transplantation.

Haploidentical bone marrow transplantation (BMT) is associated with a high risk of severe graft-versus-host disease (GVHD). While pan-T cell depletion of the graft is the most effective means of preventing severe GVHD, it is associated with delayed recovery of T cell function leading to fatal infections. We used two related Pseudomonas exotoxin-based immunotoxins, anti-Tac(Fv)-PE38 and anti-Tac(Fv)-PE38KDEL, that both target the IL-2 receptor on activated T cells, to specifically deplete alloreactive lymphocytes against haploidentical stimulators. The functional capacity of the remaining lymphocytes was tested in proliferative assays against the original haploidentical stimulator and pooled cells from other mismatched donors (third party). We varied the recombinant toxin concentration and schedule to determine the optimum conditions for selective depletion. In 10 experiments, the mean residual reactivity after depletion was 7.6 +/- 1.4% against the haploidentical stimulator and 64.2 +/- 5% against the third party, expressed as a percentage of the undepleted response to the same stimulators. Depletion was shown to be specific for mixed lymphocyte culture (MLC)-activated lymphocytes. The immunotoxin did not affect CFU-GM growth of normal BM cells. This selective depletion of haploidentical alloreactivity could be used to prevent GVHD while conserving immune recovery following haploidentical BMT.

ADP Ribose Transferases↗
PubMed1996

Early reactivation of cytomegalovirus and high risk of interstitial pneumonitis following T-depleted BMT for adults with hematological malignancies.

Reactivation of cytomegalovirus (CMV) is a major cause of morbidity and mortality following allogeneic bone marrow transplantation (BMT) and early reactivation with increased risk of interstitial pneumonia (IP) has been reported in T cell-depleted BMT. In 21 patients at risk for CMV reactivation following T cell-depleted BMT for hematological malignancies, CMV reactivation was the main cause of transplant-related mortality: 19 (90.5%) reactivated CMV and five (26% actuarial) developed fatal IP. Median reactivation time post-BMT was 22 days for IP and 39.5 days for non-IP patients (P = 0.04). Patients developing IP showed no increase in lymphocyte count following CMV reactivation. There was a trend for higher pp65 antigen load at reactivation in patients who relapsed with CMV or progressed to IP. Donor lymphocyte transfusions (DLT) were given to pre-empt leukemic relapse and viral infection (15 patients) or to treat IP in two. Nine of 19 patients reactivated CMV before receiving DLT on day 30 post-BMT and DLT were ineffective in established IP. However DLT from seropositive donors did not cause IP. These results confirm the relationship of donor T cell function with CMV reactivation following BMT. They suggest that patients receiving T cell-depleted BMT should be protected from CMV disease with prophylactic rather than pre-emptive treatments.

Adolescent↗
PubMed1995

Detailed deletion mapping of chromosome 9p and p16 gene alterations in human borderline and invasive epithelial ovarian tumors.

We used PCR amplification of tandem repeats to study the pattern of allelic loss in borderline and invasive ovarian epithelial tumors using 12 primer pairs to generate a detailed deletion map of chromosome 9p. In the invasive ovarian carcinomas, there were three regions displaying high frequency of loss of heterozygosity (LOH) ranging from 31-38%. In contrast, LOH was a rare event among the borderline ovarian tumors, with one region revealing a rate of 20% and the remaining regions only 0-8% LOH. Therefore, allelic loss does not seem to be important for the development of borderline ovarian tumors. We also examined p16 gene expression and mutations in ovarian cancer cell lines and invasive and borderline ovarian tumor tissues. Southern blot analysis revealed no losses of the p16 gene in either the invasive or borderline ovarian tumors. However, the ovarian carcinoma cell lines showed a 50% homozygous deletion rate. SSCP analysis detected a mobility shift in only one (borderline) tumor. Since the primary invasive ovarian tumors did not show any deletions or mutations, it appears that p16 does not play a role in the pathogenesis of these tumors.

Carrier Proteins↗
PubMed1995

Rat thimet oligopeptidase: large-scale expression in Escherichia coli and characterization of the recombinant enzyme.

The coding sequence for rat testis thimet oligopeptidase (TOP) (EC 3.4.24.15) was placed under the control of the T7 polymerase/promoter system. Cultures of Escherichia coli transfected with the resulting plasmid expressed the enzyme as a soluble cytoplasmic protein. Medium-scale cultures allowed isolation of the enzyme in quantities of tens of milligrams. The availability of the recombinant enzyme permitted the determination of such chemical properties as epsilon 280 (48,960), zinc content (2 atom/molecule) and available thiol content (8-10/molecule) for TOP. The recombinant enzyme showed the catalytic activities previously reported for the naturally occurring enzyme, so that we can now conclude with confidence that these are all due to TOP and there is no need to postulate the existence of separate 'Pz-peptidase' or 'endo-oligopeptidase A' enzymes.

Alkylating Agents↗
PubMed1995

Thimet oligopeptidase specificity: evidence of preferential cleavage near the C-terminus and product inhibition from kinetic analysis of peptide hydrolysis.

The substrate-size specificity of human thimet oligopeptidase (EC 3.4.24.15) was investigated with oligomers of glycyl-prolyl-leucine (GPL)n where n = 2, 3, 4 and 5. These peptides were cleaved only at Leu-Gly bonds to give GPL as the single final product. Hydrolysis was most rapid with (GPL)3 and slowest with (GPL)5. The more water-soluble oligomers of Gly-Hyp-Leu showed the same trend. (Gly-Hyp-Leu)6 was not hydrolysed, consistent with the previous finding that substrates larger than 17 amino acids are not cleaved by thimet oligopeptidase. The cleavage of (GPL)3 to GPL fitted a sequential first-order model. First-order kinetics were unexpected as the initial substrate concentration was greater than Km. The anomaly was also seen during the cleavage of bradykinin and neurotensin, and in these cases first-order behaviour was due to potent competitive inhibition by the C-terminal product. The sequential mechanism for (GPL)3 breakdown by thimet oligopeptidase does not discriminate between initial cleavages towards the N- or C-terminus. As isoleucine is an unfavourable residue in P1, substrates were made in which selected leucine residues were replaced by isoleucine. GPL--GPI--GPL (where--represents the bond between the tripeptide units) was resistant to hydrolysis and GPI--GPL--GPL was cleaved only at the -Leu-Gly- bond. Experiments with isoleucine-containing analogues of (Gly-Hyp-Leu)4 showed that thimet oligopeptidase preferred to cleave these peptides near the C-terminus.

Amino Acid Sequence↗
PubMed1995

Immunoglobulin E antibodies to papaya proteinases and their relevance to chemonucleolysis.

STUDY DESIGN: Levels of four papaya cysteine proteinases were determined in Chymodiactin, a pharmaceutical preparation of chymopapain (EC 3.4.22.6) used in chemonucleolysis for the treatment of sciatica. Twelve sera known to contain immunoglobulin E antibodies to Chymodiactin were assayed for immunoglobulin E antibodies to these enzymes. OBJECTIVES: The goal of the study was to determine what contribution each of the four proteinases makes to the allergic response that occasionally occurs during injection of a damaged intervertebral disc with chymopapain preparations. SUMMARY OF BACKGROUND DATA: The occurrence of an allergic reaction during chemonucleolysis implies prior sensitization to components of the injected enzyme solution. The latex of the unripe fruit of the papaya plant Carica papaya, from which chymopapain is purified, contains another three immunologically distinct cysteine proteinases: 1) caricain (EC 3.4.22.30), 2) glycyl endopeptidase (EC 3.4.22.25), and 3) papain (EC 3.4.22.2). METHODS: A dot-blot immunoassay was developed to quantify each enzyme in Chymodiactin. Total serum immunoglobulin E levels and specific immunoglobulin E antibody levels to each of the four papaya cysteine proteinases were assayed by an enzyme-linked immunoassay in 12 sera containing immunoglobulin E antibodies to Chymodiactin. RESULTS: Chymodiactin contained 70% chymopapain, 20% caricain, 4% glycyl endopeptidase, and 0.1% papain. Immunoglobulin E antibodies to all four proteinases were found in most of the 12 sera, but in varying proportions. Antibodies to glycyl endopeptidase were predominant in eight sera, and the mean amounts of immunoglobulin E directed against each protein were: glycyl endopeptidase, 4.21 IU/ml; caricain, 2.9 IU/ml; chymopapain, 1.97 IU/ml; and papain, 1.39 IU/ml. Total serum immunoglobulin E levels showed little correlation with immunoglobulin E responses to Chymodiactin. CONCLUSIONS: The results suggested that removal of glycyl endopeptidase and caricain from pharmaceutical preparations of chymopapain may help reduce the incidence of allergic reactions during chemonucleolysis.

Chymopapain↗
PubMed1995

Families and clans of serine peptidases.

It has become clear that the proteolytic enzymes that depend upon a serine residue for their catalytic activity belong to many different families of proteins. We have attempted to group these families in "clans." A clan is defined as a group of families the members of which have a common ancestor. That is to say, they are homologous, although some of the similarities are in the "twilight zone" and the relationships cannot be proven by rigorous statistical methods. We believe we can recognize five, or perhaps six, clans of serine peptidases. In view of the separate evolutionary origins of the serine peptidases in different clans, it is not surprising to find that they may differ greatly and that indeed there are few generalizations that can be applied to all serine peptidases. In contrast, the members of a clan commonly have marked similarities.

Amino Acid Sequence↗
PubMed1995

Characterization of a mitochondrial metallopeptidase reveals neurolysin as a homologue of thimet oligopeptidase.

We have isolated a metallopeptidase from rat liver. The peptidase is primarily located in the mitochondrial intermembrane space, where it interacts non-covalently with the inner membrane. The enzyme hydrolyzes oligopeptides, the largest substrate molecule found being dynorphin A1-17; it has no action on proteins, and does not interact with alpha 2-macroglobulin, and can therefore be classified as an oligopeptidase. We term the enzyme oligopeptidase M. Oligopeptidase M acts similarly to thimet oligopeptidase (EC 3.4.24.15) on bradykinin and several other peptides, but hydrolyzes neurotensin exclusively at the -Pro+Tyr- bond (the symbol + is used to indicate a scissile peptide bond) rather than the -Arg+Arg- bond. The enzyme is inhibited by chelating agents and some thiol-blocking compounds, but differs from thimet oligopeptidase in not being activated by thiol compounds. The peptidase is inhibited by Pro-Ile, unlike thimet oligopeptidase, and the two enzymes are separable in chromatography on hydroxyapatite. The N-terminal amino acid sequence of rat mitochondrial oligopeptidase M contains 19 out of 20 residues identical with a segment of rabbit microsomal endopeptidase and 17 matching the corresponding segment of pig-soluble angiotensin II-binding protein. Moreover, the rat protein is recognized by a monoclonal antibody against rabbit soluble angiotensin II-binding protein, all of which is consistent with these proteins being species variants of a single protein that is a homologue of thimet oligopeptidase. The biochemical properties of the mitochondrial oligopeptidase leave us in no doubt that it is neurolysin (EC 3.4.24.16), for which no sequence has previously been reported, and which has not been thought to be mitochondrial.

Amino Acid Sequence↗
PubMed1995

Immunolocalization of thimet oligopeptidase in chicken embryonic fibroblasts.

An antiserum was raised against chicken thimet oligopeptidase (TOP), and specific antibodies were isolated by immunoadsorption. The specificity of the antibodies was confirmed by immunoblot analysis with the cell extract of cultured chicken fibroblasts. Subcellular localization of TOP in normal and Rous sarcoma virus-transformed chicken embryonic fibroblasts was investigated by indirect immunofluorescence staining with affinity-purified, monospecific anti-TOP antibodies. In addition to the diffuse reaction expected for a cytosolic enzyme, a punctate, cytoplasmic localization was observed. The appearance of the organelles containing TOP did not correspond to that expected for secondary lysosomes, mitochondria, endoplasmic reticulum, or Golgi apparatus, but did resemble that of endosomes. When cells were allowed to internalize Lucifer yellow before fixation, some of the organelles containing TOP were observed to contain Lucifer yellow. Moreover, those organelles containing TOP were positively immunolabeled with the anti-transferrin receptor antibody. We conclude that part of the TOP in chicken embryonic fibroblasts is located in endosomes and discuss the implications of this.

Animals↗
PubMed1995

Overexpression of the protein tyrosine phosphatase, nonreceptor type 6 (PTPN6), in human epithelial ovarian cancer.

Our current understanding of human ovarian tumorigenesis is limited by the lack of a discrete precursor lesion as well as a limited knowledge of the steps in tumor progression. Since the alterations in the regulation of the tyrosyl residues on various cellular proteins appear to be an important pathway in neoplastic transformation, it is possible that changes in the expression of the proteins that control tyrosine phosphorylation (i.e., tyrosine kinases and phosphatases) may play a role in ovarian cancer development. Protein tyrosine phosphatase, nonreceptor type 6 (PTPN6), contains two src homology 2 domains and is expressed primarily in hematopoietic and epithelial cells. Using Northern blot and immunoblotting analysis, we showed that both the PTPN6 transcripts and proteins were overexpressed two- to four-fold in 7 of the 8 ovarian epithelial carcinoma cell lines studied. In addition, we showed that there was also a two- to threefold increase in expression of the PTPN6 transcript in 10 of 11 (91%) invasive ovarian epithelial cancer tissues examined. These observations suggest that the PTPN6 gene is potentially of etiologic relevance to a majority of ovarian cancers.

Epithelium↗