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J Scharfstein

Publications and source records attributed to J Scharfstein.

At least 37 records · Page 2Linked to original sources
PubMed1994

Trypanosoma cruzi: identification of a membrane cysteine proteinase linked through a GPI anchor.

The biochemical and functional properties of T. cruzi GP50/55, a novel glycosylphosphatidylinositol (GPI)-anchored membrane antigen have been investigated. A 50-52-kDa thiol proteinase activity could be immunoprecipitated with monoclonal antibodies (mAb) directed against GP50/55 (mAb C10), different from the one reactive with mAbs against lysosomal cysteine proteinase GP57/51. Furthermore, the mAb C10-reactive proteinase corresponded to the GPI-anchored surface antigen since the proteolytic and antigenic activity partitioned to the aqueous phase after Triton X114 phase separation of phosphatidylinositol specific phospholipase C (PI-PLC)-treated parasites. Of several proteins immunoprecipitated by a polyclonal anti-lysosomal cysteine proteinase, an mAb to GP57/51 recognized a 60-kDa protein, whereas mAb C10 recognized antigens ranging between 52 and 50 kDa. The GP50/55 antigen detected by mAb C10 is expressed on the parasite surface whereas the GP57/51 antigen is mainly intracellular. The internal peptide sequence obtained from purified GP50/55 showed that it is more homologous to the prototype of the cysteine proteinases superfamily, papain, than to the two T. cruzi lysosomal cysteine proteinases so far described. Our data indicate that the T. cruzi GP50/55 is a novel GPI-anchored cysteine proteinase and may represent another isoform of this heterogeneous group of proteinases.

Animals↗
PubMed1993

Analysis and partial epitope mapping of human T cell responses to Trypanosoma cruzi cysteinyl proteinase.

Human infection with Trypanosoma cruzi (Chagas' disease) is usually accompanied by humoral and cellular immune responses to GP57/51, a major antigen that was recently identified as a prominent cysteinyl proteinase (cruzipain). The PBMC responses of 11 chronic chagasic patients and the properties of anti-cruzipain T cell lines are reported herein. GP57/51, isolated from Y strain epimastigotes (n-cruzipain) or the recombinant protein expressed in E. coli (r-cruzipain), elicited proliferative responses of variable intensity from the patient's PBMC. T cell lines were then generated using each of these antigens. These lines, which always carried the CD4+ phenotype, were reciprocally stimulated by n-cruzipain or r-cruzipain, the responses to the former being usually stronger. The analysis of cytokine production suggested that Th1-like subsets dominate the patient's responses: IFN-gamma was consistently induced on stimulation with either n-cruzipain or r-cruzipain. In contrast, IL-4 was present in very small concentrations or was undetectable. We then sought to define T cell epitopes of cruzipain using synthetic peptides spanning portions of the central (catalytic) domain and COOH-terminal extension. From a panel of 11 peptides, only one 33 mer peptide (P214) elicited a strong proliferative response on anti-cruzipain T cell lines, the intensity being comparable to that induced by r-cruzipain. Conversely, T cell lines started with P214 were responsive to either n-cruzipain or r-cruzipain, the proliferative responses again being accompanied by IFN-gamma production, but not IL-4. Interestingly, P214 is located in a conserved region of the catalytic domain of cruzipain, hence may propitiate opportunities for cross-recognition of other members of the papain superfamily. Fine epitope mapping should reveal whether structurally similar regions of host thiol-cathepsins can be potential targets for cross-reactive T cell responses during chronic human infection.

Animals↗
PubMed1993

Signal transduction in Trypanosoma cruzi: opposite effects of adenylcyclase and phospholipase C systems in growth control.

Fetal calf serum (FCS), which is mitogenic for the pathogenic protozoa T. cruzi, inhibits cAMP production in basal and forskolin-stimulated epimastigotes. It also activates phosphoinositides hydrolysis yielding diacylglycerol and inositol phosphates (Ins-P). Ins-P production is enhanced by AlF4-, GTP or beta-gamma-methylene-GTP, thus implying G proteins mediation in the phenomenon. An enzyme with phospholipase C activity which may be involved in the phospholipid metabolism was partially characterized.

Adenylyl Cyclases↗
PubMed1993

NO forms an adduct with serum albumin that has endothelium-derived relaxing factor-like properties.

Recent evidence suggests that sulfhydryl species can react with oxides of nitrogen under physiologic conditions and thereby stabilize endothelium-derived relaxing factor (EDRF) activity, but the presence of a specific in vivo thiol carrier for nitric oxide (NO) remains controversial. The single free sulfhydryl of serum albumin is the most abundant thiol species in plasma (approximately 0.5 mM) and is particularly reactive towards NO. To examine the potential role of serum albumin in endogenous nitric oxide metabolism, we synthesized S-nitroso-BSA (S-NO-BSA), a model S-nitroso-protein, and examined its effects on platelet function and coronary and systemic vascular tone in 16 mongrel dogs. Intravenous bolus S-NO-BSA markedly reduced mean arterial pressure in a dose-dependent manner and proved seven and a half-fold less potent than intravenous nitroglycerin and 10-fold less potent than intravenous S-nitroso-cysteine (half-maximal response of 75 nmol/kg compared to 10 and 7.5 nmol/kg, respectively; P < 0.05); when given by intravenous infusion (half-maximal response = 10 nmol/kg per min), however, S-NO-BSA and nitroglycerin were equipotent. Intravenous bolus S-NO-BSA had a greater duration of action than either nitroglycerin or S-nitroso-cysteine and produced marked prolongation of the template bleeding time associated with dose-dependent inhibition of ex vivo platelet aggregation (half-maximal response approximately 70 nmol/kg). Intracoronary S-NO-BSA increased coronary blood flow (mean +/- SEM) less effectively than nitroprusside, acetylcholine, or S-nitroso-cysteine (165% +/- 24% vs. 315% +/- 82%, 483% +/- 55%, or 475% +/- 66%, respectively; P < 0.05) although with much longer duration of action. On a molar basis, S-nitroso-cysteine proved more effective than S-nitroso-BSA, nitroprusside, or acetylcholine as an epicardial coronary vasodilator. Thus, serum albumin reacts with oxides of nitrogen to form a stable S-nitroso-thiol with properties reminiscent of authentic EDRF supporting the view that protein associated thiol may participate in the action and metabolism of EDRF.

Acetylcholine↗
PubMed1993

Use of Trypanosoma cruzi purified glycoprotein (GP57/51) or trypomastigote-shed antigens to assess cure for human Chagas' disease.

With the exception of assays for the detection of antibodies promoting complement-mediated lysis of Trypanosoma cruzi, serologic tests have generally failed to assess the effectiveness of chemotherapy for Chagas' disease. Conventional serology, although useful for the diagnosis of infection, is not capable of determining which patients have been cured. Here we demonstrate that a high proportion of antibodies detected by conventional serology (using fixed epimastigotes or trypomastigotes or crude extracts obtained therefrom) are directed against the carbohydrate residue galactosyl alpha 1- > 3 galactose (Gal alpha 1- > 3 Gal), a determinant also recognized by antibodies from noninfected healthy volunteers. In a study of 14 cured patients with long-term followup, we found that the persistently positive reactions detected using conventional serology were largely eliminated following immunoadsorption with melibiose. Because of their wide distribution among microorganisms of intestinal and pulmonary microflora, these carbohydrate determinants may keep stimulating lymphocytes previously primed by T. cruzi Gal alpha 1- > 3 Gal epitopes, thereby accounting for false-positive results in cured patients. Consistent with this proposition, enzyme-linked immunosorbent assays performed with two distinct T. cruzi antigen preparations that lack the Gal alpha 1- > 3 Gal epitope, namely purified GP57/51 and trypomastigote-shed antigens, were indeed capable of determining a cure after chemotherapy, albeit to a different degree. Collectively, the data indicate that conventional immunoassays prepared with highly specific T. cruzi antigens can be useful in the assessment of a cure after chemotherapy.

Acute Disease↗
PubMed1992

Molecular approaches to antithrombotic therapy.

Several new approaches to direct and indirect inhibition of thrombin formation or activity promise to extend the benefits of pharmacologic antithrombotic interventions, both prophylactically and in acute therapy. Preliminary studies have found the new agents to be more specific and hemostatically safer than warfarin or heparin.

Amino Acid Chloromethyl Ketones↗
PubMed1992

Inhibitors of the major cysteinyl proteinase (GP57/51) impair host cell invasion and arrest the intracellular development of Trypanosoma cruzi in vitro.

Peptidyl diazomethane (PDAM) derivatives, a class of irreversible inhibitors for cysteine proteinase, were screened for the ability to impair Trypanosoma cruzi invasion and intracellular development in primary cultures of heart muscle cells (HMC). T. cruzi GP57/51, a purified cysteinyl proteinase, and the substrate Z-Phe-Arg-NHMec were used to determine inhibition rate constants (k'+2) by continuous kinetic assays. The k'+2 values ranged from 25,400 to 2,800. The best inhibitors of GP57/51 had bulky hydrophobic residues in the P1 position (in addition to P2), the S1 sub-site specificity of the enzyme being thus similar to mammalian cathepsin L. The effects of these PDAM on parasite infectivity were then investigated. The ability to invade HMC was markedly impaired when trypomastigotes were briefly exposed to 10 microM of Z-(S-Bzl)Cys-Phe-CHN2. Striking effects were observed when PDAM were added to HMC cultures that had been previously infected with trypomastigotes: Z-(S-Bzl)Cys-Phe-CHN2 with an IC50 of 0.4 microM, and less markedly Z-Phe-Phe-CHN2 and Z-Tyr-Phe-CHN2 (or Z-Phe-Tyr-CHN2) blocked amastigote replication as well as their transformation into trypomastigotes, thereby arresting intracellular development. Bz-Phe-Gly-CHN2, in contrast, failed to display antiparasite activity. Direct characterization of the target cysteinyl proteinase was sought, by incubating viable amastigotes or infected HMC with Z-[125I]Tyr-Phe-CHN2. Affinity labeling implicated GP57/51 as the major cysteinyl proteinase target for this probe. We propose that T. cruzi intracellular development is critically dependent on GP57/51 (cruzipain). Selective inhibitors for this cysteinyl proteinase may have therapeutic potential.

Animals↗
PubMed1991

Further characterization of Trypanosoma cruzi GP57/51 as the major antigen expressed by differentiating epimastigotes.

Study of Trypanosoma cruzi metacyclogenesis under chemically defined conditions showed that the expression of a group of acidic polypeptides with a molecular weight of 45-50 kDa is markedly increased on adhesion of epimastigotes to the culture vessels. Immunochemical analysis revealed that these developmentally regulated polypeptides are structurally related to, and possibly homologous with, a major T. cruzi antigen, namely, GP57/51, a glycoprotein that has recently been characterized as a cysteine proteinase. The differentiation of epimastigotes into metacyclic trypomastigotes is accompanied by a 2- to 5-fold reduction in the concentration of GP57/51 antigen as determined by radioimmunoassays using monoclonal antibodies. Two-dimensional polyacrylamide gel electrophoretic analysis of metabolically labelled parasites indicated that this antigen is synthesized as a precursor with a molecular weight of 60 kDa, which is then processed to a level of 45-50 kDa via the formation of at least one intermediate processing product. The observation that expression of GP57/51-related products increases during epimastigote differentiation suggests an important role for this proteinase during the life cycle of T. cruzi.

Animals↗
PubMed1991

Interactions between CD3 and Thy1 T cell activation pathways: blockade of CD3-mediated T lymphocyte activation induced by immobilized anti-Thy1 antibodies.

Resting murine T cell activation induced by either CD3 complexes or Thy1 molecules was investigated in vitro, using surface-bound anti-CD3 mAb as the stimulus. One mitogenic anti-Thy 1 mAb (G7) lost mitogenicity when presented to T cells immobilized on a plastic surface, even in the presence of phorbol ester. Moreover, T cell activation induced by immobilized anti-CD3 was potently blocked by coimmobilized anti-Thy 1 mAb. Nonmitogenic anti-Thy 1 mAb also blocked CD3-induced activation when coimmobilized with anti-CD3. Control experiments showed that anti-Thy 1 specifically blocked T cell activation, even in the presence of measurable and functional concentrations of plastic-bound anti-CD3. Coimmobilized anti-Thy 1 potently blocked IL2 secretion stimulated by anti-CD3. Addition of exogenous rIL2 completely prevented anti-Thy 1-mediated blockade. On the other hand, while completely blocking T cell proliferation, immobilized anti-Thy 1 only partially blocked secretion of IL3-like activity by the T cells. One IgM anti-Thy 1 mAb (2A3) induced secretion of IL3-like activity by T cells when immobilized in the absence of bound anti-CD3. These results indicate that extensive aggregation of Thy 1 molecules delivers a potent negative signal which antagonizes CD3-mediated T cell activation and growth.

Animals↗
PubMed1990

Structural and functional identification of GP57/51 antigen of Trypanosoma cruzi as a cysteine proteinase.

Purified GP57/51, a Trypanosoma cruzi glycoprotein earlier identified as a major antigen in infected humans, was subjected to N-terminal sequence analysis. Alignment of the first 30 amino acids revealed that its N-terminal region is virtually identical to that reported for a cysteine-proteinase isolated from the Tulahuen strain, including the presence of active site cysteine at position 25. The finding of serine at position 24 of GP57/51 (Y strain) has further increased the homology between this protozoan antigen with other members of the eukaryotic family of cysteine proteases, including human cathepsin L. Functional analysis of GP57/51 indicated that the antigen is indeed an active thiol proteinase, which is active across a wide pH range (5-7.5). This was shown using either human IgG or gelatin substrates co-polymerized into polyacrylamide gels prepared for electrophoresis, and also by enzyme assays peformed with the synthetic substrate Z-phe-arg-NMec. The enzyme was activated by thiol containing reagents, and was strongly inhibited by low concentrations of E-64 (IC50 0.1 microM), cystatin (IC50 1 microM), leupeptin (IC50 0.1 microM) and antipain (IC50 0.1 microM). Monoclonal antibodies directed against distinct epitopes of GP57/51 absorbed the hydrolytic activity from purified preparations, demonstrating that the antigenic and enzymatic activities were indeed expressed by the same molecular entities. The subcellular localization of immunoreactive molecules was investigated by electron microscopy; immunogold staining was conspicuously found in vesicles belonging to the endosomal-lysosomal system, in tissue culture trypomastigotes as well as in epimastigotes. The possibility that this highly antigenic protease is actively secreted and/or leaked out of damaged parasites is under investigation; its release to tissues and to the circulation may contribute to pathology, considering that it (i) can degrade proteins across a wide pH range and (ii) stimulates immune T cells from chronic chagasic patients.

Amino Acid Sequence↗
PubMed1990

Identification and partial characterization of Trypanosoma cruzi antigens recognized by T cells and immune sera from patients with Chagas' disease.

Trypanosoma cruzi antigenic specificities involved in human T-cell and antibody responses were compared in chronic chagasic patients affected with cardiomyopathy (C) or with the indeterminate form (I), the asymptomatic form of chronic Chagas' disease. T-cell Western blotting (immunoblotting) was performed to identify the most active antigens in epimastigote extracts (EPI-Ag). The patterns of peripheral blood mononuclear cell (PBMC) and T-cell proliferative responses induced by fractions blotted to nitrocellulose were heterogeneous, but the computation of their frequency distribution disclosed some important antigen specificities. Molecules ranging from 100 to 150 kilodaltons (kDa) were frequently stimulatory to PBMC from I patients (5 of 8 cases) and were less so when confronted with C patient (1 of 7 cases) lymphocytes. In contrast, both groups of patients actively responded to fractions ranging from 48 to 57 and 28 to 32 kilodaltons (kDa). The Western immunoblotting patterns of antibody reactivity displayed by 17 C and 15 I patients were also similar, yielding outstanding staining in the molecular mass ranges of 70 to 80 and 43 to 57 kDa. The latter antigen complex was recognized by 100% of the 32 chronic Chagas' disease serum specimens tested and closely corresponded to the migratory position recognized by T cells of most patients tested. The identification of the active molecules contained in the 43- to 57-kDa region was sought, with a focus on GP57/51, an antigen with well-established serodiagnostic properties. Immunoblotting analysis of EPI-Ag with a monoclonal antibody to GP57/51 confirmed its presence within the predicted molecular weight region. Highly purified GP51 was then used to demonstrate directly its capacity to promote specific PBMC proliferative responses in vitro. Data computed from a survey with 12 patients have shown a linear correlation (r = 0.93) between PBMC responses to EPI-Ag and to purified GP51, suggesting that the immune response to this particular glycoprotein may be an important component of human immune responses against T. cruzi.

Animals↗
PubMed1986

Trypanosoma cruzi: characterization and isolation of a 57/51,000 m.w. surface glycoprotein (GP57/51) expressed by epimastigotes and bloodstream trypomastigotes.

We have recently described the application of a purified glycoprotein of 25,000 Mr (GP25) of Trypanosoma cruzi in serodiagnosis of Chagas' disease. Purified GP25 lacks appreciable immunogenicity in some animal species, in spite of being generally antigenic to parasitized hosts. The underlying cause for these contrasting observations has not been determined, but it may relate to the drastic extraction conditions used in the original isolation procedure, and possible damage inflicted to the native form of this antigen. This report describes the molecular properties of a GP25-related primary antigen, and a fast performance liquid chromatographic (FPLC) procedure to attain its isolation under gentle conditions. The expression of GP25-related antigen by epimastigotes or bloodstream forms was investigated with a high-affinity monoclonal antibody to GP25, SC11G10, as well as with monospecific antisera to GP25. Immunochemical analysis of Nonidet P-40 lysates supplemented with protease inhibitors indicated that GP25 is not synthesized as such; instead, a 57,000 Mr component (GP57) was identified as the primary antigen product. Partial enzymatic conversion to GP25 was observed when inhibitors were deliberately omitted from cell extracts. Most significantly, GP57 was established as the primary biosynthetic product in [35S]-labeled bloodstream trypomastigotes after immunoprecipitation with SC11G10 antibody. This analysis when applied to metabolically labeled epimastigotes has consistently revealed a minor antigen component of 51,000 Mr (GP51), in addition to GP57. The former was identified as the antigenically related product exposed at the parasite cell surface after external radioiodination of viable trypanosomes. Access to the native form of this widely distributed surface glycoprotein should stimulate the investigation of functional and structural aspects of its immunologic activity.

Animals↗
PubMed1985

Chagas' disease: serodiagnosis with purified Gp25 antigen.

Development of a highly specific test system for the diagnosis of Chagas' disease (CD) was sought using Gp25, a surface glycoprotein recently isolated from Trypanosoma cruzi culture forms. Radioimmunoprecipitation assays were performed to screen 567 sera for IgG antibodies to Gp25. Correct diagnosis was attained in 97.8% of the 321 sera collected from chagasic patients in several endemic areas of South America. Sera from patients with various clinical forms of chronic disease displayed similar levels of antibodies (Abs) to Gp25. Moreover, there was little cross-reactivity when assayed against 246 sera from non-chagasic individuals, including 105 samples from individuals infected with unrelated trypanosomatids. Cross-reactivity was found in two such sera; these were used to identify a minor protein contaminant as the nonspecific antigen. Residual cross-reactive molecules were eliminated from Gp25 by affinity purification on Concanavalin A (Con A) columns. We recommend this antigen-antibody system for use in routine screening of blood donors.

Adult↗