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D Snary

Publications and source records attributed to D Snary.

At least 19 recordsLinked to original sources
PubMed1989

Cloning and expression of a trypomastigote-specific 85-kilodalton surface antigen gene from Trypanosoma cruzi.

An 85-kDa trypomastigote-specific surface antigen gene from Trypanosoma cruzi has been identified by screening a genomic library in lambda gt10 with trypomastigote and epimastigote cDNA. The 1.3-kb genomic clone (pTt34) hybridizes to a single trypomastigote mRNA of 3.7 kb and to multiple bands in genomic Southern blots. Dot-blot experiments show that there are 5-10 copies of this sequence per haploid genome, and these are arranged in a non-tandem manner. pTt34 has been expressed as an anthranilate synthetase fusion protein in Escherichia coli, and inclusion bodies have been used to raise antiserum in rabbits. This antiserum immunoprecipitates a cell surface trypomastigote-specific protein of 85 kDa. The DNA and predicted amino acid sequences of pTt34 are given. Four further clones obtained from a PvuII/HpaI partial genomic library in pUC13 have extended the sequence of the 3' end of pTt34; each of these clones has regions of sequence divergence and each could represent a different member of the gene family.

Amino Acid Sequence↗
PubMed1988

Sequence homology of surface membrane proteins of Babesia rodhaini.

Four surface membrane proteins of Babesia rodhaini have previously been shown to induce a degree of protective immunity, and to carry both unique and cross-reactive determinants. cDNA clones for two of the genes coding for these proteins have been isolated and used as probes to isolate a single large genomic DNA fragment which contained all four genes. DNA sequence of two of the genes and their predicted amino acid sequences confirmed that the proteins had hydrophobic sequences at their N- and C-termini, an observation consistent with their proposed cell surface location. Homologies in both amino acid and nucleotide sequences were found at the 3'and at the 5' ends, but considerable sequence variations existed elsewhere in the genes and their products. The genes coding for these four proteins were tandemly arranged along a single relatively short length of chromosome, and such structures, because of their sequence homologies, probably could have arisen by gene duplication. The extensive variation suggested that there may be a functional need for these proteins to be different or capable of varying, although computer analysis implied that the extent of this variation may be constrained by structural requirements. This variation could be indicative of a role for these proteins in the host-parasite relationship or immune evasion.

Amino Acid Sequence↗
PubMed1986

An antigenic determinant common to both mouse red blood cells and several membrane proteins of the parasitic protozoa Babesia rodhaini.

Four membrane proteins of Babesia rodhaini have been identified by monoclonal antibodies. These proteins are differentiated by serological criteria and by tryptic peptide maps but contain common antigenic determinants. In vitro translation of parasite polyadenylated RNA followed by immunoprecipitation demonstrates that these common determinants are located within the primary sequence of the protein and do not result from post-translational modification. Part of the common amino acid sequence of these proteins is also present within a protein found in mouse red blood cells. Such common structures could potentially play an important role in the host-parasite relationship. The presence of sequence homology within a group of proteins from Babesia rodhaini could be further evidence for the atypical genetic rearrangements proposed to take place within Babesia species.

Amino Acid Sequence↗
PubMed1986

Prevalence of antibodies to 72-kilodalton glycoprotein (GP72) in patients with Chagas' disease and further evidence of zymodeme-associated expression of GP72 carbohydrate epitopes.

Three competitive inhibition enzyme-linked immunosorbent assays were developed to examine the expression of the 72-kilodalton glycoprotein (GP72) and of a GP72 carbohydrate epitope in Trypanosoma cruzi strains and clones. A total of 148 strains and clones of known isozyme phenotype (principal zymodeme, Z) were tested. With monoclonal antibody 8G2B9 the enzyme-linked immunosorbent assay confirmed that the majority of Z1 strains and clones derived from them had undetectable levels of the carbohydrate epitope identified by antibody 8G2B9. This epitope was, however, readily detectable in all Z2, Z2(h), and Z3 strains and clones (P less than 0.001; 148 strains and clones tested). Zymodeme-associated differences in GP72 expression were not apparent from the enzyme-linked immunosorbent assay with monoclonal antibody WIC 226.4 (raised against periodate-treated GP72) or from that with rabbit anti-GP72 antiserum (84 or 119 strains and clones tested, respectively). Mice infected with culture-form metacyclic trypomastigotes of Z1, Z29, and Z3 or with blood-form trypomastigotes of Z1 and Z3 developed antibodies to affinity-purified GP72, showing that at least some GP72 epitopes are neither zymodeme specific nor stage specific. A total of 128 serum samples from patients with acute or clinically classified chronic Chagas' disease were assayed for immunoglobulin G (IgG) or IgM anti-GP72 antibodies. During the acute phase anti-GP72 IgM antibodies were elevated, whereas anti-GP72 IgG antibodies were low. There were no significant differences in anti-GP72 antibody levels among chronic-phase patient groups. Anti-GP72 antibodies were detected irrespective of the geographical origin of patients and irrespective of whether acute-phase blood parasitemias were due to Z1 (four patients) or Z2 (two patients).

Animals↗
PubMed1985

Comparative compositions of cell surface glycoconjugates isolated from Trypanosoma cruzi epimastigotes.

Cell surface glycoconjugates of epimastigotes of Trypanosoma cruzi have been isolated and analyzed to give their amino acid and carbohydrate compositions. Those which have been investigated are a complex of three closely associated glycoproteins, GP24, GP31, GP37, and a lipopeptidophosphoglycan. The GP24-GP31-GP37 complex has an unusual amino acid composition with very low levels of hydrophobic amino acids, it contains 56% (w/w) carbohydrate, with mannose, galactose and glucosamine (presumably N-acetyl) being present in approximately equal quantities. The lipopeptidophosphoglycan also has low levels of hydrophobic amino acids and contains equal levels of mannose and galactose together with lesser amounts of (N-acetyl) glucosamine. The glycoconjugates are contrasted and compared with two other previously characterised cell surface glycoproteins (GP25 and GP72) from T. cruzi.

Amino Acids↗
PubMed1985

Phenotypic diversity of cloned lines of Leishmania major promastigotes.

In vitro cultured promastigotes of virulent (V) and avirulent (A) cloned lines of Leishmania major, and the parental isolate LRC-L137, were examined with respect to morphology, cell size, growth rate, and apparent DNA content. Growth rates of all lines were comparable and both virulent (V121, LRC-L137) and avirulent parasites (A12, A52, A59) exhibited a progressive decrease in apparent DNA content with time in culture, as measured by incorporation of Hoechst Dye 33342. The four cloned lines and the parental isolate showed differences in the content of morphological variants and in the mean body length. Morphologically, there were similarities between A12 and A52 and between A59 and V121. Promastigote populations were also examined for the expression of the target antigen of a previously characterized monoclonal antibody, WIC-79.3. This antibody binds to a membrane antigen that is also present in culture supernatants of Leishmania of A1 serotype. Three different assays with culture supernatants all showed that V121, A59, and A12 were high producers with LRC-L137 and A52, low producers. Similar variation in expression of the 79.3 target antigen was detected in intact organisms of the various lines by immunofluorescence with flow cytometry. No simple correlation was found between the expression or release of the WIC-79.3 target antigen and virulence. The virulence or avirulence of all cloned lines for BALB/c mice remained stable. The data are discussed in terms of differentiation stages of L. major promastigotes and the continuing search for morphological and biochemical markers of virulence.

Animals↗
PubMed1985

Receptors and recognition mechanisms of Trypanosoma cruzi.

The current state of knowledge on receptor and recognition interactions which take place during the life-cycle of Trypanosoma cruzi is reviewed. Evidence suggests that carbohydrate plays a central role in these recognition mechanisms. Lectin-sugar interactions appear to be involved in uptake of the parasite by host cells including macrophages and a protein on the surface of trypomastigote which binds N-acetyl glucosamine on the host cell has been implicated in host cell invasion. Sugars on a 72,000 molecular weight glycoprotein on epimastigotes have also been implicated in colonization of the gut of the insect vector and in control of the morphological changes which take place in the insect gut.

Acetylglucosamine↗
PubMed1984

Monoclonal antibodies demonstrate similarity of surface antigens on different clones of Schistosoma mansoni schistosomula.

Previous work has demonstrated wide variation in the susceptibility of different clones of Schistosoma mansoni to resistance in mice induced by a small schistosome infection. Eight monoclonal antibodies directed against individual surface antigens of juvenile schistosomes (schistosomula) were used to determine the distribution of these antigens among clones of schistosomula. All eight antigens were detected on the surface membranes of the 32 clones of schistosomula tested. This result implies that polymorphism of schistosomula surface antigens is very unlikely to be the cause of the variation in susceptibility of different clones to resistance in mice. Absence of heterogeneity among surface antigens of schistosomula may facilitate experimental vaccination aimed against these putative target antigens.

Animals↗
PubMed1984

Cryptic epitope explains the failure of a monoclonal antibody to bind to certain isolates of Trypanosoma cruzi.

A mouse monoclonal antibody, WIC 29.26 Ab, has previously been characterized as recognizing a carbohydrate epitope on a 72,000 m.w. glycoprotein (GP72) expressed on the surface of Trypanosoma cruzi epimastigotes and metacyclic trypomastigotes. This molecule has been implicated as a receptor in the control of parasite transformation, and when used as an immunogen in mice, partially protects against T. cruzi infection. In previous experiments in which a radioimmunoassay was used, WIC 29.26 Ab was found to react with approximately 50% of T. cruzi strains and clones derived from a variety of sources. In this study, we attempted to determine whether the WIC 29.26 Ab-nonreactive isolates lack the entire GP72 or merely lack the epitope recognized by this monoclonal antibody. WIC 226.4 Ab, a monoclonal antibody raised against periodate-treated GP72, reacted in an immunofluorescence assay with all strains and clones studied, including those which had not reacted with WIC 29.26 Ab. Likewise, two polyvalent rabbit sera, directed specifically against GP72, bound to all T. cruzi isolates tested. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of detergent lysates of surface-labeled epimastigotes immunoprecipitated with WIC 29.26 Ab showed that the epitope bound by this antibody was present in all but one of the parasites that were surface-nonreactive, as well as in all those that were surface-reactive. WIC 29.26 Ab precipitated a single 72K Mr band from most strains and clones, but in several cases 79K Mr and 66K Mr bands were seen. Isolates from both the surface-reactive and the surface-nonreactive groups showed the latter pattern. These results demonstrate that GP72, or similar electrophoretic variants--and with one exception, the carbohydrate epitope bound by WIC 29.26 Ab--are present in the surface membrane of all strains and clones tested. This observation suggests that in intact epimastigotes of the surface-nonreactive isolates, the epitope is not accessible because of structural changes in the molecule itself or because of differences in the membrane environment of GP72.

Animals↗
PubMed1984

Trypanosoma cruzi: continuous cultivation with murine cell lines.

Y strain parasites (Wellcome stock) were cloned as epimastigotes by limiting dilution and one clone, Wel Tryp A2 was used to infect cell cultures and irradiated mice. The fibrosarcoma line, M4, was susceptible to infection with Wel Tryp A2 trypomastigotes but, after the first round of intracellular infection, proliferation and differentiation, failed to support parasite growth in long-term continuous culture. In contrast, a muscle-derived cell line, S2, produced regular waves of extracellular trypomastigotes and amastigotes during 80 days of continuous culture reported here. These infected S2 cultures have remained in continuous culture for up to 18 months over a 5-year period and have yielded in excess of 10(8) parasites/20 ml culture at 4-6 day intervals. Extracellular amastigotes produced in these cultures have a close morphological, immunological and biochemical analogy to intracellular parasites derived from rodents, and have maintained an absolute dependence on host cells for their growth and proliferation. These amastigotes produced low-grade chronic infections in normal CBA/T6 mice but produced acute, fatal infections with high parasitaemia in mice of the same inbred strain given 900 rad. whole-body irradiation prior to infection. Although present findings relate to a single parasite clone, the parent stock from which the clone was derived showed similar growth characteristics when co-cultured with S2 cells.

Animals↗
PubMed1984

Quantitative differences in the expression of a 72,000 molecular weight cell surface glycoprotein (GP72) in Trypanosoma cruzi zymodemes.

A high affinity monoclonal antibody, 8G2 B9, was used to assess the expression of a 72,000 m.w. glycoprotein ( GP72 ) in isoenzyme-typed T. cruzi strains ( zymodemes ). Western blotting analysis of T. cruzi clones showed that 8G2 B9 bound strongly to GP72 and also suggested that this antigen was absent or weakly detectable in T. cruzi zymodeme 1 (Z1) strains. Purified 8G2 B9 was radiolabeled with 125I and used in an inhibition radioimmune binding assay to compare the quantities of GP72 in different zymodemes . Ninety-six T. cruzi strains were assayed, of which 36 were Z1, 36 were Z2, five were Z3 , and 19 were Z2 (heterozygous). Most (64%) Z1 strains lacked detectable GP72 , whereas this antigen was always detected Z2 and Z2 (heterozygous) strains. There was an 18-fold difference between geometric mean values for the quantities of GP72 (expressed as nanograms per milligram total cell protein) in Z1 and Z2 strains (Z1, 36 ng/mg; Z2, 639 ng/mg; p less than 0.001). There were also significant differences between the geometric mean values for Z2 and Z2 (heterozygous) strains, i.e., 639 ng/mg and 1648 ng/mg, respectively (p less than 0.001). GP72 was detected in four of the Z3 strains in quantities ranging from 740 to 3640 ng/mg. The absolute amounts of antigen in GP72 -positive strains were low, comprising less than 1% of the total cell protein. The specificities of two other anti- GP72 monoclonal antibodies, 7C6 D7 and WIC 29.26, were compared with 8G2 B9. Both antibodies completely inhibited the binding of 8G2 B9 to GP72 in solid phase immunoassays, suggesting that they reacted with the same antigenic determinants. The results show that monoclonal antibody-based assessments of the expression of GP72 correlate with zymodeme classification, and they also suggest that the monoclonal antibodies recognize major antigenic determinants on GP72 . It should be possible to use 8G2 B9 as an immunologic marker to additionally investigate the clinical significance of T. cruzi zymodemes and the biologic significance of GP72 .

Animals↗
PubMed1984

Genetically engineered vaccines: problems and promises.

Determinants of important protective antigens from a variety of viral, bacterial and parasite pathogens have been successfully cloned and expressed in novel hosts using recombinant DNA techniques. The use of these cloned determinants in the development of new vaccines, including immunologically defined subunit vaccines, genetically defined live attenuated vaccines, and synthetic peptide vaccines is described.

Animals↗
PubMed1983

Studies on the structure of a phosphoglycoprotein from the parasitic protozoan Trypanosoma cruzi.

A glycoprotein (GP72) has been isolated from Trypanosoma cruzi and found to contain 41% protein, 49% carbohydrate and 10% phosphate. All phosphate was covalently attached to the carbohydrate which contained the following sugars: ribose, xylose, fucose, galactose, mannose, glucose and glucosamine. The carbohydrate side chains were linked to protein by fucose, xylose and N-acetylglucosamine; 50% of the total N-acetylglucosamine was involved in glycoprotein linkages. Two classes of carbohydrate side chains were detected. One class comprised 15% of the total carbohydrate and contained glucosamine, mannose and galactose; some of these chains were phosphorylated. The other class comprised 85% of the total carbohydrate and contained xylose, ribose, fucose, galactose, mannose, glucosamine and phosphate; these chains were antigenic and reacted with a monoclonal antibody with specificity for the whole glycoprotein.

Acetylglucosamine↗
PubMed1983

Cell surface glycoproteins of Trypanosoma cruzi: protective immunity in mice and antibody levels in human chagasic sera.

Two cell surface glycoproteins from Trypanosoma cruzi have been compared for their ability to protect mice against an acute lethal infection. One of these, a 90,000 glycoprotein found on all stages of the parasite, protected against both bloodstream and metacyclic trypomastigote challenge. A 72,000 glycoprotein found only on insect-derived stages of T. cruzi protected only against a metacyclic trypomastigote challenge. Antibody against both of these glycoproteins was present in human chagasic sera.

Animals↗
PubMed1983

Monoclonal antibodies for serotyping Leishmania strains.

Mouse monoclonal antibodies raised against Leishmania tropica major were found to precipitate with the excreted factor produced by this and other leishmanial species. We suggest that a classification system for Leishmania, based on selective precipitation reactions between monoclonal antibodies and the excreted factor, would remove many ambiguities that currently exist.

Antibodies, Monoclonal↗
PubMed1983

A monoclonal antibody with specificity for Trypanosoma cruzi, central and peripheral neurones and glia.

Of 26 hybridomas secreting anti-Trypanosoma cruzi antibodies, two reacted with murine brain and spinal cord extracts but not other tissues. Both antibodies (5H7 and 3H3) had identical isotypes (IgM) and specificities as judged by western blotting. Antigens of molecular weight 58,000 and 37,000 in mouse brain and spinal cord, and 58,000 and 35,000 in T. cruzi were detected. Different tissue antigens were recognized by a previously reported T. cruzi mammalian neural tissue cross-reacting monoclonal antibody CE5; on immunofluorescence 5H7 stained some glia, and some central and peripheral neurones in rat tissue sections. Pooled sera from mice chronically infected with T. cruzi competed with binding of 5H7 to T. cruzi antigens.

Animals↗