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Biomedical subjects

S Z Shapiro

Publications and source records attributed to S Z Shapiro.

At least 19 recordsLinked to original sources
PubMed1999

Detection of human immunodeficiency virus type 1 after infection of unstimulated peripheral blood mononuclear cells.

Application of a highly sensitive PCR-based reverse transcriptase (RT) assay to the analysis of the infection of CD4+ cell lines with human immunodeficiency virus type 1 (HIV-1) demonstrated that virus production can be detected as early as 24 h after infection. Most of the signal at 24 h was due to virus production, as it could be substantially reduced by prior treatment with the RT inhibitor zidovudine. Virus production at 24 and 48 h was unaffected by the protease inhibitor indinavir. Infection of unstimulated peripheral blood mononuclear cells (PBMC) with a macrophage-tropic HIV-1 isolate yielded increasing virus production for 2-3 weeks, while infection with a T-cell line-tropic isolate yielded only low and sporadic virus production. Productive infection of unstimulated PBMC by the macrophage-tropic virus required functional Gag matrix and Vpr proteins; therefore, the monocyte-derived macrophage is probably the virus-producing cell in these cultures.

CD4-Positive T-Lymphocytes↗
PubMed1997

A broad cytotoxic T lymphocyte response to influenza type B virus presented by multiple HLA molecules.

The HLA restriction and epitope specificity of cytotoxic T lymphocytes (CTL) involved in recovery from influenza type B infection have not been extensively characterized. Here lymphocytes obtained from a healthy individual contained virus-specific CTL restricted by class I HLA molecules, HLA-A1, A2, B7 and B8, and the class II HLA molecules, HLA-DR1 and DR3. Four conserved viral epitopes were predicted from allele-specific motifs for peptides interacting with HLA-B8 and HLA-DR1. Bulk CTL recognized three 9mer HLA-B8-restricted peptides from nucleoprotein, residues 30-38, 263-271 and 413-421, and a 13mer HLA-DR1-restricted peptide from hemagglutinin, residues 308-320. The epitopes presented by HLA-A1, HLA-B7 and HLA-DR3 remain undefined. Peptide-specific CTL lines recognized influenza type B virus-infected cells indicating the peptides are representative of naturally processed epitopes. A hemagglutinin peptide-specific CD4 CTL clone expressed approximately 200 molecules of perforin mRNA/cell, suggestive of a functional perforin pathway for target cell lysis. The results indicate a broad CTL response composed of both CD8 CTL and CD4 CTL recognizing viral epitopes presented by multiple HLA molecules.

Antigen Presentation↗
PubMed1994

Failure of immunization with trypanosome endocytotic vesicle membrane proteins to provide nonvariant immunoprotection against Trypanosoma brucei.

Purified trypanosome endocytotic vesicles were subjected to Triton X-114 phase separation to obtain a fraction enriched in putative parasite receptors for adsorptive endocytosis. Rabbits immunized with this material produced antibodies that recognized many parasite proteins, including nonvarying epitopes on the parasite's endocytotic surface, the flagellar pocket membrane, as well as on membranes of endosomes and lysosome-like structures. These antibodies were unable to stimulate in vitro complement-mediated lysis of trypanosomes, and in an in vitro test of parasite growth inhibition they actually marginally enhanced parasite proliferation. No effect was observed on the parasite prepatent period or parasitemia in mice injected with antibody purified from the rabbit antisera, but their survival with the infection was significantly shortened. Finally, little difference was detected in parasitologic or hematologic parameters between immunized and control rabbits upon challenge with T. brucei infection.

Animals↗
PubMed1994

Human serum-sensitive Trypanosoma brucei rhodesiense: a comparison with serologically identical human serum-resistant clones.

Trypanosoma brucei rhodesiense clones, which are susceptible to lysis by normal human serum, were isolated from 3 different human serum-resistant clones originally derived from strain ETat 1.10. Serologically, these pairs of serum-sensitive and serum-resistant clones displayed the same variant surface glycoprotein (VSG) on their surface. Acquisition of human serum sensitivity correlated with susceptibility to lysis by human high density lipoprotein, a trypanocidal factor in normal human serum. Analysis of these paired populations by two-dimensional gel electrophoresis of whole trypanosomes and various subcellular fractions failed to reveal any differences in mobility of VSG and other proteins. Northern blot analysis of mRNAs from serum-sensitive and serum-resistant clones showed no differences when probed with a previously described resistance-specific probe. In addition, the ethanolamine membrane transport system and the overall membrane lipid fluidity did not reveal any detectable biochemical or biophysical differences in membrane properties. If resistance to lysis is indeed mediated by membrane changes at the enzymatic or structural level, the data presented suggest that the gene product(s) responsible for this change in human serum sensitivity may be present in very small quantities.

Animals↗
PubMed1993

Canine IgA glomerulonephropathy.

Three young intact male dogs housed together in a canine blood donor facility developed immune complex glomerulonephropathy within 2 years of each other. All three had membranoproliferative glomerulonephritis with varying clinical presentation and progression. Two dogs had subendothelial, and one dog subepithelial, electron microscopic dense deposits. Immunoperoxidase staining indicated that the primary antibody involved in the glomerular disease of these three dogs was IgA. The nature of the electron dense deposits was further studied by eluting and identifying immunoglobulin from affected kidneys of one dog. The primary antibody identified had a molecular weight greater than 300,000 Da and was determined to be IgA. Although IgA glomerulonephropathy is a common cause of glomerular disease in humans, this study represents the first documentation of the clinical syndrome of IgA glomerulonephropathy in the dog.

Animals↗
PubMed1993

Glycosome-associated tyrosine-phosphorylated protein in Trypanosoma brucei.

Phosphorylation of protein at tyrosine residues is an important mechanism for regulating protein function in eukaryotic cells. In this report we have identified by immunoblotting the target for tyrosine phosphorylation in the protozoan parasite Trypanosoma brucei as a doublet band of protein with molecular masses of 200 and 220 kDa. Ultrastructurally, the tyrosine-phosphorylated protein was localized to the microbody-like organelles unique to kinetoplastid protozoa, called glycosomes. Inhibition of multiplication by the tyrosine kinase inhibitor genistein appeared to have very different kinetics in procyclic and blood-stream stage parasites. This is consistent with a glycosomal location for the target of tyrosine kinase as these life cycle stages differ substantially in their dependence on glycolysis which occurs in this organelle.

Animals↗
PubMed1992

Analysis of host components in hydatid cyst fluid and immunoblot diagnosis of human Echinococcus granulosus infection.

To improve serodiagnosis of cystic hydatidosis, immunoblotting studies were performed to look for a highly specific parasite antigen(s). First, commercially available hydatid cyst fluid antigen preparations were characterized by SDS-PAGE and by immunoblotting with sera specific for parasite and host animal proteins. One preparation, designed for use in complement fixation tests, did not appear to be suitable for immunoblotting because of the low concentrations of parasite antigens. Several host proteins, including serum albumin and IgG, were detected in the cyst fluid. Sera from patients with Echinococcus granulosus infections and other parasitic diseases were examined by immunoblotting for antibodies against specific cyst fluid parasite antigens. Several parasite antigens were variably recognized. Only one antigen, a 40 kDa protein, was recognized by all E. granulosus-infected patients. Reactivity against this antigen was also observed in all sera from E. multilocularis, cysticercosis, and schistosomiasis patients as well as in some filariasis cases. Two E. granulosus antigens, molecules of 12.5 and approximately 17 kDa, were only recognized by antibodies from some E. granulosus patients.

Animals↗
PubMed1992

Identification of an acute-phase reactant in murine infections with Trypanosoma brucei.

A 42-kDa protein appeared at a much higher concentration in plasma from Trypanosoma brucei-resistant (C57BL/6) mice after infection than in plasma from trypanosome-susceptible (C3H/He) mice. This protein was purified by sequential steps of gel filtration, protein A-Sepharose affinity chromatography, isoelectric focusing, and ammonium sulfate precipitation. The purified protein was identified as a subunit of the acute-phase reactant haptoglobin. Causes of elevated plasma haptoglobin and its implications for resistance to trypanosomiasis are discussed.

Animals↗
PubMed1990

Trypanosoma brucei: a membrane-associated protein in coated endocytotic vesicles.

Membrane proteins were isolated from purified Trypanosoma brucei coated endocytotic vesicles by phase separation with Triton X-114. The largest abundant membrane protein was a doublet band with a molecular mass of about 77 kDa. A specific antiserum was prepared against this protein by immunization with antigen bands excised from sodium dodecyl sulfate-polyacrylamide gels. Immunoblot analyses with this antiserum showed that the 77-kDa protein was present in other T. brucei, in T. congolense, and in T. vivax bloodstream-stage parasites but absent from procyclic (tsetse fly midgut)-stage trypanosomes. Antigenically related molecules of 58, 300, and 15.5 kDa were also detected. The 300- and 15.5-kDa molecules were not in purified coated vesicles; they were detected in whole bloodstream- and procyclic-form T. brucei organisms. Immunofluorescent studies localized the antigen to the region between the flagellar pocket and the nucleus of bloodstream-form parasites. Ultrastructurally, the antigen was detected on membranes of endosomes and lysosome-like structures that contained endocytosed markers.

Animals↗
PubMed1989

Acquired resistance to ixodid ticks induced by tick cement antigen.

Antisera from guinea pigs made resistant to infestation with an ixodid tick of east and central Africa, Rhipicephalus appendiculatus, were used to identify the tick antigens they recognized by immunoblotting. Most of the antigens were found in tick salivary glands and in tick attachment cement. Antisera from R. appendiculatus-resistant guinea pigs also recognized some salivary-gland antigens in ticks of other species (R. pulchellus, R. evertsi, Amblyomma variegatum and A. gemma). Antibodies against the most strongly recognized R. appendiculatus antigen, a 20-kDa molecule, were only poorly reactive with similar-sized molecules in the other ticks. A 94-kDa antigen, which appeared to have broader cross-reactivity, was purified from R. appendiculatus attachment cement, and a monospecific rabbit serum was raised against it. This antiserum clearly recognized a molecule of similar molecular weight in R. pulchellus and R. evertsi. Intravenous inoculation of rabbits with the purified molecule elicited delayed-type hypersensitivity to the antigen. The hypersensitive rabbits demonstrated resistance to feeding of R. appendiculatus ticks but slight enhanced feeding of R. pulchellus ticks. These results are discussed with respect to their relevance for artificial induction of tick-feeding resistance.

Animals↗
PubMed1989

Biochemical characterization of activation-associated bovine class I major histocompatibility complex antigens.

Utilizing a 'sandwich' ELISA assay we have been able to demonstrate that mAb W6/32, B1G6 and IL-A19 are reactive with three different monomorphic determinants on bovine class I major histocompatibility complex (MHC) molecules. Sequential immunoprecipitations performed with the mAb revealed that class I molecules on PBM comprise a single population with respect to reactivity with the mAb in that the beta 2m-associated proteins bear all three epitopes. By contrast, TCGF-driven lymphoblasts and cells transformed by Theileria parva (Tp) additionally express molecules of Mr 45000 bound to beta 2m which are recognized by mAb B1G6 and IL-A19 but not by W6/32. These two subclasses of molecules were further distinguished on the basis that, when tunicamycin was added to cultures in the preparation of cells for analysis, mAb W6/32 precipitated class I heavy chains of Mr 39000 while the extra molecules detected only by mAb B1G6 and IL-A19 were of Mr 37000 and 39000. On thymocytes, the mAb W6/32-non-reactive class I molecules are present in low amounts and are expressed by cells in the medulla area, unlike BoT1 (analogous to human CD1) molecules which are expressed by the cortical cells. Our studies also revealed that the supposed beta 2m-specific mAb B1G6 does not recognize the beta 2m-associated molecules (BoT1) precipitated by mAb TH97A and thus the specificity of mAb B1G6 in cattle is for an epitope on bovine beta 2m which is strongly influenced by the nature of the heavy chain with which the beta 2m is associated.

Animals↗
PubMed1989

Analysis of Propionibacterium acnes-induced non-specific immunity to Trypanosoma brucei in mice.

Mice treated with dead Propionibacterium acnes (previously called Corynebacterium parvum), up to 30 days before infection with any of three strains of Trypanosoma brucei, were more able to limit the level of first-wave parasitaemia than untreated controls. Reduced parasitaemia was not due to enhanced phagocytosis of input parasites and was associated with a dramatic reduction in the proportion of multiplying T. brucei in the blood of treated as compared to control mice. For 4 days after P. acnes treatment, T. brucei growth-inhibitory molecules, assayed by their effect on T. brucei multiplication under axenic culture conditions, were detected in the serum of recipient mice. The molecules were released by macrophages collected from the peritoneal cavity of P. acnes-treated mice, and similar molecules were produced in vitro by macrophages from normal mice after incubation with P. acnes. Accessory studies suggested that the molecules were breakdown products of P. acnes and were unlikely to be responsible for the long-term in-vivo effects of the P. acnes treatment. It was also shown that monokines and lymphokines which are likely to be induced by in-vivo P. acnes treatment, i.e. IL-1, IL-2, TNF alpha, INF alpha, INF beta, INF gamma, PGE1, PGE2, PGF2 alpha and biological mediators present in Con-A and LPS-induced spleen cell supernatants (collected 20, 40, 60 or 80 h after mitogen stimulation) had no influence on T. brucei growth under axenic culture conditions over a wide range of concentrations. The studies suggest that the P. acnes effect was not due to a direct interaction of these biological mediators with the T. brucei. We suggest that the reduction in T. brucei parasitaemia in P. acnes-treated mice reflects secondary physiological effects of one or more unidentified biological mediators.

Animals↗
PubMed1987

Elimination of the detection of an artefactual 65 kDa keratin band from immunoblots.

Contamination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) samples with skin keratin has produced a recurrent problem of artefactual bands when highly sensitive detection methods, such as silver staining, have been used. Such an artefact also can be a problem in immunoblots because some sera appear to contain antibodies that react with a human skin keratin. A simple method for the removal of these antibodies with keratin immobilized on an affinity chromatography substrate is described.

Animals↗
PubMed1987

A simple method for the production of specific antiserum to protein encoded in cloned genes. Immunization with precipitin lines.

A simple technique for raising specific antisera to protein encoded by cloned genes is described. The procedure involves preparation of an antiserum to Escherichia coli beta-galactosidase and the use of that serum to immunoprecipitate a fusion protein in a crossed immunoelectrophoresis gel followed by immunization with fusion protein precipitin arcs. An antiserum was prepared against protein encoded by an open reading frame in a dispersed repeated DNA sequence found in the protozoan Trypanosoma brucei. This serum recognized a polypeptide doublet of 33.5 and 32.5 kDa on immunoblots prepared from extracts of T. brucei. The method described should be applicable to other investigations where an immunochemical reagent against protein encoded by a cloned gene is desired.

Animals↗
PubMed1987

A life-cycle stage-specific antigen of Theileria parva recognized by anti-macroschizont monoclonal antibodies.

Four monoclonal antibodies, raised against macroschizonts of Theileria parva, were studied to characterize their antigen binding specificity. The indirect fluorescent antibody test showed that the antigen(s) recognized were present in the macroschizont stage of the parasite life-cycle but not in piroplasm, kinete or sporozoite stages. Immunoblot analysis of macroschizont stage antigens suggested that all four antibodies recognized the same antigen. This was a molecule which varied in molecular mass between different parasite stocks, ranging from 68,000 to 95,000 Da. The antigen was localized by immunoelectron microscopy to the surface of the intracellular macroschizonts.

Animals↗