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Biomedical subjects

T W Pearson

Publications and source records attributed to T W Pearson.

At least 73 records · Page 4Linked to original sources
PubMed1985

Comparison of African trypanosomes of different antigenic phenotypes, subspecies and life cycle stages by two-dimensional gel electrophoresis.

High resolution two-dimensional polyacrylamide gel (2D gel) electrophoresis and autoradiography were used to analyze the protein gene products of African trypanosomes biosynthetically labelled with [35S]methionine. Using cloned populations of parasites it was found that: antigenically different bloodstream trypanosomes from the same serodeme differed only in their variant surface glycoproteins; Trypanosoma brucei, T.b. rhodesiense and T.b. gambiense subspecies could be distinguished on the basis of differences in expressed proteins; transformation from bloodstream trypomastigotes to procyclic epimastigote culture forms was accompanied by loss of variant surface glycoproteins and several other qualitative and quantitative changes in minor proteins. The results indicate that 2D gel analysis may allow improved classification of African trypanosomes (based on the observation of hundreds of protein markers) and may also provide a general technique for the identification of lifecycle stage specific markers.

Animals↗
PubMed1985

Rapid preparative scale purification of myristylated variant surface glycoproteins from African trypanosomes.

A rapid method for the preparative-scale purification of variant surface glycoproteins (VSG) from African trypanosomes has been developed to investigate the recently described myristylated form of this molecule. This high-performance liquid chromatography (HPLC) procedure gives high yields and can be achieved in as little as 1 h from the preparation of a cell lysate in 0.1% trifluoroacetic acid (TFA). Solubilization of myristylated VSG in 0.1% TFA was shown to be much more effective than in neutral or zwitterionic detergents. Surface iodination of bloodstream trypanosomes and subsequent lysis in TFA showed that no proteolytic degradation of VSG occurred during solubilization and purification. Biosynthetic labelling of trypanosomes with [3H]myristic acid and purification of VSG by the described method, demonstrated that the VSG possessed covalently linked fatty acid and can therefore be defined as the membrane form of VSG. Evidence was provided that the myristic acid was covalently associated with the C-terminal portion of the VSG via a hydroxyester bond. Solubilization of the myristylated VSG by 0.1% TFA could be interpreted to indicate that the fatty acid was not embedded in the trypanosome membrane.

Animals↗
PubMed1985

Identification of an antigen specific to Trypanosoma congolense by using monoclonal antibodies.

Using indirect immunofluorescence assays on acetone-fixed smears of a series of different parasites, we have shown that two monoclonal antibodies bind specifically to Trypanosoma congolense organisms. The antibodies bind to both bloodstream trypomastigotes and procyclic culture forms of the parasite and are thus not stage specific. Immunoprecipitation and immunoblot analysis showed that both monoclonal reagents bound a protein of approximately 31,000 m.w. This antigen appeared to be located on the plasma membrane of T. congolense, but the epitope was not exposed on the surface of living bloodstream or procyclic organisms. The antigen was detectable on acetone-fixed organisms or in trypanosome lysates in enzyme-linked immunosorbent assays and may therefore by useful as a species-specific marker in field assays for epidemiologic and clinical investigations.

Animals↗
PubMed1984

Purification of major variable-surface glycoproteins from African trypanosomes by reverse-phase high-performance liquid chromatography.

Reverse-phase high-performance liquid chromatography (RP-HPLC) was used in a one-step procedure to purify and analyze several different major variable-surface glycoproteins (VSGs) from lysates of African trypanosomes. RP-HPLC was used to fractionate lysates of trypanosomes and the VSG localized to the major peak of the elution profile using a rabbit antiserum to the cross-reacting determinant of the VSG. Polyacrylamide gel electrophoresis of HPLC fractions showed that the purity of isolated VSGs was equivalent to or better than that attained using conventional purification procedures. The elution positions of purified VSGs from a variety of cloned trypanosomes were identical, indicating the presence of a common hydrophobic feature on the surface of these highly polymorphic antigens. Preliminary experiments have shown that purification of VSG from trypanosome lysates may be scaled up to preparative levels. The results show that RP-HPLC is a useful procedure for rapid preparation of highly purified trypanosome VSGs and that analysis of their various molecular forms will be facilitated by the application of HPLC methods.

Animals↗
PubMed1984

Amino terminal sequence homology among variant surface glycoproteins of African trypanosomes.

Amino terminal amino acid sequences were determined for eight variable surface glycoproteins purified from successive parasitemias of cloned Trypanosoma brucei. These sequences were compared by sequence alignment analysis with each other and with amino terminal sequences of variable surface glycoproteins from other clones of T. brucei, T. congolense and T. equiperdum. Contrary to previously held views, a remarkable degree of sequence homology was found among all sequences. In several cases the homology was greater between glycoproteins from different species than between glycoproteins isolated from successive parasite peaks, suggesting a common primordial gene for trypanosome variable surface glycoproteins. This amino terminal homology, taken together with homologies found in other regions of variant surface glycoproteins strongly suggests that their tertiary structures are conserved.

Amino Acid Sequence↗
PubMed1983

Antibody responses induced by immunization of inbred mice susceptible and resistant to African trypanosomes.

We tested the ability of inbred mice that were either susceptible (strain A/J) or resistant (strain C57BL/6 and A/J X C57BL/6 hybrids) to African trypanosomes to produce specific antibodies to trypanosome antigens in the absence of living parasites. This experiment was carried out to eliminate the influence of trypanosome growth or metabolism on immune responsiveness. Mice were immunized with keyhole limpet hemocyanin or solubilized Trypanosoma brucei gambiense, and serum antibodies were measured in solid-phase radioimmunometric assays after primary and challenge injections. Both susceptible and resistant mice showed increases in keyhole limpet hemocyanin-specific or trypanosome-specific immunoglobulin M and immunoglobulin G after immunization. When immunized with trypanosome antigens, resistant mice made qualitatively and quantitatively superior specific immunoglobulin M responses, particularly to the trypanosome major variable surface glycoprotein. Susceptible A/J mice produced good specific antibody responses, although these were predominantly of the immunoglobulin G isotypes. These results show that A/J and C57BL/6 mice respond differentially in terms of immunoglobulin isotype and repertoire in response to injected antigens. The possibility that this differential antibody response influences susceptibility to African trypanosomes is discussed.

Animals↗
PubMed1981

Immunodepression in trypanosome-infected mice. VI. Comparison of immune responses of different lymphoid organs.

Mitogen stimulation of cells from various lymphoid organs of C3H/He mice chronically infected with an isolate of Trypanosoma congolense was studied at different time intervals after infection, using concanavalin A (Con A) and lipopolysaccharide (LPS). At the same time, changes in the percentages of T, B and null lymphocytes in these organs were determined by immunofluorescence staining. The responses of T and B lymphocytes in the spleen were totally depressed, and the cellular composition was drastically altered by day 14 after infection. Unlike the spleen, the lymph nodes showed minor changes in their T and B lymphocyte responses and cell composition during the course of the infection, except the B cell response and composition which were altered late in the infection. The thymus and bone marrow did not show any appreciable changes in their mitogen responses and cell composition throughout the infection. The peripheral blood lymphocytes showed reduced B cell responses. Spleen cells from chronically infected mice suppressed lymphocyte stimulation induced in normal spleen and lymph node cell populations by Con A, LPS and allogeneic stimulator cells. Lymph node cells from the same group of mice did not exhibit any such suppressor activity. In the experimental system used here, the spleen is the primary site of immune depression, and other lymphoid organs such as the lymph nodes and thymus are very little affected.

Animals↗
PubMed1981

Intracellular monosaccharide and amino acid concentrations and activities and the mechanisms of insulin action.

Current amino acid and monosaccharide transport models are based on an assumption which equates the intracellular chemical activity of a solute with its concentration. This assumption was tested for alpha-aminoisobutyric acid and 3-O-methylglucose in a giant cell, the amphibian oocyte, by using recently developed cryomicrodissection and internal reference phase techniques. We found the following. (i) alpha-Aminoisobutyric acid and 3-O-methylglucose activities were much greater in cytoplasm than was suggested by concentration data; i.e., activity coefficients were higher than in ordinary water solutions. This is attributable to the inaccessibility of considerable water as solvent (solute exclusion). (ii) Solute concentrations varied regionally as follows: nucleus > > animal cytoplasm > vegetal cytoplasm. Insulin increased the nucleus/cytoplasm concentration asymmetry, apparently by increasing cytoplasmic solute exclusion. (iii) Nuclear activity coefficients more closely resembled those of ordinary saline solutions so that nucleus/ extracellular concentration ratios reflected transmembrane activity gradients better than did cytoplasm (or whole cell)/extracellular ratios. (iv) Mediated passive alpha-aminoisobutyric acid and 3-O-methylglucose transport were constituent oocyte membrane properties. Membrane active transport was initiated with time (in the presence of substrate) and by insulin. (v) Increased temperature mimicked insulin in enhancing transmembrane alpha-aminoisobutyric acid activity gradients and increasing the nucleus/cytoplasm concentration asymmetry. These results indicated that concentration data are a misleading measure of cellular amino acid and monosaccharide activity; some consequences of this observation were explored. A model is proposed in which cell water has reduced solvent capacity or is compartmentalized (considered less likely) and is susceptible to physiological modulation. The model accounts for many observations in small cells, suggesting generality of the exclusion phenomenon and a previously unrecognized metabolic control mechanism.

3-O-Methylglucose↗
PubMed1981

Trypanosome variable surface antigens: studies using two-dimensional gel electrophoresis and monoclonal antibodies.

Variable surface antigens of cloned populations of African trypanosomes were studied using 2-dimensional gel electrophoresis and monoclonal antibodies. Two-dimensional gel maps showed that the only major differences in protein profiles of the non-nuclear materials from different clones of solubilized trypanosomes were attributable to the variable surface antigens and that purification doesn't alter these molecules, at least with respect to charge and apparent m.w. Purified variable surface antigens were used as immunogens and a number of monoclonal antibodies were derived using cell-fusion technology. In radioimmunoassay, each of the monoclonal reagents was shown to be specific for the immunizing antigen, and in immunofluorescence tests using acetone-fixed trypanosomes, each bound specifically to the clone from which the antigens were purified. Only 20% of the monoclonal reagents bound to living trypanosomes, however, providing evidence for both exposed and nonexposed antigenic sites on the variable surface antigens. Those reagents derived to a single antigen bound to different nonoverlapping antigenic sites, which were on the protein portion of the molecules and not the carbohydrate moieties. The data show that monoclonal antibodies are ideal probes for studying localization of antigenic sites on the antigen molecules and for studying antigen synthesis, glycosylation, and architecture in relation to the cell surface. In addition, the finding of exposed and nonexposed antigenic sites on trypanosome variable antigens allows a possible explanation for the role of the antigens in pathogenesis of African trypanosomiasis.

Animals↗
PubMed1981

The in vitro response to mitogens of leucocytes from cattle infected with Trypanosoma congolense.

The in vitro proliferative response of bovine leucocytes from peripheral blood to LPS, PWM and Con A was monitored during the course of infection with T. congolense. The stimulatory effect of LPS and PWM on PBL was significantly decreased in infected cattle while the reduction of stimulatory response to Con A was less marked. There was a high background proliferation of unstimulated leucocytes from infected cattle. This increase in background counts correlated with the parasitaemia in that both features were abolished by treatment with the trypanocidal drug Berenil (4,4'-diamidino-diazoaminobenzene-diacetamide acetone). Co-cultivation of trypanosomes with PBL from normal cattle did not depress the responses of the latter to mitogens, and trypanosomes maintained in a cell-free medium failed to incorporate 14C-thymidine under the conditions used.

Animals↗
PubMed1980

Methods for derivation and detection of anti-parasite monoclonal antibodies.

We describe detailed methods for derivation and cloning of myeloma hybrids which secrete antibodies specific for antigens of protozoan parasites. The methods were designed to enable the derivation of large numbers of specific monoclonal antibodies and to give high cloning efficiencies of desired hybrids. Although special attention is paid to derivation and detection of anti-parasite antibodies, the methods can be applied to many different antibody-antigen systems. Using the described methods we have isolated more than 90 myeloma hybrids which secrete antibodies specific for antigens of African trypanosomes and Theileria parasites, thus illustrating their effectiveness.

Animals↗
PubMed1980

The bovine lymphoid system: II. Derivation and partial characterlzation of monoclonal antibodies against bovine peripheral blood lymphocytes.

We have raised monoclonal antibodies to produce reagents specific for bovine lymphocyte subpopulations. Spleen cells from mice immunized with bovine peripheral blood lymphocytes were fused with X63-Ag8 myeloma cells and eleven myeloma-hybrids which secreted antibody specific for bovine lymphocytes were doubly cloned. Five of the hybrids secreted antibodies which bound to the majority of bovine lymphocytes. Two of these antibodies were specific for polymorphic antigens. One antibody bound to B lymphocytes and serum IgM molecules. The remaining five bound to subpopulations of lymphocytes. Four monoclonal antibodies bound only to bovine cells while six also bound to lymphocytes from other bovidae. None bound to human lymphocytes. We discuss the difficulty of correlating the specificities of monoclonal antibodies to functional lymphocyte subpopulations in outbred animals where few other defined markers are available.

Animals↗
PubMed1979

Cell-mediated immunity to Theileria-transformed cell lines.

In East and Central Africa the protozoan parasite Theileria parva causes a disease of cattle called East Coast fever (ECF). In Kenya alone between 60,000 and 85,000 cattle die from ECF every year. Infected animals can recover from ECF either naturally or after treatment with tetracyclines or menoctone and are subsequently able to resist challenge with the homologous strain of parasite. That this acquired resistance is due to cell-mediated rather than humoral immunity has been suspected but never decisively shown. A major difficulty in studying immunity to ECF has been the lack of inbred animals for studying Theileria-specific immunity in the absence of allogeneic histocompatibility barriers. We have avoided this problem by measuring cell-mediated immune responses in a syngeneic system in vitro. Unidirectional mixed lymphocyte cultures (MLC) were set up using bovine peripheral blood lymphocytes (PBL) as responder cells and autologous cell lines transformed in vitro by T. parva as stimulator cells. In these cultures, DNA synthesis was induced in PBL from both normal and Theileria-immune animals. However, cytotoxic lymphocytes were induced only in cultures containing responder lymphocytes from Theileria-immune cattle. The results show that Theileria-transformed cells express antigens which are recognized by effector cells and provide evidence that cell-mediated cytotoxic mechanisms function in immunity to ECF.

Animals↗