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Biomedical subjects

K N Brown

Publications and source records attributed to K N Brown.

At least 55 records · Page 3Linked to original sources
PubMed1983

Intraerythrocytic development and antigenicity of Plasmodium falciparum and comparison with simian and rodent malaria parasites.

Using sorbitol-synchronised cultures and metabolic labelling with [35S]methionine, the stage specificity of polypeptides synthesised by the intraerythrocytic stages of Plasmodium falciparum was studied. We confirmed that the synthesis of many polypeptides is restricted to defined morphological stages of parasite development, while other polypeptides are synthesised more or less throughout the cycle. The synthesis of at least 6 polypeptides was confined to the period of differentiation of mature trophozoites to schizonts and merozoites. Polypeptides synthesised by a cloned long-term passage isolate were very similar to those of a recently cultured uncloned isolate. Comparison of polypeptides synthesized during differentiation of mature trophozoites to schizonts and merozoites by P. falciparum with those of P. chabaudi and P. knowlesi showed that while P. chabaudi and P. knowlesi synthesised a 250 000 molecular weight polypeptide at this stage the apparently equivalent polypeptide of P. falciparum was of significantly lower molecular weight being 200 000. Using a surface immunoprecipitation technique, it was shown that this 200 000 mol. wt. polypeptide was accessible to antibodies on the surface of erythrocytes infected with mature trophozoites and schizonts. A 150 000 mol. wt. polypeptide was also accessible to antibodies. By comparing polypeptides synthesised during the differentiation of mature trophozoites to schizonts and merozoites with those recovered in the ring stage parasites after schizogony and erythrocyte invasion, it was shown that this 200 000 mol. wt. polypeptide and 140 000 and 120 000 mol. wt. polypeptides were not taken into the erythrocyte by the invading merozoite. The importance of these polypeptides in terms of the parasite biology and in the induction and expression of immunity to malaria is discussed.

Animals↗
PubMed1982

Stage specific protein and nucleic acid synthesis during the asexual cycle of the rodent malaria Plasmodium chabaudi.

The asexual, intraerythrocytic stage of Plasmodium chabaudi develops synchronously in CBA mice. This in vivo synchrony has been exploited in in vitro pulse-labelling experiments to investigate the stage specificity of macromolecular synthesis by malaria parasites. Groups of mice were infected on day 0 with P. chabaudi and on day 3, individual mice were killed at three hour intervals, and the parasitised blood labelled in vitro for 2 h with radioactive precursors of protein or nucleic acid synthesis. By taking 11 samples covering one and one-third parasite division cycles, it was shown that the synthesis of many parasite polypeptides was restricted to defined morphological stages of parasite development. Other polypeptides were synthesised more or less continuously during the growth cycle. The synthesis of at least 6 polypeptides was confined to schizont or merozoite differentiation. RNA synthesis was shown to increase in rate steadily during parasite growth and to fall sharply during merozoite invasion. Approximately 40% of DNA synthesis was shown to occur during trophozoite growth, but the majority (60%) was confined to a short 4-6 h period at or just before schizogony.

Animals↗
PubMed1982

Identification of a schizont- and species-specific surface glycoprotein on erythrocytes infected with rodent malarias.

Erythrocytes infected with mature trophozoites of Plasmodium chabaudi and reticulocytes infected with P. berghei were labelled metabolically in vitro with [35S]methionine. The labelled cells were incubated with normal and immune serum and washed to remove unbound antibody. Solubilisation of the antibody-coated cells in detergent was followed by co-precipitation of antibody/antigen complexes and analysis of the immunoprecipitates by SDS-PAGE and fluorography. One major parasite polypeptide of 250 000 daltons was found to be exposed to antibody in both species. A labelled band of the same molecular weight could be identified by immunoprecipitation and SDS-PAGE analysis of P. chabaudi-infected cells that had been surface-labelled with periodate/NaB3H4. This molecule also incorporated [3H]glucosamine in short term cultures of mature parasitised erythrocytes. The results suggest that a 250 000 dalton glycoprotein which is synthesised only by late trophozoites or schizonts is exposed either on the surface of the infected erythrocyte, the surface of the merozoite, or both. Furthermore, the exposed portion of the molecule was not immunologically cross-reactive in the two Plasmodium species, but some cross reaction was detectable in total parasite lysates. The significance of these findings to protective immunity is discussed.

Animals↗
PubMed1982

The binding of antibodies from Plasmodium berghei-infected rats to isoantigenic and parasite-specific antigenic sites on the surfaces of infected reticulocytes.

Ferritin-labelling techniques at the ultrastructural level have shown that antiserum from August rats immune to P. berghei infection contains antibodies which bind to the surfaces of parasitized reticulocytes but not to uninfected cells. Two antibody specificities have been demonstrated by comparing antisera i absorbed with infected reticulocytes, ii absorbed with uninfected reticulocytes, and iii unabsorbed. Ferritin labeling was much increased with antiserum preabsorbed with uninfected reticulocytes, and also with heat-inactivated serum, indicating a blocking effect on parasite-specific antibody binding by cold-reacting anti-erythrocyte isoantibodies known to be present. Energy-dependent aggregation, shedding and endocytosis of labelled material was observed at the surfaces of unfixed infected reticulocytes.

Animals↗
PubMed1982

Monoclonal antibodies that protect in vivo against Plasmodium chabaudi recognize a 250,000-dalton parasite polypeptide.

Twenty monoclonal antibodies have been prepared to the erythrocytes from CBA/Ca mice infected with the rodent malaria Plasmodium chabaudi. By immunofluorescence, 15 of these antibodies recognized parasite antigens expressed only during the development of mature trophozoites to schizonts and merozoites, 2 recognized parasite antigens that were expressed throughout most of the intraerythrocytic cycle, and 3 recognized the membranes of all infected and uninfected erythrocytes. By immunoprecipitation of [35S]methionine-labeled, parasitized erythrocytes, parasite antigens recognized by all of the antiparasite antibodies were characterized. Eleven precipitated a 250,000-dalton parasite polypeptide which was synthesized and expressed late in the intraerythrocytic cell cycle and which appeared to be the major coat protein of the merozoites. In passive protection experiments, transfer of hyperimmune serum before infection with the parasite resulted in a delay in the rise of parasitemia, reduction in peak parasitemias, and a delay in the clearance of the parasitemia. Two monoclonal antibodies to the 250,000-dalton polypeptide had a similar but not as marked effect on parasitemia when given as a single dose before infection. When mixed and administered throughout the course of infection, their effects were greater. They had no influence on the course of Plasmodium berghei KSP11 parasitemia. Monoclonal antibodies to other parasite antigens and normal erythrocyte antigens failed to have a significant and reproducible effect on P. chabaudi parasitemia. The results suggest that this 250,000-dalton malaria parasite antigen may be important in the induction and expression of antibody-mediated immunity to malaria.

Animals↗
PubMed1981

Enzyme-linked immunosorbent assay compared with immunoprecipitation for serotyping Neisseria meningitidis and its use in demonstrating serotype polymorphism.

Rabbit antisera generated against 11 prototype strains of group B Neisseria meningitidis were tritiated by using the enzyme-linked immunosorbent assay and the Ouchterlony immunoprecipitation technique. The relative insensitivity of the immunoprecipitation technique in detecting antibody compared with the enzyme-linked immunosorbent assay was demonstrated. Cross-reactions within the group and among the 11 serotypes were characterized with respect to established serological techniques. Serotype polymorphism among the strains of N. meningitidis was demonstrated with the enzyme-linked immunosorbent assay.

Agglutination Tests↗
PubMed1981

Erythrocyte destruction and protective immunity to Malaria: enhancement of the immune response by phenylhydrazine treatment.

Malaria infection is characterized by extensive destruction of erythrocytes. In addition, the surface membrane of parasitized erythrocytes becomes biochemically and antigenically modified. Thus during infection the host immune system is exposed to massive amounts of modified erythrocytes on a scale not normally considered in conventional immunological experiments. The haemocytoxic drug phenylhydrazine hydrochloride has been used to mimic, in otherwise normal animals, the effect of the modification and destruction of erythrocytes which occurs in malaria. The experiments demonstrated that protective immunity to Plasmodium berghei KSP11 infection in rats and mice is significantly enhanced by this treatment, that this effect generates memory, can be transferred with spleen cells, and can have both enhancing and suppressive action on the protective immune response.

Animals↗
PubMed1980

Cold isohaemagglutinins in Plasmodium berghei-infected rats reacting with parasitized reticulocytes.

Significant levels of cold IgM and IgG isohaemagglutinins were detected in the serum of rats infected with Plasmodium berghei KSP 11. Peak titres occurred 15 days after initial infection at the time when the parasitaemia was dropping rapidly, or 7 days after a second challenge infection. Infected reticulocytes were much more sensitive to agglutination than uninfected cells, but absorption experiments demonstrated isoantigenicity in the determinants involved. This result indicated that the presence of the parasite resulted in exposure of membrane isoantigens normally masked. Agglutination could be inhibited with fractions with pIs 7.7-7.8 obtained from parasitized reticulocytes. Formaldehyde and glutaraldehyde-fixed infected cells each gave distinct agglutination reactions, different from unfixed cells.

Agglutination Tests↗
PubMed1979

Morphological stabilization of capsules of group B streptococci, types Ia, Ib, II, and III, with specific antibody.

Antibody prepared to the type-specific capsular polysaccharides of group B streptococci was used to demonstrate a stabilizing effect on the capsular glycocalyx. This permitted visualization by electron microscopy of the size of the capsule relative to the rest of the bacterial cell, and clear differences in the dimensions of untreated and antibody-treated capsular material were noted. Antibodies produced against group B streptococci types Ia, Ib, II, and III were used to demonstrate morphologically that the organization and extent of the capsular glycocalyx more closely resembles its natural state on stabilization by reaction with specific antibody.

Animals↗
PubMed1979

The possible role of isoantigens in protective immunity to malaria.

The possibility that autoimmune responses to modified red cell antigens might be involved in protective immunity to malaria was investigated in Plasmodium berghei infection of August rats. Animals rendered anaemic by phenylhydrazine treatment at the time of immunization showed significantly greater protection than rats given antigen alone, or phenylhydrazine alone. Adoptive transfer experiments indicated that this enhanced response could be transferred with spleen cells.

Animals↗
PubMed1979

Synthesis of Plasmodium knowlesi polypeptides in a cell-free system.

Preliminary results are presented concerning the characterization of Plasmodium knowlesi antigens. RNA with messenger activity has been extracted from P. knowlesi and translated in a rabbit reticulocyte cell-free system. The products of this system have been analysed both before and after precipitation with specific P. knowlesi antiserum and many polypeptides ranging in molecular weight from over 100 000 to approximately 20 000 have been found. A good correlation was found between these results and those obtained by McColin with in vitro labelling of P. knowlesi with ((3)H)-isoleucine. The major polypeptide in both systems had a molecular weight of about 50 000-55 000. These results indicate the parasite origin of the antigens.

Animals↗
PubMed1978

Penetration of antimicrobials into tissue culture cells and leucocytes.

When exposed to HeLa cells in tissue culture for 72 hr., antimicrobials could be categorised into three groups characterised by cell associated concentrations much lower (ampicillin, cephalexin, cloxacillin, flucloxacillin, streptomycin and trimethoprim, all 14% or less), much higher (tetracycline and polymyxins) or approximating to those extracellularly (erythromycin, lincomycin, fusidic acid and gentamicin). For kanamycin, neomycin and sulphonamides, cell associated levels were between 24 and 47% and for penicillin G and cephaloridine were 66% of those extracellularly. With mouse peritoneal macrophages and human peripheral blood leucocytes cell associated levels for representative antibiotics were all lower after 3 hr. exposure than in the tissue culture cells. However, studies on the rate of release of cell associated antibiotic and of the effects of surface active agents indicated that the differences between cell types were due to loss of cell association during washing procedures to remove extracellular antibiotic. The effects of bactericidal antibiotics on survival of bacteria phagocytosed by mouse macrophages suggested that the cell association observed in tissue culture cells represented true intracellular penetration rather than mere binding to the cell surface. Within families of antibiotics, alterations to the molecule change cell penetration and the variations observed can not be explained merely in terms of simple diffusion, molecular size, dissociation constants, lipid solubility or protein binding.

Animals↗
PubMed1977

Translation in a reticulocyte cell-free system of RNA isolated from blood and culture forms of Trypanosoma brucei.

RNA with messenger activity has been extracted from both blood and culture (insect mid-gut) forms of Trypanosoma brucei and translated in a reticulocyte cell-free system. The products of this cell-free system have been compared, and many common polypeptides demonstrated. A major polypeptide of 58000--65000 molecular weight was made when both blood and culture form RNA was added to the cell-free system. Antiserum raised against purified variant antigen from a cloned variant (MIAG 099) was used to detect specific products of this system. A major polypeptide of approximately 58000-65000 molecular weight was precipitated when the homologous trypanosome (MIAG 099) blood form RNA was used in the cell-free system. No such polypeptide was precipitated when RNA from a heterologous strain culture or blood form was used in the system. Competition experiments, in which excess purified variant antigen was addded after incubation but before addition of specific antiserum, confirmed that the polypeptide of 58000--65000 molecular weight is the variant antigen.

Animals↗