RESUMO
In our report "Activation of Raf as a result of recruitment to the plasma membrane" (3 June, p. 1463) (1), panels E and F of figure 1 on page 1464 were incorrect. The correct photographs appear below. In addition, the [See figure in the PDF file] second sentence of the legend to figure 1 should have read, "The Raf constructs were tagged at the COOH-terminus with a Glu-Glu epitope (MEYMPME) (24) for c-Raf, or at the NH(2)-terminus with both the Glu-Glu and the Myc (MEQKLISEEDL) (23) epitopes for RafCAAX"; the next-to-the-last sentence of the legend to figure 1 should have read, "The c-Raf constructs in (A through D) are Glu-Glu-tagged and were detected by using an anti Glu-Glu antibody, and the RafCAAX and Raf6QCAAX constructs used in E and F were detected by using the antibody to Raf COOH-terminal peptide"; and the third sentence of note 26 should have read, "After blocking with 5% milk in phosphate-buffered saline (M-PBS), cells were incubated with a mouse monoclonal antibody to Glu-Glu or a rabbit polyclonal antibody to a 20-amino acid COOH-terminal peptide of Raf-1 (Santa Cruz Biotechnology, Santa Cruz, California), washed, and incubated with donkey antibodies to mouse or rabbit IgG combined with Texas Red (Jackson) in M-PBS, washed, and mounted in FITC-Guard (Testog)."
Assuntos
Vacinas Antimaláricas , Pesquisa , Órgãos Governamentais , Humanos , Cooperação Internacional , National Institutes of Health (U.S.) , Estados Unidos , Organização Mundial da SaúdeRESUMO
The circumsporozoite (CS) protein of the human malaria parasite Plasmodium falciparum may be the most promising target for the development of a malaria vaccine. In this study, proteins composed of 16, 32, or 48 tandem copies of a tetrapeptide repeating sequence found in the CS protein were efficiently expressed in the bacterium Escherichia coli. When injected into mice, these recombinant products resulted in the production of high titers of antibodies that reacted with the authentic CS protein on live sporozoites and blocked sporozoite invasion of human hepatoma cells in vitro. These CS protein derivatives are therefore candidates for a human malaria vaccine.
Assuntos
Formação de Anticorpos , Antígenos de Superfície/imunologia , Malária/prevenção & controle , Plasmodium falciparum/imunologia , Proteínas de Protozoários , Vacinas/imunologia , Animais , Antígenos de Superfície/genética , Carcinoma Hepatocelular , Linhagem Celular , Clonagem Molecular , Reações Cruzadas , DNA Recombinante , Escherichia coli/genética , Humanos , Neoplasias Hepáticas , Camundongos , Plasmodium/imunologia , Plasmodium falciparum/genética , Plasmodium falciparum/fisiologiaRESUMO
Schizonts of the malaria parasite Plasmodium falciparum synthesize a 195 kDa surface glycoprotein (gp195) that is processed into several smaller products including one of 83 kDa, which, in the case of the Camp strain, is sequentially processed into 73 and 67 kDa products. gp195 and its processing intermediates larger than 83 kDa were not precipitated from culture supernates, but the 83 and 73 kDa products were precipitated by three monoclonal antibodies (McAbs). The 83 and 73 kDa products were affinity purified from culture supernates by adsorbing to McAb 7B2 coupled to Affigel 10 and eluting either with 0.2 N acetic acid, pH 2.8, or with 3 M potassium isothiocyanate (KSCN). The epitope recognized by McAb 7B2 was denatured by acid elution but could be regenerated by treating with 8 M urea followed by dialysis. The implications of renaturing antigens to regenerate epitopes should be considered in studies on the purification, function and immunogenicity of malaria antigens.
Assuntos
Antígenos de Protozoários/análise , Glicoproteínas/análise , Plasmodium falciparum/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antígenos de Protozoários/isolamento & purificação , Cromatografia de Afinidade , Epitopos/análise , Epitopos/isolamento & purificação , Glicoproteínas/isolamento & purificação , Humanos , ImunoensaioRESUMO
The subcellular location of the major malarial glycoprotein in erythrocytes infected with schizonts of Plasmodium falciparum has been studied by two methods. In the first, glycoproteins were labelled with [3H]glucosamine or [3H]isoleucine during in vitro culture. Trypsin treatment of intact infected erythrocytes caused no major qualitative or quantitative changes in [3H]glucosamine labelled glycoproteins or [3H]isoleucine labelled proteins separated by sodium dodecylsulfate polyacrylamide gel electrophoresis. However, in the presence of Triton X-100 the labelled glycoproteins and proteins were completely cleaved by trypsin. In the second method, two monoclonal antibodies which specifically immunoprecipitate the major 195 kDa glycoprotein failed to react on indirect immunofluorescence with intact non-fixed schizont-infected erythrocytes, but reacted strongly with saponin released schizonts indicating specificity for the surface of mature intracellular parasites. Immunoelectronmicroscopy using ferritin-conjugated secondary antibody confirmed the location of the epitope(s) recognized by these monoclonals on the surface of intracellular parasites. Ferritin particles were not associated with knob-bearing erythrocyte membranes. The results indicate that only a small proportion or none of the 195 kDa glycoprotein is on the surface of the infected erythrocyte and that the largest proportion is expressed on the surface of mature intraerythrocytic parasites.
Assuntos
Glicoproteínas/metabolismo , Malária/sangue , Proteínas de Membrana/metabolismo , Plasmodium falciparum/metabolismo , Animais , Anticorpos Monoclonais , Aotus trivirgatus , Membrana Eritrocítica/metabolismo , Eritrócitos/parasitologia , Malária/parasitologia , Plasmodium falciparum/crescimento & desenvolvimento , Frações Subcelulares/metabolismoRESUMO
Plasmodium falciparum-parasitized erythrocytes obtained from continuous in vitro cultures were fractionated over metrizamide density gradients. Late developmental stage schizonts were isolated from uninfected erythrocytes and other intracellular blood stage forms (rings and trophozoites) by centrifugation through 15% metrizamide. Schizonts comprised, on the average, 85% of the total number of cells recovered from the top fraction of the gradient with the remaining cells being predominantly uninfected erythrocytes. The mean percentage of schizonts recovered was 27% relative to the estimated number of mature forms present before fractionation. The concentrated schizonts were viable based upon their ability to complete schizogony and undergo a new cycle of reinvasion and schizogony in vitro. In addition, in these short term synchronous cultures the newly infected cells were able to incorporate radiolabeled hypoxanthine. This function was inhibited by the addition of chloroquine to these 45- to 46-hour cultures. Metrizamide-purified schizonts should be useful for further study and characterization of the unique metabolic, biochemical, and immunological properties of the malaria parasite.
Assuntos
Centrifugação com Gradiente de Concentração , Centrifugação Isopícnica , Metrizamida , Plasmodium falciparum/isolamento & purificação , Eritrócitos/parasitologia , Plasmodium falciparum/parasitologiaRESUMO
Murine resident peritoneal adherent cells bound and ingested Trypanosoma rhodesiense in the presence of specific rat or mouse antiserum. Serum which mediated this phenomenon was obtained as early as 3 days after mice were immunized with gamma-irradiated parasites, with peak levels of activity obtained on day 7. A second injection of gamma-irradiated trypanosomes resulted in a secondary elevation in activity. Fresh normal serum, as a source of complement, enhanced phagocytosis in the presence of otherwise suboptimal antiserum concentrations. P388D1 cells, which like peritoneal macrophages possess Fc and complement receptors, also bound trypanosomes in the presence of antiserum. This in vitro model reflects events that occur in vivo in hosts immunized against T. rhodesiense.
Assuntos
Anticorpos/imunologia , Macrófagos/imunologia , Fagocitose , Trypanosoma/imunologia , Animais , Líquido Ascítico/citologia , Vacina BCG , Linhagem Celular , Proteínas do Sistema Complemento/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Complemento , Receptores FcRESUMO
The isolation and characterization of a new serodeme of Trypanosoma brucei rhodesiense is described. A clone of organisms derived from a human infection produced chronic infections in mice. Additional clones of differing antigenic specificities were isolated from peaks of parasitemia which occurred in these mice. The variable antigen types (VATs) of these clones were determined by agglutination, immunofluorescence, and protection of actively immunized mice. Thirteen distinct VATs were isolated and designated Walter Reed Army Trypanozoon antigenic types. The described methodology and reagents, together with the chronicity of the infection produced in mice by this serodeme, provide a model for further study of immunopathology and antigenic variation in African trypanosomiasis. The use of these reagents in determining the incidence of VATs in an endemic area may allow an assessment of the feasibility of immunoprophylaxis.
Assuntos
Sorotipagem , Trypanosoma cruzi/imunologia , Tripanossomíase Africana/imunologia , Animais , Doença Crônica , Epitopos , Imunofluorescência , Humanos , Quênia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Coelhos , Ratos , Trypanosoma cruzi/isolamento & purificaçãoRESUMO
Parasitologic, hematologic, and immunologic parameters were monitored in intact (nonsplenectomized), adult chimpanzees infected with a "chimp-adapted" strain of Plasmodium falciparum. Following primary and secondary injections of 10(9) P. falciparum-infected erythrocytes, each chimpanzee developed a low grade parasitemia (up to 1,000/mm3) and maintained the infection without evidence of eliminating the parasites. Hematologic and serum biochemical values, as well as the majority of immunologic parameters tested, remained unaltered in infected chimpanzees. However, 2 weeks after infection T cells from infected chimpanzees demonstrated an enhanced response in vitro to stimulation with the mitogen PHA, and monocyte phagocytic activity for antibody-coated erythrocytes (Fc-mediated phagocytosis) increased significantly. During malarial infection, apes developed a strong T cell proliferative response to P. falciparum antigens and monocytes showed enhanced phagocytic activity for P. falciparum-infected erythrocytes in the absence of immune serum. These results suggest that cellular immune mechanisms, especially macrophage activation, may help control, but not eliminate, P. falciparum malaria in chimpanzees.
Assuntos
Malária/parasitologia , Animais , Linfócitos B/fisiologia , Feminino , Imunoglobulina G/análise , Contagem de Leucócitos , Ativação Linfocitária , Macrófagos/fisiologia , Malária/imunologia , Masculino , Monócitos/fisiologia , Pan troglodytes/parasitologia , Fagocitose , Plasmodium falciparum , Linfócitos T/fisiologiaRESUMO
Human sera obtained from persons infected with Plasmodium falciparum were tested by a standard indirect fluorescent antibody (IFA) technique using antigen obtained from long term in vitro cultures of two strains of P. falciparum, and antibody in high titer was reproducibly detected. Sera from uninfected persons had undetectable or very low titers of antibody. The use of cultured parasites offers a convenient, stable source of antigens from different P. falciparum strains without requiring their adaptation to primates. Differences observed in IFA titers obtained by reacting immune serum with two different P. falciparum strains suggests the need for further evaluation of strain specificity.
Assuntos
Antígenos , Imunofluorescência , Malária/diagnóstico , Plasmodium falciparum/imunologia , Animais , Anticorpos/análise , Diagnóstico Diferencial , Epitopos , HumanosRESUMO
The extent to which human antibodies involved in functional immunity react with antigenic determinants varying between different isolates or strains of the human malaria parasite Plasmodium falciparum will influence the design of vaccines against malaria. We identified nine immune sera from Cambodian refugees which blocked in vitro invasion of erythrocytes by merozoites of the Camp strain of P. falciparum and agglutinated Camp strain merozoites. However, none of these sera blocked invasion of erythrocytes by merozoites of the FCR-3 strain. We conclude that antibodies in these human sera recognized antigenic determinants present on the surface of viable merozoites of the Camp strain but not the FCR-3 strain. These parasite strains and in vitro assays can be used to analyze strain-specific functional immunity in humans.
Assuntos
Anticorpos/imunologia , Eritrócitos/parasitologia , Plasmodium falciparum/imunologia , Adolescente , Adulto , Aglutinação , Criança , Pré-Escolar , Cloroquina/farmacologia , Diálise , Humanos , Soros Imunes/imunologia , Pessoa de Meia-Idade , Plasmodium falciparum/fisiologia , Especificidade da EspécieRESUMO
In November 1977, 627 soldiers belonging primarily to the First Battalion, 82nd Airborne Division, stationed at Fort Bragg, were sent to the Canal Zone, Panama, for jungle warfare training. A medical surveillance program incorporating pre- and post-evaluations over a 6-month period with dermatologic examinations, questionnaires, and serologic tests was established. Ten cases of cutaneous leishmaniasis (1.6/100 men) were diagnosed by positive Leishmania culture. The demonstrated lack of sensitivity and specificity of the indirect fluorescent antibody test and the direct agglutination test render these serological methods useless as diagnostic screening methods in the early stages of this disease.
Assuntos
Leishmaniose/etiologia , Medicina Militar , Clima Tropical , Adolescente , Adulto , Humanos , Leishmaniose/diagnóstico , Masculino , Zona do Canal do PanamáRESUMO
The kinetoplast DNA (kDNA) from promastigotes of Leishmania responsible for Old and New World cutaneous and visceral disease was characterized to determine if species and strains causing similar or different diseases could be identified. Restriction enzymes were used to digest kDNA into fragments that were separated into characteristic banding patterns after electrophoresis in agarose or linear gradient polyacrylamide gels. Hybridization was conducted with a 32P-kDNA probe and kDNA fragments transferred from agarose gels to nitrocellulose paper. Leishmania species causing cutaneous diseases in the New and Old Worlds all had different kDNA digest patterns. Visceralizing Leishmania from the New and Old Worlds also had different kDNA restriction fragment patterns although Leishmania donovani parasites with similar fragment patterns were isolated from several humans from central Kenya. Nucleotide sequences were shared among kDNA networks from L. donovani, Leishmania d. chagasi, Leishmania d. infantum, Leishmania tropica, and Leishmania major as determined by hybridization with a 32P-kDNA probe from L. donovani. However, no hybridization was detected between the L. donovani 32P-kDNA probe and kDNA from Leishmania aethiopica or Leishmania braziliensis panamensis. Leishmania characterization results for the same isolates from the published literature were compared and kinetoplast DNA analysis was found to be one of the most sensitive procedures for species and strain identification.
Assuntos
DNA/genética , Leishmania/classificação , Leishmaniose Visceral/parasitologia , Leishmaniose/parasitologia , DNA/análise , Enzimas de Restrição do DNA , Eletroforese em Gel de Poliacrilamida , Humanos , Leishmania/genética , Leishmania/ultraestrutura , Organoides/ultraestruturaRESUMO
The in vitro growth of Plasmodium falciparum in red cells containing haemoglobin E (HbE) was studied at oxygen concentration of 5 to 20%, with and without antioxidants. Under all conditions, parasite growth decreased as the concentration of HbE increased as compared with growth in red cells containing only HbA. The decreases were proportionately greatest at the highest oxygen concentration. The antioxidant vitamin C partially reversed the decreases in growth observed in HbE-containing cells at 20% oxygen. South-east Asian refugees with HbAE or HbEE had high antimalarial IFA titres, indicative of exposure to malaria more frequently than did refugees with HbAA. The decreased growth of P. falciparum in HbE-containing red cells may reduce the severity of malaria infections, conferring a survival advantage and thus increasing the numbers of individuals with HbE in local areas of South-east Asia with high incidences of malaria.