RESUMEN
We report here cloning and expression of full length mitochondrial HSP60 gene of Brugia malayi adult worm (mtHSP60bm), purification of the gene product by affinity chromatography, its in silico 3D structure and the sequence homology of the protein with Escherichia coli GroEL/ES and human HSP60. The ATP binding pocket of human HSP60 and mtHSP60bm were analyzed and compared using in silico models. The distribution of HSP60 in different life-stages of the parasite was determined using antibodies raised against recombinant mtHSP60bm (rmtHSP60bm). mtHSP60bm was present in all life-stages of the parasite except third stage infective larvae, in which it could be induced by heat-shock, and showed high degree of homology with E. coli GroEL/ES. The ATP binding pocket of HSP60 in humans, E. coli and B. malayi were also found structurally conserved. This similarity between human and mtHSP60bm might be useful in understanding the host-parasite interactions. This is the first ever report on distribution, cloning, sequence homology and ATP binding site of mtHSP60bm.
Asunto(s)
Adenosina Trifosfato/metabolismo , Brugia Malayi/metabolismo , Chaperonina 60/química , Chaperonina 60/genética , Aedes , Animales , Sitios de Unión , Brugia Malayi/genética , Brugia Malayi/aislamiento & purificación , Chaperonina 60/aislamiento & purificación , Chaperonina 60/metabolismo , Cromatografía de Afinidad , Clonación Molecular , ADN Complementario/genética , ADN de Helmintos/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Gerbillinae , Interacciones Huésped-Parásitos , Humanos , Inmunización , Masculino , Conformación Molecular , Datos de Secuencia Molecular , Murinae , ARN de Helminto/genética , ARN de Helminto/aislamiento & purificación , Homología de SecuenciaRESUMEN
This is the first report of successful immunization of experimental monkeys against a human malaria parasite, Plasmodium falciparum. Of the five owl monkeys (Aotus trivirgatus) used in this pilot study, two served as controls and the other three were immunized with P. falciparum antigen consisting primarily of mature segmenters containing fully developed merozoites. Two injections of antigen emulsified with Freund's complete adjuvant were administered intramuscularly 3 weeks apart. Three weeks after the second vaccination, all monkeys were challenged with the homologous strain of P. falciparum. The control monkeys died with high levels of parasitemia within 2 weeks of challenge. The three immunized monkeys survived and showed strong protection against P. falciparum. These results are encouraging for the possible future development of an effective vaccine against human malaria.
Asunto(s)
Haplorrinos , Malaria/prevención & control , Plasmodium falciparum/inmunología , Vacunación , Animales , Modelos Animales de EnfermedadRESUMEN
Of six fractions of human plasma tested, only Cohn's fraction IV-4 was effective for the replacement of whole plasma from monkey (Macaca mulatta) for the in vitro culture of Plasmodium knowlesi. The effects observed on multiplication and morphology of parasites suggest a specific role of some substance or substances in fraction IV-4.
Asunto(s)
Sustitutos del Plasma , Plasmodium/crecimiento & desarrollo , Animales , HaplorrinosRESUMEN
A chloroform extract of Cohn's fraction IV-4 of human plasma successfully replaced whole fraction IV-4 for the intracellular in vitro culture of Plasmodium knowlesi. We are now able to report the successful replacement of monkey plasma by stearic acid.
Asunto(s)
Medios de Cultivo , Sustitutos del Plasma , Plasmodium , Ácidos Esteáricos , Animales , Proteínas Sanguíneas , Cloroformo , Eritrocitos/metabolismo , Glucosa/metabolismo , Haplorrinos , Humanos , Técnicas In Vitro , Ácidos Linoleicos/metabolismo , Ácidos Oléicos/metabolismo , Ácidos Palmíticos/metabolismo , Conejos , RatasRESUMEN
Owl monkeys (Aotus trivirgatus griseimembra) were effectively immunized against a human malaria parasite, Plasmodium falciparum. Two injections of antigen, primarily mature segmenters with fully developed merozoites, mixed with adjuvant (6-O-stearoyl-N-acetylmuramyl-L-alanyl-D-isoglutamine and liposomes) were administered intramuscularly at a 4-week interval. Approximately 2 weeks after the second vaccination, the monkeys were challenged with the homologous strain of P. falciparum. All immunized monkeys survived the challenge. The substitution of Freund's complete adjuvant is an encouraging step toward the development of an effective and safe vaccine for human malaria.
Asunto(s)
Acetilmuramil-Alanil-Isoglutamina/inmunología , Adyuvantes Inmunológicos , Glicopéptidos/inmunología , Malaria/prevención & control , Plasmodium falciparum/inmunología , Vacunación , Animales , Haplorrinos , LiposomasRESUMEN
A Plasmodium falciparum polypeptide (46 kDa) associated with the infected erythrocytes of all asexual stages as well as immature gametocytes was identified by the monoclonal antibody (Mab) 30B8.3. The expression of this protein was not dependent upon the knobby phenotype and was detected in parasites grown either in human or Aotus erythrocytes. The antigen was heatstable, did not label with [14C]glucosamine, and was not sensitive to periodate oxidation. Immunofluorescent staining patterns of Mab 30B8.3 on in vitro cultured parasites varied from punctate (rings and trophozoites) to patchy (trophozoites and schizonts) fluorescence. The Mab 30B8.3 antigen was not detected on the infected erythrocyte surface by conventional wet-mount IFA procedure. However, when parasites were cultured in the presence of Mab 30B8.3, the epitope was detected by the monoclonal antibodies present in the culture medium. Differential extraction of the polypeptide from infected erythrocytes and immune electron microscopy of cryosectioned parasites localized the 30B8.3 epitope primarily on membranes of Maurer's clefts within the infected erythrocyte's cytosol. This 46 kDa polypeptide is unique because it seemed to be an integral membrane protein of the Maurer's clefts/vesicles and it was not secreted into the culture medium nor deposited on the infected erythrocyte membrane. Previous studies indicate that several parasite proteins, excreted extracellularly or deposited on infected erythrocyte membrane, are found to be associated with Maurer's cleft membranes and vesicles. The 46 kDa polypeptide described in this study may play an important role in the transport of the parasite antigens.
Asunto(s)
Antígenos de Protozoos/análisis , Membrana Eritrocítica/parasitología , Péptidos/análisis , Plasmodium falciparum/análisis , Animales , Anticuerpos Monoclonales/inmunología , Antígenos de Protozoos/inmunología , Epítopos/análisis , Epítopos/inmunología , Membrana Eritrocítica/ultraestructura , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Microscopía Electrónica , Fenotipo , Plasmodium falciparum/inmunología , Plasmodium falciparum/ultraestructuraRESUMEN
Over 70% of rhesus monkey peripheral mononuclear cells were isolated on sodium metrizoate-ficoll gradients with greater than 98% purity. Rhesus blood contained 47.8% active E, 58.2% total E, and 30.2% EAC rosette forming cells. Optimal conditions for mitogen studies were determined using phytohemagglutinin, concanavalin A, pokeweed mitogen, lipopolysaccharide and streptolysin O.
Asunto(s)
Activación de Linfocitos , Linfocitos/inmunología , Mitógenos/farmacología , Formación de Roseta , Animales , Separación Celular , Centrifugación por Gradiente de Densidad , Concanavalina A/farmacología , Haplorrinos , Lipopolisacáridos/farmacología , Macaca mulatta , Fitohemaglutininas/farmacología , Mitógenos de Phytolacca americana/farmacologíaRESUMEN
The response of spleen and peripheral blood lymphocytes (PBL) to mitogen stimulation with phytohemagglutinin (PHA), Concanavalin A (ConA), and pokeweed mitogen (PWM) was determined for owl monkeys (Aotus trivirgatus) experimentally infected with the Vietnam-Oak Knoll (FVO) and the Uganda-Palo Alto (FUP) strains of Plasmodium falciparum. PBL from Panamanian Aotus monkeys with less than 25% FVO infection responded normally to mitogen stimulation; however, increased parasitemia of 25--50% resulted in a significant suppression of ConA responsiveness. Colombian Aotus monkeys infected with the PUF strain also developed a suppression to ConA stimulation but with a lower parasitemia (10--25%). When the parasitemia became greater than 50% in these animals, PHA, ConA, and PWM responses were significantly decreased in cultures of PBL. Spleen cells from all acutely infected Aotus monkeys were suppressed to PHA and ConA, but not PWM stimulation. Changes in mitogen responsiveness of experimentally infected Aotus monkeys are similar to those reported for humans with acute falciparum malaria.
Asunto(s)
Linfocitos/inmunología , Malaria/inmunología , Mitógenos/farmacología , Bazo/inmunología , Animales , Aotus trivirgatus , Concanavalina A/farmacología , Haplorrinos , Técnicas In Vitro , Lectinas/farmacología , Plasmodium falciparumRESUMEN
Cultures of Plasmodium falciparum with parasitemias of 3-5% were exposed to 4 mM colchicine for 24 hours. Synchrony was observed 48 hours after treatment, and the cultures remained synchronous for more than two replicative cycles. The percentage of ring stage parasites reached peaks of over 90% at 72 and 120 hours. The percentage of schizonts reached a peak of 70% at 144 hours. Parasitemias in both colchicine-treated and untreated control cultures reached peaks of over 20% 120 hours after colchicine treatment.
Asunto(s)
Colchicina/farmacología , Eritrocitos/parasitología , Parasitología/métodos , Plasmodium falciparum/crecimiento & desarrollo , Animales , Medios de Cultivo , Humanos , Factores de TiempoRESUMEN
Two Aotus trivirgatus griseimenbra monkeys which had been immunized with the merozoite-enriched FUP strain of Plasmodium falciparum were protected against a primary challenge with the homologous strain. The results described here show that these two monkeys were protected against a subsequent challenge with a heterologous strain (FVO) of P. falciparum. The unimmunized control monkey died of FVO infection by day 18.
Asunto(s)
Inmunización , Malaria/inmunología , Plasmodium falciparum/inmunología , Animales , Aotus trivirgatus , HaplorrinosRESUMEN
Plasma from units of human blood collected in CPDA-1 preservative were compared with human serum in cultures of Plasmodium falciparum. No significant differences in parasite growth and multiplication were seen between cultures containing serum or plasma. The wider availability of plasma makes it an attractive alternative to serum, especially in large scale cultures.
Asunto(s)
Plasma , Plasmodium falciparum/crecimiento & desarrollo , Animales , Sangre , Medios de Cultivo , Humanos , Factores de TiempoRESUMEN
Compound 48/80 inhibited the growth of protozoa, bacteria, and fungi but had no effect on the multiplication of viruses. All susceptible organisms were inhibited by 10 microgram/ml of crude compound 48/80, and some were inhibited by as little as 0.1 microgram/ml. Against Tetrahymena pyriformis, this drug was seven times more potent than quinine. Separation of compound 48/80 into different fractions indicated that some antimicrobial activity could be separated from the histamine-liberating activity. It was found that compound 48/80 is not surface active at 500 microgram/ml.
Asunto(s)
Bacterias/efectos de los fármacos , Eucariontes/efectos de los fármacos , Hongos/efectos de los fármacos , p-Metoxi-N-metilfenetilamina/farmacología , Animales , Presión Sanguínea/efectos de los fármacos , Cromatografía en Gel , Liberación de Histamina/efectos de los fármacos , Técnicas In Vitro , Masculino , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Ratas , Tensión Superficial , Virus/efectos de los fármacosRESUMEN
The relationship among geographic origin, phenotype, karyotype, and susceptibility of owl monkeys to 2 strains of Plasmodium falciparum was investigated. Owl monkeys from Columbia and Panama were both susceptible to fatal infections with the Asian FVO (Vietnam-Oak Knoll) strain of P. falciparum. However, when inoculated with the African FUP (Uganda-Palo Alto) strain, most Colombian owl monkeys developed fatal or potentially fatal (bled out with parasitemias of over 25%) infections, but Panamanian monkeys generally survived. Colombian and Panamanian monkeys that spontaneously recovered from malaria infection were phenotypically indistinguishable from those which died. Karyotype analysis revealed that animals considered in this study were either Karyotype II (54 chromosomes) or II (53 chromosomes). Karyotype differences between individual monkeys did not correlate with increased susceptibility or resistance to malaria. Thus, the country of origin of owl monkeys appears to play a more important role in host susceptibility to malaria infection than karyotype.
Asunto(s)
Aotus trivirgatus/genética , Cromosomas , Haplorrinos/genética , Malaria/veterinaria , Enfermedades de los Monos/genética , Animales , Colombia , Cariotipificación , Malaria/genética , Panamá , Perú , Fenotipo , Plasmodium falciparumRESUMEN
An extract of red blood cells was prepared from monkey blood. Red cell extract (RCE) of Aotus monkeys was beneficial for the in-vitro cultivation of 2 strains of P. falciparum. The active material(s) in Aotus RCE was not sedimented by centrifugation at 34,800 g for 1 hr. Rhesus monkey RCE improved the growth of P. knowlesi for in-vitro cultivation through 4 schizogonous cycles (88 hr).
Asunto(s)
Eritrocitos , Plasmodium/crecimiento & desarrollo , Extractos de Tejidos/farmacología , Animales , Aotus trivirgatus , Medios de Cultivo , Eritrocitos/parasitología , Plasmodium falciparum/crecimiento & desarrolloRESUMEN
Postembedding immunoelectron microscopy, using pooled serum samples from a recent vaccination experiment involving Aotus monkeys, was used to localize immune targets in Plasmodium falciparum-infected erythrocytes and free merozoites. Serum samples from Aotus monkeys, protected completely by immunization with the P. falciparum merozoite surface coat precursor protein, identified immune targets on the surface of free and intracellular merozoites as well as the cytoplasm, plasma membrane, and parasitophorous vacuole membrane of immature schizonts. Serum samples from unprotected monkeys, which had been immunized with a complex of 143-kDa, 132-kDa, and 102-kDa polypeptides reacted specifically with the rhoptries of immature schizonts and mature merozoites.
Asunto(s)
Antígenos de Protozoos/análisis , Sueros Inmunes/inmunología , Plasmodium falciparum/inmunología , Animales , Antígenos de Superficie/análisis , Cebidae , Microscopía Electrónica , Plasmodium falciparum/ultraestructura , VacunaciónRESUMEN
The unavailability of in vitro or in vivo experimental systems has been the major factor hampering the progress of research studies on Theileria sergenti, causative agent of theileriosis, a major disease of cattle in Japan. We report the first successful propagation of T. sergenti in SCID mice into which uninfected bovine erythrocytes (Bo-RBC) were supplied periodically. The infectivity of T. sergenti proliferated in an SCID mouse was ascertained by successful transfer of infection into another SCID mouse into which uninfected Bo-RBC were supplied periodically.
Asunto(s)
Modelos Animales de Enfermedad , Eritrocitos/parasitología , Ratones SCID , Theileria/crecimiento & desarrollo , Theileriosis/sangre , Animales , Bovinos , Masculino , Ratones , Especificidad de la EspecieRESUMEN
Lymphocytes from healthy adult cats were separated into T and B cell-enriched subfractions by centrifuging rosetted cells on sodium metrizoate/Ficoll gradients. The responsiveness of unseparated lymphocytes (T + B), and T and B cell-enriched subfractions to stimulation with mitogens (phytohemagglutinin-P (PHA-P), concanavalin A (ConA), and pokeweed mitogen (PWM) was tested. Cultures of unseparated lymphocytes and those enriched in T cells showed similar responsiveness of PHA, ConA, and PWM stimulation; however, only a weak response to ConA and PWM was observed in B cell-enriched cultures. The mitogenic effects of PHA-P, ConA, and PWM on feline lymphocytes appeared to be due primarily to T-cell activation.