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Biomedical subjects

R J Howard

Publications and source records attributed to R J Howard.

At least 235 records · Page 13Linked to original sources
PubMed1987

Transport of an Mr approximately 300,000 Plasmodium falciparum protein (Pf EMP 2) from the intraerythrocytic asexual parasite to the cytoplasmic face of the host cell membrane.

The profound changes in the morphology, antigenicity, and functional properties of the host erythrocyte membrane induced by intraerythrocytic parasites of the human malaria Plasmodium falciparum are poorly understood at the molecular level. We have used mouse mAbs to identify a very large malarial protein (Mr approximately 300,000) that is exported from the parasite and deposited on the cytoplasmic face of the erythrocyte membrane. This protein is denoted P. falciparum erythrocyte membrane protein 2 (Pf EMP 2). The mAbs did not react with the surface of intact infected erythrocytes, nor was Pf EMP 2 accessible to exogenous proteases or lactoperoxidase-catalyzed radioiodination of intact cells. The mAbs also had no effect on in vitro cytoadherence of infected cells to the C32 amelanotic melanoma cell line. These properties distinguish Pf EMP 2 from Pf EMP 1, the cell surface malarial protein of similar size that is associated with the cytoadherent property of P. falciparum-infected erythrocytes. The mAbs did not react with Pf EMP 1. In one strain of parasite there was a significant difference in relative mobility of the 125I-surface-labeled Pf EMP 1 and the biosynthetically labeled Pf EMP 2, further distinguishing these proteins. By cryo-thin-section immunoelectron microscopy we identified organelles involved in the transit of Pf EMP through the erythrocyte cytoplasm to the internal face of the erythrocyte membrane where the protein is associated with electron-dense material under knobs. These results show that the intraerythrocytic malaria parasite has evolved a novel system for transporting malarial proteins beyond its own plasma membrane, through a vacuolar membrane and the host erythrocyte cytoplasm to the erythrocyte membrane, where they become membrane bound and presumably alter the properties of this membrane to the parasite's advantage.

Animals↗
PubMed1987

Localization and resection of gastrinomas in Zollinger-Ellison syndrome.

From 1971-1986, 24 patients were diagnosed as having Zollinger-Ellison syndrome (ZES) and 22 patients had laparotomy. Of this group, gross tumor was identified in 15 of 22 patients. Ten of 15 patients had resection of their gastrinomas with the specific aim of curing the disease. This group had responded favorably to either cimetidine or ranitidine before operation. Preoperative transhepatic portal venous sampling (PVS) with gastrin determinations was performed in six patients; three patients had this procedure twice. The tumor was correctly localized by PVS in five of six patients. In four of six patients, the tumor was easily found at surgery. In two of six patients (33%) PVS was vital to intraoperative decisions. Criteria for biochemical cure are normal periodic fasting gastrin and secretin infusion tests. Of the 10 patients who had resection for potential cure, two patients failed within 48 hours of surgery on the basis of an elevated fasting serum gastrin level in one patient and a positive secretin infusion test in the other patient. Eight patients were considered cured with follow-up from 6 months through 15 years. Of the eight cured patients, the tumors were located as follows: four were extraintestinal and extrapancreatic, four were in the duodenal wall, one patient had a tumor located in the uncinate process of the pancreas, and one tumor was located in a lymph node along the lesser curve of the stomach. Two patients had mobilization of the pancreas and duodenum for a "blind" pancreatoduodenectomy based on preoperative PVS (2 procedures each patient). In one patient a 3-mm gastrinoma was enucleated from the posterior uncinate process. The second patient had pancreatoduodenectomy with findings of two duodenal wall gastrinomas. Both patients remained cured of ZES beyond 2 years. It is concluded that PVS does indeed locate some tumors before operation, even those not easily found at surgery. ZES can be cured by an aggressive approach combining preoperative tumor localization and tumor resection. Of the eight patients biochemically and perhaps biologically cured, follow-up was greater than four years in five patients, greater than two years in two patients, and beyond six months in one patient.

Duodenal Neoplasms↗
PubMed1987

Ultrastructure of the erythrocytic stages of Plasmodium malariae.

This report describes the fine structure of the erythrocytic stages of Plasmodium malariae. Erythrocytic parasites from a naturally acquired human infection and an experimentally infected chimpanzee were morphologically indistinguishable and structurally similar to other primate malarias. New findings included observations of highly structured arrays of merozoite surface coat proteins in the cytoplasm of early schizonts and on the surface of budding merozoites and the presence of knobs in the membranes of Maurer's clefts. Morphological evidence is presented suggesting that proteins are transported between the erythrocyte surface and intracellular parasites via two routes: one associated with Maurer's clefts for transport of membrane-associated knob material and a second associated with caveolae in the host cell membrane for the import or export of host- or parasite-derived substances through the erythrocyte cytoplasm.

Adolescent↗
PubMed1987

Cytoadherence of Plasmodium falciparum-infected erythrocytes to human melanoma cell lines correlates with surface OKM5 antigen.

OKM5 antigen and thrombospondin are currently under investigation as potential receptors on the surface of human monocytes, endothelial cells, and melanomas responsible for the cytoadherence of Plasmodium falciparum-infected erythrocytes. We have studies the binding capacity of six human melanoma cell lines and related this property to the cytoplasmic and surface expression of the OKM5 antigen and thrombospondin by using indirect immunofluorescence assays on methanol-fixed and nonfixed melanomas. The presence of OKM5 antigen was detectable only in the melanoma lines which bound P. falciparum-infected erythrocytes. Thrombospondin was present in the cytoplasm of all the melanoma lines but was not detectable on the surface of any cells. Our work demonstrates a direct correlation between surface OKM5 antigen and cytoadherence in vitro. While our results do not exclude thrombospondin as a mediator of cytoadherence to endothelial cells in vivo, they showed no correlation between the presence of thrombospondin and the ability of melanoma cell lines to cytoadhere in vitro.

Animals↗
PubMed1987

Thrombospondin binding by parasitized erythrocyte isolates in falciparum malaria.

Toward understanding the pathogenesis of vascular sequestration in falciparum malaria, we investigated binding of Plasmodium falciparum parasitized erythrocyte isolates to thrombospondin and other adhesive proteins. Blood samples with rings from 12 patients with falciparum malaria were cultured 30 hr until parasites were mature trophozoites and schizonts. All parasitized erythrocyte isolates bound to thrombospondin, but not to fibronectin, laminin, vitronectin, or factor VIII/von Willebrand factor. Parasitized erythrocyte binding varied among isolates, ranging from 192 to 6,725 per mm2, average 2,953. There was good correlation between trophozoite plus schizont % parasitemia and thrombospondin binding (r = 0.884, P less than 0.001). In two patients with stupor, 3,642 and 2,864 parasitized erythrocytes bound per mm2, in proportion to parasitemia, suggesting cerebral malaria is not due to increased binding affinity. These results indicate there is a conserved function among isolates from this geographic region, known to be antigenically diverse at the parasitized erythrocyte membrane surface. These results support the hypothesis that specific binding to an endothelial receptor, possibly involving thrombospondin, plays a role in vascular sequestration in falciparum malaria.

Adult↗
PubMed1986

Research toward malaria vaccines.

Malaria exacts a toll of disease to people in the Tropics that seems incomprehensible to those only familiar with medicine and human health in the developed world. The methods of molecular biology, immunology, and cell biology are now being used to develop an antimalarial vaccine. The Plasmodium parasites that cause malaria have many stages in their life cycle. Each stage is antigenically distinct and potentially could be interrupted by different vaccines. However, achieving complete protection by vaccination may require a better understanding of the complexities of B- and T-cell priming in natural infections and the development of an appropriate adjuvant for use in humans.

Antigens, Protozoan↗
PubMed1986

Parasite-infected-cell-agglutination and indirect immunofluorescence assays for detection of human serum antibodies bound to antigens on Plasmodium falciparum-infected erythrocytes.

Two methods are described for detecting the binding of serum antibodies from adults in an endemic malarious area (The Gambia) to surface antigens on Plasmodium falciparum-infected erythrocytes. An antibody-mediated parasite-infected-cell-agglutination assay (without secondary antibody) and an indirect immunofluorescence assay employing an anti-Fc secondary reagent were used to detect bound antibody. The surface of erythrocytes containing mature parasites bound antibody, but the surface of uninfected cells or cells containing early parasite stages did not react. Serum from 'non-immune' Europeans did not agglutinate infected erythrocytes, however, in the immunofluorescence test with anti-Ig and anti-F(ab')2 secondary reagents we could detect the binding of IgG antibody from 'non-immune' European serum to a small proportion of infected cells. In contrast to the results with freshly collected isolates, antibodies from sera of Gambian adults did not bind to the surface of infected cells from five different culture-adapted isolates of P. falciparum. These assays are suitable for studies on the antigenic diversity of erythrocyte antigens in natural infections and specific antibody responses to these antigens in infected patients.

Antibodies↗
PubMed1986

Inhibitory effects of histidine analogues on growth and protein synthesis by Plasmodium falciparum in vitro.

The human malaria parasite Plasmodium falciparum synthesizes several proteins that are unusually rich in histidine. We therefore screened histidine analogues for their capacity to inhibit in vitro parasite growth. Analogues were added to cultures of ring-stage parasites, and parasite morphological development was assessed by light microscopy after a 22-hr culture. Inhibition of morphological development was identified as the appearance of condensed or pycnotic parasites rather than mature trophozoites. Inhibition of parasite protein synthesis was assessed by radioactivity counting of [3H] isoleucine incorporated into acid-insoluble products and by sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography of [3H]histidine-labeled malarial proteins. 2-F-L-Histidine and 2-I-D, L-histidine exerted the most pronounced inhibitory effects, the fluoro-analogue being the more effective of the two. At a 0.125 mM concentration, both compounds inhibited parasite growth and 2-F-L-histidine also inhibited protein synthesis. At a 1.0 mM concentration, 2-azido-L-histidine, alpha-methyl-L-histidine and WR 177589A also inhibited P. falciparum growth and protein synthesis. Twenty other histidine analogues, including 5-F-L-histidine and 5-I-L-histidine, showed little or no effect under these conditions. The inhibitory histidine analogues may be of interest for antimalarial chemotherapy if they should prove to have greater effect on P. falciparum protein synthesis than on host protein synthesis.

Animals↗
PubMed1986

Antigens induced on erythrocytes by P. falciparum: expression of diverse and conserved determinants.

Red blood cells that are infected with the malaria parasite Plasmodium falciparum express new antigens on their surface. In a study of these antigens in the erythrocytes of naturally infected children in the Gambia, an antibody-mediated agglutination assay revealed an extreme degree of antigenic diversity. Serum samples from each of ten children in the convalescent stage of malaria infection reacted with infected cells from the same child but generally not with infected cells from the other children. The Gambian children's erythrocytes also expressed shared determinants: sera from Gambian adults often reacted with the surface of infected cells from all of the children and were shown by adsorption and elution experiments to contain antibodies that recognized several isolates. Conserved determinants exposed on infected erythrocytes may be important for development of antimalarial immunity either naturally or through vaccination.

Adult↗
PubMed1986

Statistical analysis of statural growth following kidney transplantation.

Factors affecting the growth rates of 59 children and adolescents for the first 2 years following kidney transplantation were evaluated. The factors assessed were age at transplantation, renal function, prednisone dosage, donor source, and prior history of transplantation. The observed growth velocity was expressed as the percentage, of the growth velocity predicted by bone age. Normal growth (greater than or equal to 80%) was exhibited by 37% of the patients and 22% had accelerated growth (greater than or equal to 100%). The chronologic age at transplantation did not correlate significantly with growth when bone age was used as the reference for expected velocity. Males grew better than did females. There was a unique sex/race interaction with black males growing most rapidly. Better renal function, the ability to lower prednisone dosage, alternate day prednisone administration, and a decreasing diastolic blood pressure were positively correlated with better growth rates after transplantation. The donor source and prior history of transplantation did not significantly influence growth rate.

Adolescent↗
PubMed1986

Statistical analysis of statural growth following kidney transplantation.

Factors affecting the growth rates of 59 children and adolescents for the first 2 years following kidney transplantation were evaluated. The factors assessed were age at transplantation, renal function, prednisone dosage, donor source, and prior history of transplantation. The observed growth velocity was expressed as the percentage of the growth velocity predicted by bone age. Normal growth (greater than or equal to 80%) was exhibited by 37% of the patients and 22% had accelerated growth (greater than or equal to 100%). The chronological age at transplantation did not correlate significantly with growth when bone age was used as the reference for expected velocity. Males grew better than did females. There was a unique sex/race interaction with black males growing most rapidly. Better renal function, the ability to lower prednisone dosage, alternate day prednisone, and a decreasing diastolic blood pressure were positively correlated with better growth rates after transplantation. Donor source and prior history of transplantation did not significantly influence growth rate.

Adolescent↗
PubMed1986

Plasmodium falciparum and P. knowlesi: initial identification and characterization of malaria synthesized glycolipids.

This is the first report establishing the existence of glycolipids synthesized by plasmodia, in particular Plasmodium falciparum. Trophozoites, schizonts, gametocytes, and gametes were metabolically labeled in vitro with [3H]glucosamine, [3H]galactose, [3H]glucose, [3H]mannose, [3H]fucose, [32P]inorganic phosphate, or [35S]sulfate, and total lipid extracts analyzed by high-performance thin-layer chromatography and autoradiography or fluorography. Parasites incorporated [3H]monosaccharides into distinctly different series of molecules previously undescribed. Three properties of [3H]glucosamine labeled molecules indicate they are glycolipids. First, labeled molecules have lipid solubility properties. Second, mobility on thin-layer chromatography was characteristic of glycolipids. Third, following acid hydrolysis, [3H]glucosamine was recovered from a total lipid extract of labeled parasites demonstrating that glucosamine is a constituent of some of these lipid molecules. Most of these glycolipids are neutral and alkali labile. The majority of these glycolipids differs from several synthesized phospholipids. None of these glycolipids was sulfated. Plasmodial glycolipid synthesis occurs concomitantly with glycoprotein synthesis, and both increase during schizogony. Many of these glycolipids appear to be identical among three strains of P. falciparum and between two species, P. falciparum and P. knowlesi. In contrast, there are stage specific differences in glycolipid synthesis among rings, schizonts, gametocytes, and a mixture of gametes plus zygotes of P. falciparum, examples of both erythrocytic and vector forms of the parasite.

Animals↗
PubMed1986

Identification and analysis of epimastigote surface and metabolic proteins in Trypanosoma cruzi.

Metabolic and surface membrane proteins of four epimastigote-stage Trypanosoma cruzi clones were analyzed by one and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No major inter-clonal differences were observed in the metabolic protein patterns, indicating that these proteins are highly conserved. However, marked quantitative and qualitative differences were observed in surface-labeled protein patterns following both one and two-dimensional electrophoretic analyses. Inter and intra-clonal differences in antigenic properties also were demonstrated by immunoprecipitation of the surface proteins with sera from animals immunized or infected with various T. cruzi stocks. Thus, a wide spectrum of both phenotypic and antigenic diversity exists in T. cruzi which may be relevant to problems of the diagnosis and immunotherapy of Chagas' disease.

Animals↗
PubMed1986

Identification of isolate-specific proteins on sorbitol-enriched Plasmodium falciparum infected erythrocytes from Gambian patients.

We have compared the surface radio-iodinated proteins of uninfected and Plasmodium falciparum-infected erythrocytes from natural infections of human patients. Cryopreserved infected blood from Gambian children with falciparum malaria was thawed, cultured to the middle trophozoite stage, and surface radio-iodinated. Trophozoite-infected cells were enriched about 10-fold on a Percoll gradient newly designed to separate cells based on their differential permeability to sorbitol. Infected blood was radio-iodinated and erythrocytes from the fraction enriched in parasitized cells and uninfected erythrocytes from the same sample obtained from the gradient and compared by SDS-PAGE and autoradiography. In each sample, parasitized erythrocytes contained one or more polypeptides of very high molecular weight (Mr 250 000-300 000) that were not found on uninfected erythrocytes from the same patient. These proteins were isolate-specific in size and number, suggesting that natural isolates contain a variable number of different P. falciparum phenotypes for this surface protein. In addition, these radio-iodinated surface proteins could not be extracted from the host cell membrane by the non-ionic detergent Triton X-100, but were extracted by SDS. The properties of these proteins suggest they are the equivalent for natural infections of the strain-dependent antigen previously described (Leech, Barnwell, Miller & Howard, 1984) on the surface of P. falciparum-infected Aotus erythrocytes. In addition, we observed a second parasite-dependent modification of labelled proteins on infected erythrocytes with the appearance of a new band of Mr 30 000. There were also variations in the pattern of radio-isotope labelled proteins on uninfected erythrocytes from different patients.

Animals↗