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Biomedical subjects

R J Howard

Publications and source records attributed to R J Howard.

At least 253 records · Page 14Linked to original sources
PubMed1986

Sialoglycoproteins and sialic acids of Plasmodium knowlesi schizont-infected erythrocytes and normal rhesus monkey erythrocytes.

The effects of malaria infection on RBC sialic acids and sialoglycoproteins were studied with asexual blood-stage infections of Plasmodium knowlesi in rhesus monkeys. Glycoprotein radio-isotope labelling methods were used to compare the sialoglycoproteins of normal RBC and P. knowlesi schizont-infected RBC (SI-RBC). Tritiation of glycoproteins from SI-RBC with the standard sialidase + galactose oxidase/NaB3H4 method or standard periodate/NaB3H4 method was significantly decreased when compared to normal RBC. However, tritium uptake into glycoproteins was normal when SI-RBC were treated with 5-fold higher concentrations of both enzymes in the first labelling method, or with a 5-fold increase in the molar ratio of periodate to sialic acid in the second method. The mobility of tritiated host cell glycoproteins on SDS-polyacrylamide gels was identical for SI-RBC and normal RBC. New bands, possibly glycoproteins, of 230, 160, 90, 52, and 30 kDa were detected after labelling SI-RBC by the modified periodate/NaB3H4 method. Sialic acid analysis of normal rhesus monkey RBC (62 micrograms/10(10) RBC) revealed that 46% of the total sialic acid was N-glycolylneuraminic acid, 33% was N-acetyl-9-O-acetylneuraminic acid, and the remainder N-acetylneuraminic acid. SI-RBC collected either directly from infected monkeys or after in vitro culture of ring-infected RBC in horse serum, had increased total sialic acid (126 or 115 micrograms/10(10) RBC, respectively). The sialic acid content of infected RBC must increase during parasite development since RBC infected with ring-stage P. knowlesi had the same content as normal RBC. There was no significant difference in the ratio of the three sialic acids of SI-RBC and normal RBC. In contrast, the uninfected RBC from infected blood of different monkeys showed marked variation in sialic acid composition and generally had a lower sialic acid content than normal RBC.

Animals↗
PubMed1986

Sialic acid analysis and tritium-labelling of sialoglycoproteins of mouse erythrocytes infected with Plasmodium berghei.

Schizont-infected red blood cells (SI-RBC) from Plasmodium berghei-infected mice contain between 2 and 10 times as much sialic acid as uninfected RBC from the same blood (99-550 micrograms/10(10) RBC versus 33-65 micrograms/10(10) RBC). Total RBC samples from infected animals containing up to 63% ring- and trophozoite-infected cells had identical sialic acid contents to purified RBC samples (of less than 3% parasitaemia) from the same blood (52-64 micrograms/10(10) RBC). We conclude that RBC containing immature parasites have the same sialic acid content as uninfected RBC from infected blood and that total cellular sialic acid increases during maturation to the schizont stage. Uninfected RBC from infected blood had 25-50% as much sialic acid as normal mouse RBC (33-65 micrograms/10(10) RBC versus 126 micrograms/10(10) RBC). There were no qualitative changes in RBC sialic acids, all RBC samples having 60-70% N-acetylneuraminic acid, 30-40% N-acetyl-9-O-acetylneuraminic acid and 5-10% N-glycolylneuraminic acid. The quantitative changes we observed during infection must reflect changes in murine sialoglycoconjugates, as we have shown elsewhere that Plasmodia do not synthesize or contain sialic acids. Since the sialic acid composition of mouse serum glycoconjugates is quite different to that of the RBC fractions studied here, the quantitative data suggest that part of the sialic acids of the uninfected RBC has been transferred to SI-RBC. With higher molar ratios of periodate to substrate than generally used, we were able to radio-isotopically label normal murine sialoglycoproteins on SI-RBC and purified uninfected RBC from infected blood by the periodate/NaB3H4 method. Several new proteins were then tritiated with SI-RBC but these proteins may be intracellular and could even lack sialic acid.

Animals↗
PubMed1986

Homologous genes encode two distinct histidine-rich proteins in a cloned isolate of Plasmodium falciparum.

Two genes encoding distinct histidine-rich proteins in a Plasmodium falciparum clone exhibit high levels of homology, suggesting they have originated by duplication and divergence from a common ancestral sequence. Both genes have a similar interrupted structure and an exon that encodes closely related tandem repeats of very high histidine and alanine content. The most common repeat encoded by one gene, the hexapeptide Ala-His-His-Ala-Ala-Asp, differs in the sixth position from the most common repeat encoded by the other gene, the hexapeptide Ala-His-His-Ala-Ala-Asn. The divergence of the repeat domains is greater than that of the flanking regions, which exhibit 85-90% homology, including untranslated sequences. This suggests the tandem repeats are relatively labile elements within the genome that may provide the parasite with a means of rapid evolutionary change.

Amino Acid Sequence↗
PubMed1986

Secretion of a malarial histidine-rich protein (Pf HRP II) from Plasmodium falciparum-infected erythrocytes.

Plasmodium falciparum-infected erythrocytes (IRBCs) synthesize several histidine-rich proteins (HRPs) that accumulate high levels of [3H]histidine but very low levels of amino acids such as [3H]isoleucine or [35S]methionine. We prepared a monoclonal antibody which reacts specifically with one of these HRPs (Pf HRP II) and studied the location and synthesis of this protein during the parasite's intracellular growth. With the knob-positive Malayan Camp strain of P. falciparum, the monoclonal antibody identified a multiplet of protein bands with major species at Mr 72,000 and 69,000. Pf HRP II synthesis began with immature parasites (rings) and continued through the trophozoite stage. The Mr 72,000 band of Pf HRP II, but not the faster moving bands of the multiplet, was recovered as a water-soluble protein from the culture supernatant of intact IRBCs. Approximately 50% of the total [3H]histidine radioactivity incorporated into the Mr 72,000 band was extracellular between 2 and 24 h of culture. Immunofluorescence and cryothin-section immunoelectron microscopy localized Pf HRP II to several cell compartments including the parasite cytoplasm, as concentrated "packets" in the host erythrocyte cytoplasm and at the IRBC membrane. Our results provide evidence for an intracellular route of transport for a secreted malarial protein from the parasite through several membranes and the host cell cytoplasm.

Animals↗
PubMed1986

Antigenic diversity and size diversity of Plasmodium falciparum antigens in isolates from Gambian patients. I. S-antigens.

Ring-stage asexual parasites of P. falciparum were collected from six Gambian children and the S-antigens radiolabelled by 3H-glycine uptake during in vitro culture up to rupture of infected cells and merozoite release. Ouchterlony double diffusion of boiled culture supernatants against a panel of adult Gambian sera identified one S-antigen precipitin arc for five isolates and two precipitin arcs for one isolate. Five of the six isolates were serologically distinct. Analysis of S-antigens by comparison of SDS-polyacrylamide gel electrophoresis patterns of heat-treated soluble proteins revealed a more complex pattern of 3H-labelled S-antigens that was different for each isolate. There were between two and six different 3H-labelled bands for each isolate in the size range of molecular weight 137 000 to 285 000. This result confirms the large size range of S-antigens identified with culture adapted P. falciparum. Several bands were relatively weakly labelled with 3H-glycine, suggesting that natural isolates contain one or two predominant S-antigen phenotypes and several other S-antigen phenotypes expressed by minor parasite subpopulations. Immunoprecipitation was performed using a panel of sera from Gambian adults, or, acute and 3 week convalescent sera from the same patients used for S-antigen radiolabelling. Adult sera generally immunoprecipitated some of the S-antigens in each isolate, including antigens that must represent extremely minor parasite subpopulations since they could not be seen in the patterns of non-immunoprecipitated heat-stable proteins. Sera from convalescent children were generally negative on immunoprecipitation, even with the homologous isolate. In one case we observed the acquisition of specific immunoprecipitating antibody to one of the homologous S-antigens during the convalescent period. The antigenic and structural complexity of S-antigens in natural isolates that have not been submitted to the selection pressure of adaptation for in vitro culture is clearly greater than for culture adapted P. falciparum.

Adult↗
PubMed1986

Antigenic diversity and size diversity of P. falciparum antigens in isolates from Gambian patients. II. the schizont surface glycoprotein of molecular weight approximately 200 000.

A panel of monoclonal antibodies has been shown previously to identify both serologically diverse and serologically conserved epitopes on a major polymorphic surface protein of P. falciparum schizonts from culture-adapted isolates. The molecular nature of the antigen recognized by eight of these monoclonal antibodies was studied with three isolates analyzed directly from patients in The Gambia. Malarial (glyco) proteins were labelled by biosynthetic uptake of 3H-glucosamine or 3H-leucine during culture of ring-stage parasites from infected blood to the late-trophozoite/early-schizont stage (26-30 h). Those monoclonal antibodies which reacted positively with an isolate by indirect immunofluorescence also immunoprecipitated a single 3H-leucine or 3H-glucosamine labelled antigen of mol. wt approximately 200 000 from Triton X-100 extracts of the same isolate. Monoclonal antibodies which did not react by indirect immunofluorescence failed to immunoprecipitate this antigen. Although each of the three isolates studied in detail was very similar serologically with the panel of monoclonal antibodies specific for this mol. wt approximately 200 000 antigen, this protein could be distinguished with each isolate on the basis of its apparent size on SDS-polyacrylamide gel electrophoresis. The specifically immunoprecipitated antigen had a mol. wt of 204 000, 197 000 or 202 000, depending on the isolate. Size diversity of this malarial glycoprotein was also detected with seven other Gambian P. falciparum isolates. We conclude that natural isolates of P. falciparum express a major 3H-glucosamine labelled glycoprotein of mol. wt Mr approximately 200 000 which exhibits size diversity and expresses antigenically conserved as well as diverse epitopes as defined by the panel of monoclonal antibodies.

Antibodies, Monoclonal↗
PubMed1986

Membrane-associated electron-dense material of the asexual stages of Plasmodium falciparum: evidence for movement from the intracellular parasite to the erythrocyte membrane.

Electron-dense material (EDM) appears at the parasite plasma membrane with trophozoites of several strains of Plasmodium falciparum cultured in vitro. The EDM is also seen associated with unit membrane-bounded Maurer's clefts in K+ P. falciparum-infected erythrocytes. The cytoplasmic clefts lack the EDM with K- parasites. Some EDM have the same density and appearance as the material located under knobs at the erythrocyte membrane. The EDM at the parasite plasma membrane is absent with schizonts when expression of new knobs at the erythrocyte membrane appears to have ceased. This electron microscopic study suggests that the parasite-derived EDM is transported from the parasite plasmalemma to the erythrocyte membrane via Maurer's clefts in the erythrocyte cytoplasm.

Animals↗
PubMed1986

Inhibition of Plasmodium falciparum growth by IgG antibody produced by human lymphocytes transformed with Epstein-Barr virus.

Supernatants from Epstein-Barr virus (EBV)--stimulated B lymphocytes obtained from two adult Gambians who were partially immune to malaria markedly inhibited the growth of Plasmodium falciparum in vitro (55-95% inhibition). When 22 separate colonies were derived by micromanipulation from one of these primary cultures and their supernatants assayed, the degree of inhibition correlated with levels of IgG fluorescent antibody and total IgG. The inhibitory anti-P. falciparum IgG immunoprecipitated an antigen of mol. wt 195,000, identified as the major schizont surface glycoprotein by dual biosynthetic labelling with 3H-glucosamine or 35S-methionine. Other studies on the analogous schizont surface protein of rodent malarias have shown that this antigen stimulates protective immunity. Production of this inhibitory antibody by adult Gambians may therefore contribute to their immunity to malaria. Human antibodies produced by EBV-stimulated B lymphocytes may be used to identify other important P. falciparum antigens and have potential applications for immunotherapy.

Adult↗
PubMed1986

Parasitized erythrocyte antigens and thrombospondin adhesion in the immunology and pathogenesis of falciparum malaria.

Falciparum-parasitized erythrocytes develop new antigens on the erythrocyte surface. Both antimalarial antibody and thrombospondin bind to surface antigens or ligands on the surface of the infected red cell. Diverse antigens may be involved in parasite evasion of host immunity, but conserved molecules may be required for pathogenesis. There is a high degree of antigenic diversity at the infected erythrocyte surface among isolates. This diversity may underlie the potential of falciparum malaria to repeatedly reinfect the same person. There is also evidence for an antigenically conserved, possibly functionally conserved, determinant on the infected red cell surface. Antibody to a conserved determinant on the infected red cell surface may be associated with immunity to falciparum malaria. A conserved membrane surface determinant may be involved in parasitized erythrocyte sequestration. Thrombospondin binding in vitro is specific to mature trophozoites and schizonts, the same stages which sequester in vivo. Specific adhesion to thrombospondin is common among laboratory strains of falciparum-parasitized erythrocytes bearing knobs and among all isolates of falciparum tested. The numbers of parasitized erythrocytes bound to thrombospondin in vitro increases with parasitemia. Specific binding of parasitized erythrocytes to an endothelial receptor and to thrombospondin, which itself interacts with clotting factors, may play a role in sequestration and vascular obstruction.

Antigens, Protozoan↗
PubMed1985

Surgical infection stratification system for intra-abdominal infection. Multicenter trial.

One hundred eighty-seven patients treated for established intra-abdominal infection in five medical centers were studied using the surgical infection stratification system. This system combines an anatomic category with a numerical estimate (acute physiology score [APS]) of deviation from normal of 33 routine laboratory tests or physical findings. Overall mortality was 24% and the rate of treatment "success" with a single operation and single course of antibiotics was 48%. Eighty percent of deaths occurred with infection present. Multivariate analysis disclosed that APS, malnutrition, and age were most noteworthy for predicting survival or death. Intra-abdominal infection carries substantial mortality and morbidity despite advances in understanding the underlying pathophysiologic condition. Additional clinical studies are needed. Uniform reporting criteria, including the use of an objective severity scoring system, are suggested for future reports concerning intra-abdominal infection.

Abdomen↗
PubMed1985

Evaluation of percutaneous transhepatic balloon dilatation of benign biliary strictures in high-risk patients.

During the period from 1979 through 1984, 17 patients with benign biliary strictures underwent percutaneous transhepatic balloon dilatation. All patients presented with either hyperbilirubinemia and acute cholangitis, a history of intermittent chills and fever, or both. Balloon dilatation was most successful in those patients with intrahepatic strictures, sclerosing cholangitis, strictured biliary enteric anastomoses, and distal common bile duct strictures with retained calculi. Two patients with postsurgical common bile duct injury had treatment failure 3 and 18 months after dilatation and subsequently underwent elective hepaticojejunostomy. Although our longest treatment success is now more than 4 1/2 years in an 83 year old woman who is 15 years posthepaticojejunostomy, the mean follow-up of the entire group has only been 2 years. On the basis of our early experience and that of others, we now recommend an initial attempt at balloon dilatation in most patients with postsurgical benign biliary strictures. Further technical advances and longer follow-up in present and future series may certainly broaden the appeal of this nonoperative procedure.

Adult↗
PubMed1985

Immunochemical analysis of surface membrane antigens on erythrocytes infected with non-cloned SICA[+] or cloned SICA[-] Plasmodium knowlesi.

The SICA[-] or non-agglutinable phenotype of Plasmodium knowlesi schizont-infected erythrocytes has been defined serologically but not biochemically. Similarly, non-cloned SICA[+] or agglutinable parasites have been shown serologically to express SICA or variant antigen(s) but the number and nature of such antigens have not been defined. Here we describe the immunochemical analysis of surface antigen expression on [125I]lactoperoxidase-labelled erythrocytes infected either with a SICA[-] clone or with non-cloned SICA[+] parasites using the methods developed for identification of variant antigens with cloned SICA[+] parasites. No 125I-labelled antigens in the size range Mr 190 000-225 000 were specifically immunoprecipitated from erythrocytes infected with the SICA[-] clone, even using homologous antisera produced by multiple infections or immunizations. Further, no 125I-labelled proteins of this size were seen in detergent extracts of the SICA[-] parasites that were not also seen with uninfected cells. We conclude that the SICA[-]phenotype reflects the absence of a variant antigen at the erythrocyte surface, as predicted by the serological assays. In contrast, with the non-cloned SICA[+] parasites, a complex group of proteins, Mr 195 000-225 000, was identified by [125I]lactoperoxidase labelling of intact infected erythrocytes. These proteins are SICA antigens since they not only share the characteristic detergent solubility properties and size range of SICA antigens identified previously with SICA[+] clones, but they were only immunoprecipitated by antisera which reacted specifically with the surface of infected erythrocytes. Agglutinating sera immunoprecipitated several of these 125I-labelled antigens. Sera specific for clones derived from this non-cloned SICA[+] population failed to agglutinate, but did react by indirect immunofluorescence with 10-16% of infected cells. These sera specifically immunoprecipitated single, quantitatively minor 125I-labelled antigens in this size range. The results suggest that a population of non-cloned SICA[+] parasites contains at least 10 different variant-antigen phenotypes. Indirect immunofluorescence was also performed against a non-cloned SICA[+] population derived by antigenic variation of a SICA[+] clone in vivo. The variant population contained at least 3 antigenically distinct SICA phenotypes, indicating that antigenic variation of clones may produce populations as antigenically heterogenous as antigenic variation of uncloned lines. It is therefore likely that natural malaria isolates contain a large number of different variant antigens.

Agglutination↗
PubMed1985

Structural alteration of the membrane of erythrocytes infected with Plasmodium falciparum.

Human erythrocytes infected with five strains of Plasmodium falciparum and Aotus erythrocytes infected with three strains of P. falciparum were studied by thin-section and freeze-fracture electron microscopy. All strains of P. falciparum we studied induced electron-dense conical knobs, measuring 30-40 nm in height and 90-100 nm in diameter on erythrocyte membranes. Freeze-fracture demonstrated that the knobs were distributed over the membrane of both human and Aotus erythrocytes. A distinct difference was seen between the intramembrane particle (IMP) distribution over the knobs of human and Aotus erythrocyte membranes. There was no change in IMP distribution in infected human erythrocyte membranes, but infected Aotus erythrocytes showed an aggregation of IMP over the P face of the knobs with a clear zone at the base. This difference in IMP distribution was related only to the host species and not to parasite strains. Biochemical analysis demonstrated that a higher proportion of band 3 was bound to the cytoskeleton of uninfected Aotus erythrocytes than uninfected human erythrocytes after Triton X-100 extraction. This may account for the different effects of P. falciparum infection on IMP distribution in the two different cell types.

Animals↗
PubMed1985

Complications of Hickman-Broviac catheters.

Although Hickman-Broviac catheters have improved the care of patients with leukemia and patients using catheters for hyperalimentation, they are associated with a substantial morbidity. We experienced a 34 per cent complication rate. Some of these complications may be relieved with new therapy (streptokinase) and some of the intraoperative problems can be prevented by a more careful technique. We believe the cephalic vein cutdown technique remains the procedure of choice. Due to the morbidity of placement, every attempt should be made to save a catheter, as in type 1 infections. Type 2 infections should be the major indication for removal of the catheters.

Adolescent↗
PubMed1985

Necrotizing fasciitis.

Necrotizing infections of soft tissues are rapidly progressive infections accompanied by a high mortality. Clinical presentation involves fever, cellulitis, edema, crepitus, bullae, necrosis and sepsis. Operative findings include fascial and subcutaneous tissue necrosis with or without myonecrosis. The treatment is prompt surgical debridement. Delay in treatment results in decreased survival time. The mortality in our study was 30 per cent (20 of 33). The time from recognition of an infection by the patient or physician until operative debridement averaged three and one-half days for survivors compared with 11.7 days for nonsurvivors. These infections frequently occur in compromised hosts and the mortality is greatly increased in these patients. Patients with diabetes had a mortality of 63 per cent (five of eight). The mortality for infections of the abdomen (44 per cent) and perineum (38 per cent) is greater than for the extremities (18 per cent). The bacteriologic findings of these infections involved three combinations of organisms. We had 30 mixed infections involving two or more organisms. In addition, three patients had identical signs and symptoms caused by infection with a single organism--Vibrio species. These patients all had some type of contact with a marine environment as a predisposing cause. We also applied both the APACHE and SIS systems to these patients to evaluate the severity of the illness. Nonsurvivors presented with a mean SIS score of 8.64 compared with survivors with 3.82. Initial scores with the APACHE system for nonsurvivors was 12.0 compared with 2.14 for survivors. In both systems, scores rapidly increased at three and seven days in nonsurvivors as compared with a rapid decline in the survivors. We suggest that the best descriptive system is to simply identify the organisms and tissues involved.

Adult↗
PubMed1985

Necrotizing soft-tissue infections caused by marine vibrios.

Halophilic, noncholera marine Vibrio bacteria can cause septicemia, gastroenteritis, cellulitis, and necrotizing fasciitis. We describe six patients with necrotizing fasciitis and review 12 cases described previously. The 18 patients included 14 men and four women. Their ages ranged from 32 to 79 years (average 58.1 years). Eleven patients were older than 55 years. Nine infections were caused by V. vulnificus, three by V. parahaemolyticus, and one by V. alginolyticus. In five cases the Vibrio species was not identified. Twelve patients had associated conditions that might have made them more susceptible to these infections, such as cirrhosis, steroid therapy, hemochromatosis, and multiple myeloma. These infections usually occur in apparently insignificant wounds (puncture wounds, insect bites) exposed to sea water or fish. Treatment is by debridement and antibiotic therapy. Three patients required amputation to control the infection. Six (33.3%) of the 18 patients died.

Adult↗