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Biomedical subjects

R J Howard

Publications and source records attributed to R J Howard.

At least 271 records · Page 15Linked to original sources
PubMed1985

Two-dimensional echocardiography for diagnosing acute ascending aortic dissection.

The diagnosis of type A aortic dissection (involving the ascending aorta) can be made with two-dimensional echocardiography, but surgeons usually demand angiographic confirmation. Between January 1983 and February 1984, 10 patients presented at St. Michael's Hospital in Toronto with type A aortic dissection. Six patients underwent two-dimensional echocardiography early in their evaluation. A positive diagnosis was made in four, based upon visualization of an intimal flap or a false lumen. In the other two patients, the findings of an enlarged ascending aorta and pericardial effusion were suggestive but not diagnostic. Three patients survived operation and are alive and well (follow-up ranging from 3 to 5 months). One patient died while awaiting surgery, one died during aortography and one died during operation. All echocardiographic and angiographic findings were confirmed at surgery or at autopsy. It is concluded that two-dimensional echocardiography can diagnose ascending aortic dissection quickly, accurately, easily and without risk to the patient. In selected cases the presence of a false lumen or of an intimal flap makes it possible to proceed with operation without delay or the potential hazards of aortography.

Acute Disease↗
PubMed1984

Antigenic variation of bloodstage malaria parasites.

An antigen on the surface of erythrocytes infected with mature asexual malaria parasites has been shown to undergo antigenic variation in two malaria species. Plasmodium falciparum-infected erythrocytes from squirrel monkeys express a new antigen that is identified by reactivity with antibody from infected animals in an indirect immunofluorescence assay. Cloned P. knowlesi parasites in rhesus monkeys undergo antigenic variation of an erythrocyte surface antigen as defined by antibody-mediated cell agglutination (the SICA test) and indirect immunofluorescence. This variant antigen is a malarial protein that changes both in size (Mr 185 000-225 000) and antigenicity in cloned parasites derived by antigenic variation in vivo. Antigenic variation on the erythrocyte surface probably contributes to the capacity of malaria parasites to establish chronic infections with multiple recrudescences and to the finding that individuals can be repeatedly reinfected. The fundamental reasons for expression of these highly immunogenic antigens on the erythrocyte membrane remain obscure. Other major questions remain to be explored: the repertoire of variant antigens; the genetic basis of antigenic variation and the structural basis for the antigenic uniqueness of each variant antigen. Some properties of malarial antigenic variation indicate that control of antigenic variation in plasmodia will be quite different to antigenic variation in the African trypanosomes. The host spleen is required both for variant antigen expression and antigenic variation, and variant-specific antibody appears to induce antigenic variation rather than select pre-existing variants.

Animals↗
PubMed1984

Knob-positive and knob-negative Plasmodium falciparum differ in expression of a strain-specific malarial antigen on the surface of infected erythrocytes.

We have investigated the expression of a strain-specific malarial antigen on the surface of erythrocytes infected with knobless (K-) variants of knob-positive (K+) strains of Plasmodium falciparum. Aotus blood infected with K+ or K- parasites derived from two independent geographical isolates (Malayan camp and Santa Lucia) was surface iodinated by the lactoperoxidase method. Infected and uninfected erythrocytes were then separated by a new procedure involving equilibrium density sedimentation on a Percoll gradient containing sorbitol. Strain-specific antigens were readily identified on the surface of erythrocytes infected with either of the K+ strains by their characteristic size and detergent solubility. These proteins were not detected on the surface of erythrocytes infected with either of the K- variants nor on uninfected erythrocytes isolated from K+- or K- -infected blood. These results are consistent with a role for the strain-specific surface antigen in cytoadherence of P. falciparum-infected erythrocytes. Our findings represent the second biochemical difference (with the knob-associated histidine-rich protein) between K+ and K- P. falciparum.

Animals↗
PubMed1984

Identification of a strain-specific malarial antigen exposed on the surface of Plasmodium falciparum-infected erythrocytes.

We have identified strain-specific antigens with Camp and St. Lucia strains of P. falciparum of Mr approximately 285,000 and approximately 260,000, respectively. These strain-specific antigens were metabolically labeled with radioactive amino acids, indicating that they were of parasite origin rather than altered host components. These proteins had the properties of a molecule exposed on the surface of infected erythrocytes (IE). First, the proteins are accessible to lactoperoxidase-catalyzed radioiodination of IE. Second, the radioiodinated proteins were cleaved by low concentrations of trypsin (0.1 microgram/ml). Third, these antigens were immunoprecipitated after addition of immune sera to intact IE. Fourth, the strain-specific immuno-precipitation of these proteins correlated with the capacity of immune sera to block cytoadherence of IE in a strain-specific fashion. Fifth, the strain-specific antigen had detergent solubility properties (i.e., insolubility in 1% Triton X-100, solubility in 5% sodium dodecyl sulfate) similar to the variant antigen of P. knowlesi, which has been proven to be a malarial protein exposed on the erythrocyte surface.

Animals↗
PubMed1984

Results of kidney retransplantation.

Sixty-two patients had a second renal transplant at the University of Florida, Gainesville , between Nov 30, 1966 and June 26, 1982. Two-year graft function for all 62 patients was 53.2%, and patient survival was 83.9%. The 51 recipients of cadaveric grafts had a two-year graft survival of 47%. Eleven patients who received second grafts from related donors had a two-year graft survival of 81.8%. Source of first grafts and length of survival of first grafts did not seem to be important in determining the outcome of second grafts. Recipients of cadaveric second kidneys had a significantly better two-year graft survival if they waited longer than six months between loss of the first kidney and retransplantation (52.9%) than if they waited less than six months (35.3%). We believe the results of kidney retransplantation justify second transplants in patients who lost their first grafts.

Adult↗
PubMed1984

A proposed classification of intra-abdominal infections. Stratification of etiology and risk for future therapeutic trials.

Serious studies of ultra-abdominal infections have yielded a mortality that ranges from 3.5% (a composite of antibiotic studies evaluating 1,275 patients) to more than 60% in studies of multiple-organ failure. In reviewing these studies, it is apparent that under the umbrella of "serious intra-abdominal infection," different diseases, processes, and patients are being studied. We define a binomial classification incorporating both an anatomic and a functional approach to intra-abdominal infection. There are ten etiologic classifications of intra-abdominal infections combined with an acute physiologic score (APS), measuring severity of illness, in three gradations. In the patient with undifferentiated intra-abdominal sepsis, the patient will be in class I, II, III, or IV, with a score, A, B, or C, added. Each of the groups V-X, defining a specific infectious category, can also use the APS, which has been verified in patients in intensive care and in those with intra-abdominal infection. It is anticipated that using this approach will provide a technique to evaluate patient groups uniformly in which drug or technical approaches to the management of intra-abdominal infections are being studied. The patients being studied will be more clearly defined, and studies will be comparable from one center to another.

Bacterial Infections↗
PubMed1984

The detergent solubility properties of a malarial (Plasmodium knowlesi) variant antigen expressed on the surface of infected erythrocytes.

Four detergents have been compared for identification of the Plasmodium knowlesi variant antigen on infected erythrocytes by immunoprecipitation analysis. Erythrocytes infected with late trophozoite and schizont forms of cloned asexual parasites were labeled by lactoperoxidase-catalyzed radioiodination and extracted either with the anionic detergents sodium dodecyl sulfate (SDS) or cholate, the neutral detergent Triton X-100, or the zwitterion 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS). After addition of Triton X-100 to SDS and cholate extracts, parallel immunoprecipitations of the four extracts were performed using rhesus monkey antisera of defined agglutinability. Identical results were obtained with clone Pk1(A+), which has 125I-variant antigens of Mr 210,000 and 190,000, and with clone Pk1(B+)1+, which has variant antigens of Mr 200,000-205,000. SDS yielded maximal levels of immunoprecipitated 125I-variant antigens. Variant-specific immunoprecipitation was detected in some experiments with Triton X-100 and cholic acid but with significantly lower recovery than with SDS. CHAPS extraction did not yield the variant antigens on immunoprecipitation. The variant antigens could also be identified in Triton X-100-insoluble material by subsequent extraction with SDS, indicating that failure to recover these proteins in the Triton X-100-soluble fraction is due to failure of this detergent to extract the variant antigens rather than to degradation during extraction. We suggest that the 125I-variant antigens either have a structure that renders them intrinsically insoluble in Triton X-100, cholate, or CHAPS, or that they are associated in some way with host cell membrane components that also resist solubilization by these detergents.

Animals↗
PubMed1984

Neutrophil migration under agarose in healthy and cytomegalovirus-infected mice.

A technique of measuring directed and random migration of mouse neutrophils was developed to study these neutrophil functions in a standardized small animal population into which specific variables could be introduced. Values in healthy animals were highly reproducible; variations in random and directed migration did not exceed 9%. Mouse neutrophils had significant impairment of directed and random migration at 3, 5, and 8 days after infection with cytomegalovirus. Technical reproducibility and the ability to reflect a disease process make this model a promising one for the laboratory study of neutrophil migration.

Animals↗
PubMed1984

Identification of parasite proteins in a membrane preparation enriched for the surface membrane of erythrocytes infected with Plasmodium knowlesi.

A subcellular fraction enriched in erythrocyte membranes has been isolated from rhesus monkey erythrocytes infected with Plasmodium knowlesi. Infected cells were lysed by centrifugation through a zone of hypotonic buffer and membranes isolated by equilibrium density gradient centrifugation in the same tube. The purified membrane fraction was shown to include the erythrocyte surface membrane by several methods: electron microscopy, identification of Coomassie Blue stained erythrocyte membrane proteins, identification of band 3 with a monoclonal antibody, and identification of radioiodinated cell surface proteins. The resulting ghosts were shown to be specifically reactive with monkey sera against the variant surface antigens of P. knowlesi by indirect immunofluorescence and membrane agglutination. No reactivity was seen with a monoclonal antibody (13C11) against the intracellular schizont surface. A number of metabolically labelled parasite proteins were enriched in this membrane function, including peptides of 277, 208, 173, 153, 134, 109, 80, 60 and 48 kDa and the variant surface antigens of variable molecular mass (180-207 kDa). These proteins were distinct from the major parasite proteins of total infected erythrocytes and isolated merozoites. The major glucosamine labelled glycoprotein of the internal schizont (230 kDa) was not found in this fraction. Moreover, no fragment of this parasite glycoprotein was found in this membrane fraction, indicating that no part of this molecule is transported to the erythrocyte surface. In contrast, the variant antigen of P. knowlesi, known to be on the erythrocyte surface, could be readily identified as peptides unique to specific cloned parasite lines. We propose that the other nine parasite proteins found within this membrane fraction represent a starting point for the identification of other parasite proteins transported to the surface membrane of the infected erythrocyte.

Animals↗
PubMed1984

Localization of the major Plasmodium falciparum glycoprotein on the surface of mature intraerythrocytic trophozoites and schizonts.

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.

Animals↗
PubMed1984

N-terminal amino acid sequence of the histidine-rich protein from Plasmodium lophurae.

We have determined the N-terminal amino acid sequence of the first 25 amino acids of the histidine-rich protein (HisRP) isolated from granules of the avian malaria parasite Plasmodium lophurae. The protein was purified from cytoplasmic granules and shown to be 65.2 mol % histidine, close to the previously described value of 73 mol % histidine (Kilejian (1974) J. Biol. Chem. 249, 4650-4655). Ten of the first 25 residues were histidine, five of which formed the sequence His-His-His-His-His (positions 14-18). Also notable was the presence of eight acidic residues within the N-terminal 25 residues. HisRP contained no detectable carbohydrate. When the HisRP was biosynthetically labeled in cultured infected erythrocytes, incorporation of [3H]His greatly exceeded [3H]Ile. Labeled HisRP was not solubilized with 1% w/v Triton X-100 but could be solubilized with greater than or equal to 1% w/v sodium dodecyl sulfate. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the [3H]His labeled protein migrated as a doublet (Mr 53 000 and 50 000). Only one of these bands (Mr 53 000) comigrated with the Coomassie Blue stained protein isolated by the acid-extraction procedure from purified granules. The amino acid composition of HisRP and presence of five contiguous histidine residues in the sequence studied here suggests that other sequences of several contiguous histidine residues must exist in this molecule.

Amino Acid Sequence↗
PubMed1984

Solubilization and immunoprecipitation of 125I-labelled antigens from Plasmodium knowlesi schizont-infected erythrocytes using non-ionic, anionic and zwitterionic detergents.

Plasmodium knowlesi malaria-infected erythrocytes were radio-iodinated and several non-ionic, anionic and zwitterionic detergents were compared in their capacity to extract the labelled membrane proteins. The use of these detergents for antigen identification was tested by immunoprecipitation, after addition of Triton X-100 to some detergent extracts, using hyperimmune monkey antiserum and protein A-Sepharose. 125I-labelled antigens were specifically immunoprecipitated with all detergents tested, including the anionic detergents sodium dodecyl sulphate (SDS), deoxycholate and cholate; the zwitterions Zwittergent-312 and -314, CHAPS and Empigen BB, as well as several non-ionic detergents. The SDS-polyacrylamide gel electrophoresis patterns of 125I-labelled antigens varied after extraction with different detergents, there being no consistent pattern for detergents of a particular class. A total of 14 125I-labelled antigens were identified, 11 of them using Triton X-100. Some minor 125I-labelled antigens identified with Triton X-100 were immunoprecipitated in greater amount after extraction in other detergents. Most importantly, two antigens Mr 200 000 and 180 000 were detected only after extraction with deoxycholate or SDS.

Animals↗
PubMed1984

Plasmodium falciparum malaria: association of knobs on the surface of infected erythrocytes with a histidine-rich protein and the erythrocyte skeleton.

Plasmodium falciparum-infected erythrocytes (RBC) develop surface protrusions (knobs) which consist of electron-dense submembrane cups and the overlying RBC plasma membrane. Knobs mediate cytoadherence to endothelial cells. Falciparum variants exist that lack knobs. Using knobby (K+) and knobless (K-) variants of two strains of P. falciparum, we confirmed Kilejian's original observation that a histidine-rich protein occurred in K+ parasites but not K- variants (Kilejian, A., 1979, Proc. Natl. Acad. Sci. USA, 76:4650-4653; and Kilejian, A., 1980, J. Exp. Med., 151:1534-1538). Two additional histidine-rich proteins of lower molecular weight were synthesized by K+ and K- variants of both strains. We used differential detergent extraction and thin-section electron microscopy to investigate the subcellular location of the histidine-rich protein unique to K+ parasites. Triton X-100, Zwittergent 314, cholic acid, CHAPS, and Triton X-100/0.6 M KCl failed to extract the unique histidine-rich protein. The residues insoluble in these detergents contained the unique histidine-rich protein and electron-dense cups. The protein was extracted by 1% SDS and by 1% Triton X-100/9 M urea. The electron-dense cups were missing from the insoluble residues of these detergents. The electron-dense cups and the unique histidine-rich protein appeared to be associated with the RBC skeleton, particularly RBC protein bands 1, 2, 4.1, and 5. We propose that the unique histidine-rich protein binds to the RBC skeleton to form the electron-dense cup. The electron-dense cup produces knobs by forming focal protrusions of the RBC membrane. These protrusions are the specific points of attachment between infected RBC and endothelium.

Animals↗
PubMed1984

Assessment of the risk for broad sensitization by blood transfusions.

The risk factors associated with the production of lymphocyte antibodies were studied by evaluating the conditions of sensitization in 73 renal failure patients and by searching for lymphocyte antibodies by flow cytometry before the induction of overt sensitization by blood transfusions. In 14 patients the lymphocytotoxic antibodies were not broadly reactive and became undetectable within 5 months. These patients were mostly first transplant candidates who received transfusions prior to the rise of panel antibody reactivity. The remaining 59 patients developed broadly reactive antibodies that persisted for longer than 5 months, regardless of whether or not they were given subsequent blood transfusions. This group was made up almost exclusively of multiparous women or patients who had previously lost a kidney graft. There were 13 patients having no lymphocytotoxic antibodies who developed broad sensitization after blood transfusions. These patients were also multiparous women or previously transplanted patients, suggesting that previous exposure to alloantigens by transplants or pregnancies appeared to be a precondition for blood transfusions to induce broad sensitization. This was confirmed by detecting lymphocyte antibodies by flow cytometry in the pretransfusion serum of 9 of the 13 patients. In contrast, patients who did not make antibodies after transfusions, or those who developed temporary responses, did not have lymphocyte antibodies in their pretransfusion specimens. These findings suggest that patients with low levels of lymphocyte antibodies, not detectable by standard cytotoxicity, are at high risk of developing broadly reactive cytotoxic antibodies after blood transfusions.

Antilymphocyte Serum↗
PubMed1984

Role of regular and leukocyte-free blood transfusions in the generation of broad sensitization.

The factors associated with the development of humoral sensitization were studied prospectively in 30 previously transplanted patients immediately after graft rejection. Lymphocyte antibodies were measured both by conventional cytotoxicity in 30 panel cells and by flow cytometry in up to 10 target cells. Although lymphocyte antibodies induced by graft rejection alone were detected in 12 of 26 patients (46%), lymphocytotoxic antibodies were present in only 2 of 27 patients. Of the 25 patients without lymphocytotoxic antibodies, 13 developed them later. In all cases panel antibody reactivity developed after the patients received blood transfusions. No other factor was associated with the development of lymphocytotoxic antibodies, including transplant nephrectomy. There were 12 patients who remained negative for lymphocytotoxic antibodies even though 5 of them were transfused. The powerful role of blood transfusions in the generation of broad sensitization was further documented in 5 patients who received blood units completely depleted of leukocytes by cottonwool filtration and red cell washing. Four of these patients showed significant increases in the level of lymphocytotoxic antibodies, even when stored blood units were used. One additional patient became broadly sensitized by the transfusion of frozen blood. These results show (A) that broad sensitization may not develop if patients are not transfused after graft rejection; (B) that blood transfusions lead to broad sensitization in most (76%) pretransplanted patients; and (C) that transfusion of leukocyte-free blood may delay, but not avoid, the development of broad sensitization.

Autoantibodies↗
PubMed1984

Planned random donor blood transfusion in preparation for transplantation. Sensitization and graft survival.

Random donor blood transfusions were used to prepare 183 prospective recipients for one-haplotype living-related donor (LRD) grafts or cadaver donor (CD) grafts. Five units of packed red blood cells were administered over a 7-10 day period, and weekly sera were monitored for six weeks. Sensitization was uncommon in men and nulliparous women (8/153), was of low reactivity, and was not a barrier to transplantation. Multiparous women had a 44% frequency of sensitization on presentation and 11/24 initially lacking cytotoxicity developed reactive serum following transfusion. Single-haplotype LRD recipients had 96% one-year graft survival. CD recipients had one-year graft survival of 72%. The rate of transplantation in surviving candidates exceeded 90%, and supports the hypothesis of a protective immune response.

Blood Transfusion↗
PubMed1984

Malaria parasites do not contain or synthesize sialic acids.

The capacity of Plasmodia to synthesize sialic acids was investigated by adding radioactive acetate to short-term in vitro cultures of the intraerythrocytic asexual forms of three malaria parasites (the human malaria Plasmodium falciparum in Aotus trivirgatus erythrocytes; the simian malaria P. knowlesi in rhesus monkey erythrocytes; the rodent malaria P. berghei in mouse erythrocytes) and to cultures of extracellular zygotes of the avian malaria P. gallinaceum. Radioactive acetate was added to normal rhesus monkey erythrocytes and to cells of the murine myeloma NS-1 for comparison. Although [1-14C]-acetate labeled many proteins with each malaria parasite and the NS-1 cells, analysis of purified sialic acids revealed that only with the NS-1 cells was radioactivity incorporated into sialic acids. Furthermore, N-acetyl[6-3H]mannosamine was not incorporated into sialic acids or malarial glycoproteins when added to P. knowlesi cultures. All of the malaria parasites underwent growth or differentiation during these experiments as measured by [35S]methionine uptake into protein and by light microscopy. Extracellular parasites largely free of erythrocyte membranes were prepared to determine whether Plasmodia contain sialic acids that are not labeled by exogenous precursors. Purified merozoites of P. knowlesi and zygotes of P. gallinaceum did not contain detectable amounts of sialic acids on chemical analysis. Thus, although we could show that Plasmodia can incorporate radioactive sugars such as glucosamine, galactose and mannose into proteins, presumably glycoproteins, they do not synthesize sialic acids or sialo-glycoproteins, nor do they contain sialo-glycoconjugates of host origin.

Animals↗