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J D Haynes

Publications and source records attributed to J D Haynes.

At least 37 records · Page 2Linked to original sources
PubMed1990

Characterization of the 175-kilodalton erythrocyte binding antigen of Plasmodium falciparum.

EBA-175 is a soluble 175-kDa Plasmodium falciparum antigen that is released into culture supernatants during rupture of schizont-infected erythrocytes. EBA-175 binds to erythrocytes and binding is sialic acid-dependent. A clone expressing the gene encoding EBA-175 was obtained previously by screening a genomic DNA expression library with antibodies that had been affinity-purified from EBA-175. Antibodies were raised against a 43-amino-acid peptide (EBA-peptide 4) synthesized according to the deduced amino acid sequence. Antibodies to peptide 4 and affinity-purified antibodies specific for EBA-175 were used to characterize further EBA-175 giving the following results: (1) EBA-175 differs biochemically and immunologically from other reported malarial antigens; (2) the EBA-175s from six geographical isolates of P. falciparum are antigenically conserved; (3) EBA-175 is expressed during schizogony as a 190-kDa protein which is larger than the culture supernatant form of the antigen. The 190-kDa form of the protein is recovered from the cell pellet in schizont-infected erythrocytes and partitions to the soluble fraction when extracted with detergent; (4) release of soluble EBA-175 into the culture supernatant coincides with schizont rupture; (5) there was no observable change in pI (pI = 6.86) by isoelectric focusing between the cellular and supernatant species of the protein; and (6) release of EBA-175 into the culture supernatant is inhibited by the addition of chymostatin and leupeptin. The continued research into the role of EBA-175 during erythrocyte invasion may aid in vaccine development for malaria.

Animals↗
PubMed1990

Primary structure of the 175K Plasmodium falciparum erythrocyte binding antigen and identification of a peptide which elicits antibodies that inhibit malaria merozoite invasion.

The Plasmodium falciparum gene encoding erythrocyte binding antigen-175 (EBA-175), a putative receptor for red cell invasion (Camus, D., and T. J. Hadley. 1985. Science (Wash. DC). 230:553-556.), has been isolated and characterized. DNA sequencing demonstrated a single open reading frame encoding a translation product of 1,435 amino acid residues. Peptides corresponding to regions on the deduced amino acid sequence predicted to be B cell epitopes were assessed for immunogenicity. Immunization of mice and rabbits with EBA-peptide 4, a synthetic peptide encompassing amino acid residues 1,062-1,103, produced antibodies that recognized P. falciparum merozoites in an indirect fluorescent antibody assay. When compared to sera from rabbits immunized with the same adjuvant and carrier protein, sera from rabbits immunized with EBA-peptide 4 inhibited merozoite invasion of erythrocytes in vitro by 80% at a 1:5 dilution. Furthermore, these sera inhibited the binding of purified, authentic EBA-175 to erythrocytes, suggesting that their activity in inhibiting merozoite invasion of erythrocytes is mediated by blocking the binding of EBA-175 to erythrocytes. Since the nucleotide sequence of EBA-peptide 4 is conserved among seven strains of P. falciparum from throughout the world (Sim, B. K. L. 1990. Mol. Biochem. Parasitol. 41:293-296.), these data identify a region of the protein that should be a focus of vaccine development efforts.

Amino Acid Sequence↗
PubMed1988

Receptor-like specificity of a Plasmodium knowlesi malarial protein that binds to Duffy antigen ligands on erythrocytes.

A 135-kD parasite protein, a minor component of the Plasmodium knowlesi malaria radiolabeled proteins released into culture supernatant at the time of merozoite release and reinvasion, specifically bound to human erythrocytes that are invaded and carry a Duffy blood group determinant (Fya or Fyb), but did not bind to human erythrocytes that are not invaded and do not carry a Duffy determinant (FyFy). Specific anti-Duffy antibodies blocked the binding of the 135-kD protein to erythrocytes carrying that specific Duffy determinant. Purified 135-kD protein bound specifically to the 35-45-kD Duffy glycoprotein on a blot of electrophoretically separated membrane proteins from Fya and Fyb erythrocytes but not from FyFy erythrocytes. Binding of the 135-kD protein was consistently greater to Fyb than to Fya both on the blot and on intact erythrocytes. The 135-kD protein also bound to rhesus erythrocytes that are Fyb and are invaded, but not to rabbit or guinea pig erythrocytes that are Duffy-negative and are not invaded. Cleavage of the Duffy determinant by pretreating Fyb human erythrocytes with chymotrypsin greatly reduced both invasion and binding of the 135-kD protein, whereas pretreating Fyb erythrocytes with trypsin had little effect on the Duffy antigen, the 135-kD protein binding, or on invasion. However, instances of invasion of other enzyme-treated erythrocytes that are Duffy-negative and do not bind the 135-kD protein suggest that alternative pathways for invasion do exist.

Animals↗
PubMed1988

The pharmacokinetics of cefixime in the fasted and fed state.

Twenty healthy adult volunteers received single 400 mg oral doses of cefixime in an open, randomized, crossover study, administered twice in the fasted state and twice with a standard breakfast. The study design allowed both an evaluation of a potential food effect and also an analysis of both intrasubject and intersubject variability in the fasted and fed state. There was a small but significantly longer (approximately 1 h) time to peak concentration when the drug was given with food. Peak serum concentrations, area under the curve, and 24 h urinary recovery values were unchanged in the fed and fasted states. The terminal elimination half-life of the drug given after a meal (3.6 h) was slightly longer than that observed after dosing in the fasting condition (3.5 h). The intrasubject and intersubject variabilities were less than 12% and 33% respectively, for both area under the curve and 24 h urinary recovery, and were virtually the same for the fasted and fed occasions. Therefore, the drug may be administered with or without food.

Administration, Oral↗
PubMed1988

Identification of Plasmodium knowlesi erythrocyte binding proteins.

Plasmodium knowlesi, a malaria of Old World monkeys, invades all Duffy blood group positive human erythrocytes and various New World monkey erythrocytes except Cebus apella. We had previously identified a 135 kDa parasite protein in supernatants of P. knowlesi cultures that bound to Duffy positive but not to Duffy negative human erythrocytes [Haynes et al., J. Exp. Med. 167, 1873-1881 (1988)]. We now use New World monkey erythrocytes as a reagent to identify P. knowlesi proteins in culture supernatants that will bind to all New World monkey erythrocytes susceptible to invasion but not to C. apella erythrocytes, which are refractory to invasion. The 135 kDa protein binds to all New World monkey erythrocytes, including C. appella. Another protein of 155 kDa binds to all New World monkey erythrocytes except C. apella. The 155 kDa protein binds to Old World monkey erythrocytes, the natural host of P. knowlesi; it does not bind to human Duffy positive erythrocytes. This and the previous study are the beginning of the identification of parasite proteins of P. knowlesi that bind to erythrocytes in a receptor specific manner.

Animals↗
PubMed1988

Pharmacokinetics of cefixime in the young and elderly.

The pharmacokinetics of cefixime were compared in 12 young and 12 elderly subjects receiving 400 mg once-a-day for five days. Mean peak serum concentrations (Cmax) on days one and five in the elderly (4.90 and 5.68 mg/l) were comparable (P greater than 0.05) to those in the young subjects (3.88 and 4.74 mg/l). Serum area under the curve (AUC) values on days one and five in the elderly (41.0 and 49.5 mg.h/l) were higher (P less than 0.05) than those in young subjects (28.6 and 34.9 mg.h/l). In addition, the elimination half-life, mean residence time, average concentration, minimal concentration and renal clearance (Clr) values were significantly higher (P less than 0.05) in the elderly. A significant linear correlation (P less than 0.05) was found between the Clr of cefixime (total and unbound) and creatinine clearance. The urinary recovery (Ae0----24) and protein binding of cefixime on days one and five was similar in the elderly and young. Overall, there is no need for any dosage adjustment of the drug in the elderly.

Administration, Oral↗
PubMed1987

Monoclonal antibody characterization of Plasmodium falciparum antigens in immune complexes formed when schizonts rupture in the presence of immune serum.

When Plasmodium falciparum parasites are cultured with some immune sera, merozoites are agglutinated by antibodies to form immune clusters of merozoites and prevent their invasion into erythrocytes. Within these immune clusters of merozoites, several antigens that are normally found in the soluble fraction after detergent extraction accumulate in relatively insoluble immune complexes. From mice immunized with these immune complexes, we obtained hybridomas secreting monoclonal antibodies (mAb) that react with various immune clusters of merozoites antigens, including mAb 3D5, which recognizes a 101-kDa antigen (p101) and mAb, 5E3, which recognizes a 113-kDa antigen (p113). Both mAb reacted with antigens at the surface of schizonts, in the vacuolar space, and at the surface of merozoites before their release from schizont-infected cells. Both p101 and p113 were synthesized by mature trophozoites and young schizonts. In pulse-chase experiments, p113 was processed to 100-, 70-, 55-, and 50-kDa products. Both p101 and p113 appeared in the culture medium when schizont rupture occurred in normal culture medium but were found in immune complexes when schizont rupture occurred in the presence of immune serum. Antibodies in immune complexes, when dissociated with acid and used to probe immunoblots, reacted with affinity-purified p101 and p113. Antigens such as these, which are accessible at the parasite surface and react with antibodies present in immune serum that inhibits parasite invasion, are logical candidates to study in the search for a vaccine against the erythrocytic stages of malaria.

Animals↗
PubMed1987

Monoclonal antibody characterization of the 195-kilodalton major surface glycoprotein of Plasmodium falciparum malaria schizonts and merozoites: identification of additional processed products and a serotype-restricted repetitive epitope.

The gp195 from Camp strain parasites was characterized with eight monoclonal antibodies (MAb) that recognize different epitopes on gp195 and three of its merozoite-associated processed products. Four MAb (3H7, 3B10, 7F1, and 4G12) reacted with different epitopes on the 45-kDa glycosylated product (gp45), shown by differences in their reactivities with soluble and immunoblotted gp45. One MAb (7H10) reacted with a conformational epitope probably formed as a result of the interaction of gp45 with a nonglycosylated 45-kDa product (p45). Three other MAb (3D3, 7B11, and 7B2) reacted with different epitopes on a nonglycosylated 83-kDa product (p83), shown by differences in their reactivities against various parasite isolates in immunofluorescent antibody assays. Immunoprecipitation of antigens that were pulse-labeled with [3H]isoleucine and chased with cold isoleucine showed that p45 and gp45 were processed products of gp195 and p83 was sequentially processed into smaller fragments of 73 and 67 kDa (p73 and p67). Immunoblots showed that the 7B11 and 7B2 epitopes were present on p83, p73, and p67, but that the 3D3 epitope was present only on p83 and p73. A two-site immunoassay showed the 3D3 epitope to be repetitive. The 3D3 and 7B11 epitopes were serotype restricted (present in seven and 24 of 33 isolates, respectively), but the other five epitopes were common to all isolates tested. The gp195 and its processed products have Mr that are consistent with the Mr of a number of antigens shown previously to be associated with the immune complexes that are formed when merozoites are agglutinated by antibodies contained in some growth inhibitory immune sera.

Animals↗
PubMed1987

Pharmacokinetics of cefixime after once-a-day and twice-a-day dosing to steady state.

The pharmacokinetics of cefixime (CL 284,635; FK027), a new orally active broad-spectrum cephalosporin, were determined in 26 healthy volunteers, after multiple 200-mg twice-a-day (group 1; N = 13) or 400-mg once-a-day (group 2; N = 13) dosing for 15 days. On study days 1, 8, and 15, mean peak serum concentrations (Cmax) were 1.67, 1.75, and 1.87 micrograms/mL, respectively, for group 1 and 2.76, 3.04, and 2.67, respectively, for group 2. Over the 15-day period, mean trough serum concentrations were, on average, 0.40 and 0.08 microgram/mL for groups 1 and 2, respectively. Comparison (ANOVA) of serum and urinary excretion pharmacokinetic parameters for cefixime on days 1, 8, and 15 found no significant (P greater than .05) differences for either group except for a small but significantly (P less than .05) earlier time to reach Cmax and higher renal clearance on days 8 and 15 in group 1. These differences, however, are not clinically significant. On study days 1, 8, and 15, mean Cmax and AUC0-tau values for Group 2 were about 1.5 to 2.2 time those for Group 1. Urinary excretion of cefixime accounted for 11.9 to 14.5% and 9.9 to 12.4% of the dose in groups 1 and 2, respectively, over the 15-day study. Overall, there was no accumulation of cefixime in serum or urine nor was there a reduction in serum concentrations of urinary amounts over the 15-day dosing period when the drug was given either as a 200-mg twice-a-day or 400-mg once-a-day dosing regimen.

Adult↗
PubMed1987

Characterization of gp195 processed products purified from Plasmodium falciparum culture supernates.

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.

Animals↗
PubMed1987

Falciparum malaria parasites invade erythrocytes that lack glycophorin A and B (MkMk). Strain differences indicate receptor heterogeneity and two pathways for invasion.

To determine the ligands on erythrocytes for invasion by Plasmodium falciparum, we tested invasion into MkMk erythrocytes that lack glycophorins A and B and enzyme-treated erythrocytes by parasites that differ in their requirement for erythrocyte sialic acid. The 7G8 strain invaded MkMk erythrocytes and neuraminidase-treated normal erythrocytes with greater than 50% the efficiency of normal erythrocytes. In contrast, the Camp strain invaded MkMk erythrocytes at 20% of control and neuraminidase-treated normal erythrocytes at only 1.8% of control. Invasion of MkMk erythrocytes by 7G8 parasites was unaffected by treatment with neuraminidase but was markedly reduced by treatment with trypsin. In contrast, invasion of MkMk cells by Camp parasites was markedly reduced by neuraminidase but was unaffected by trypsin. We conclude that the 7G8 and Camp strains differ in ligand requirements for invasion and that 7G8 requires a trypsin sensitive ligand distinct from glycophorins A and B.

Animals↗
PubMed1987

Serotypic antigens of Plasmodium falciparum recognized by serotype-restricted inhibitory human sera.

Immune sera from some Cambodian refugees contain functional serotypic antibodies that inhibit invasion of erythrocytes by the Camp strain but not by the FCR-3 strain of Plasmodium falciparum. Using a new assay, the "competitive heterologous antigen assay" (CHAA), the serotypic antibodies in a pool of three inhibitory sera were characterized by the antigens they precipitated. In the CHAA, immunoprecipitation of antigens by antibodies to common or cross-reacting antigenic determinants was blocked with excess heterologous unlabeled FCR-3 antigens before 3H-labeled Camp schizont and merozoite antigens were immunoprecipitated. The predominant Camp strain serotypic antigens revealed after electrophoresis and autoradiography were the major 195 Kd glycoprotein surface antigen (gp195) and its processed products at 150, 83, 73, and possibly 45 Kd. Additional serotypic antigens were identified at 180, 130, 65, 50, and 32 Kd. It is likely that one or more of these serotypic antigens is a target for the serotypic antibodies that inhibit invasion.

Animals↗
PubMed1987

Absorption kinetics of orally administered leucovorin calcium.

Oral dose proportionality and pharmacokinetics of leucovorin [(d,l)-5-formyltetrahydrofolate (5-formyl-THF)] were studied in 30 healthy male subjects. In a randomized cross-over design, 24 fasted subjects were given 4 of a series of 5 single test doses between 20 and 100 mg, at 1-week intervals, of 5-formyl-THF as an oral solution of leucovorin calcium. Six separate subjects received 200 mg iv and po in a 2-way crossover. Blood and urine samples were collected over 24 hours for differential microbiological folate assays using Lactobacillus casei and Streptococcus faecalis. Using L casei activity to measure total serum folates, the area under the concentration-time curve from 0 to infinite time (AUC[0-infinity]) was calculated. Relative bioavailabilities were 78%, 62%, 49%, and 42% for the 40-, 60-, 80-, and 100-mg doses, respectively. Both the AUC and peak concentration (CPEAK) of total folates (consisting predominantly of the major metabolite, 5-methyltetrahydrofolate (5-methyl-THF], displayed significant deviation from linearity consistent with a saturation of folate absorption. Absolute bioavailability of the 200-mg oral dose of leucovorin based on AUC was 31% compared with that of the iv dose (6,848 vs. 22,298 ng.hr/ml, respectively). Total clearance, terminal half-life, and apparent volume of distribution of total folate at the 200-mg dose were not significantly different between the two routes of administration. Eighty-three percent of the biologically active iv dose was recovered in the urine within 24 hours, 31% as 5-methyl-THF. Twenty percent of the same oral dose was excreted in 24 hours, 16% as 5-methyl-THF. In contrast to the nondose-proportionality observed in total serum folates, AUC of the small component of S faecalis activity, which appeared earlier than 5-methyl-THF, displayed linear kinetics, suggestive of a distinct mechanism of uptake. As dose increased, S faecalis activity increased in relative proportion to L casei, indicating that saturation of the enzymatic bioconversion to 5-methyl-THF may also be occurring. In light of the demonstrated nondose-proportionality of total folates with oral leucovorin in this dose range, consideration should be given to parenteral administration in regimens employing higher doses. Oral administration should be at a level consistent with the capacity for efficient folate uptake.

Administration, Oral↗
PubMed1986

Plasmodium falciparum antigens synthesized by schizonts and stabilized at the merozoite surface when schizonts mature in the presence of protease inhibitors.

Schizonts of Plasmodium falciparum were cultured in medium containing a mixture of 10 micrograms/ml each of leupeptin, chymostatin, pepstatin, and antipain. The protease inhibitors did not inhibit macromolecular synthesis but were associated with decreased reinvasion of red cells and the accumulation of well preserved merozoites clustered around pigment granules (PCM, protease inhibitor clusters of merozoites). The parasite pellet from PCM cultures contained increased amounts of merozoite antigens, particularly at Mr 83, 73, 66, 45, and 17 kDa. The increases of the Mr 83, 73, and 45 kDa surface antigens observed in PCM had been observed also in similar merozoite clusters obtained by culturing schizonts in the presence of inhibitory antibodies. These three antigens are processed products of the abundant Mr 195 kDa schizont surface antigen. Liquid-phase double immunofluorescence of PCM demonstrated a residual red cell membrane through which monoclonal antibodies passed and reacted with the Mr 83, 73, and 45 kDa merozoite surface antigens or their precursors. The processes associated with normal reinvasion apparently involve protease(s), which plays a role(s) in the breakdown of the red cell membrane and the shedding of merozoite surface antigens. Interference with these processes by protease inhibitors is useful in increasing recoveries of merozoite antigens, as well as in elucidating mechanisms of reinvasion.

Animals↗
PubMed1986

Plasmodium falciparum antigens synthesized by schizonts and stabilized at the merozoite surface by antibodies when schizonts mature in the presence of growth inhibitory immune serum.

Some immune sera that inhibit erythrocyte invasion by merozoites also agglutinate the merozoites as they emerge from rupturing schizonts. These immune clusters of merozoites (ICM) possess a surface coat that is cross-linked by antibody and is thicker than the surface coat associated with normal merozoites (NM) obtained from cultures containing preimmune serum. Analysis of metabolically labeled ICM and NM performed by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that washed ICM possessed immune complexes containing antigens representative of schizonts and merozoites. Characteristics of the immune complexes included: a) they were not soluble in pH 8 Triton X-100, b) they were soluble at an acid pH, and c) after pH neutralization they were precipitated by using staphylococcal protein A. Merozoite antigens having Mr of 83, 73, and 45 kDa were associated with immune complexes in ICM. The 83 and 73 kDa antigens were recovered in considerably larger quantities from ICM than from NM. Schizont antigens having Mr of 230, 173 (triplet), 152 (doublet), and 31 kDa were associated with immune complexes in ICM, and a 195 kDa antigen(s) from schizonts and merozoites was also present in the immune complexes. In addition, other antigens of Mr 113, 101, 65, and 51 kDa may have been immune complexed. These 15 antigens accounted for less than 30% of the schizont and merozoite antigens recognized by the immune serum. Immune complexes probably formed between antibodies and a) surface antigens of schizont-infected erythrocytes exposed to antibody before schizont rupture, b) surface antigens of merozoites and schizonts exposed during schizont rupture, and c) soluble antigens normally released during schizont rupture. The antibody components of the immune complexes may have prevented rapid degradation or shedding of some antigens from the merozoite surface. Allowing schizonts to rupture in the presence of inhibitory antibodies (to form ICM) is a useful approach to identifying exposed targets of protective immunity against malaria.

Animals↗
PubMed1986

Decreased growth of Plasmodium falciparum in red cells containing haemoglobin E, a role for oxidative stress, and a sero-epidemiological correlation.

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.

Antibodies↗
PubMed1986

Epitope map and processing scheme for the 195,000-dalton surface glycoprotein of Plasmodium falciparum merozoites deduced from cloned overlapping segments of the gene.

DNA fragments from human malaria parasites were cloned into lambda gt11 to produce a genomic DNA expression library. A pool of monoclonal antibodies (mAbs) recognizing three domains of the 195-kDa major merozoite surface glycoprotein (gp195) reacted with seven clones expressing malaria antigens. mAbs recognizing the 83-kDa product of gp195 reacted with the clones, but mAbs recognizing a glycosylated 45-kDa and a nonglycosylated 45-kDa domain did not. Restriction enzyme mapping revealed that the clones contained overlapping segments encoding about 70% of the gene beginning at the 5' end and ending at an EcoRI restriction enzyme site 3.3 kilobase pairs downstream. The mAbs recognizing the 83-kDa domain reacted differently with the clones, allowing the mapping of three epitopes, one of which was repetitive. Affinity-purified antibodies were selected from immune monkey serum with recombinant expression proteins adsorbed to nitrocellulose filters. When used to probe electrophoretic immunoblots of parasite extracts, these antigen-selected antibodies reacted with specific sets of processed products of gp195, including those associated with the 83- and the nonglycosylated 45-kDa domains. This information, combined with the mAb epitope map, allowed a tentative scheme for processing gp195 from the Camp strain to be proposed.

Animals↗