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R T Reese

Publications and source records attributed to R T Reese.

At least 37 records · Page 2Linked to original sources
PubMed1985

Expression of Plasmodium falciparum surface antigens in Escherichia coli.

The asexual blood stages of the human malarial parasite Plasmodium falciparum produce many antigens, only some of which are important for protective immunity. Most of the putative protective antigens are believed to be expressed in schizonts and merozoites, the late stages of the asexual cycle. With the aim of cloning and characterizing genes for important parasite antigens, we used late-stage P. falciparum mRNA to construct a library of cDNA sequences inserted in the Escherichia coli expression vector pUC8. Nine thousand clones from the expression library were immunologically screened in situ with serum from Aotus monkeys immune to P. falciparum, and 95 clones expressing parasite antigens were identified. Mice were immunized with lysates from 49 of the bacterial clones that reacted with Aotus sera, and the mouse sera were tested for their reactivity with parasite antigens by indirect immunofluorescence, immunoprecipitation, and immunoblotting assays. Several different P. falciparum antigens were identified by these assays. Indirect immunofluorescence studies of extracellular merozoites showed that three of these antigens appear to be located on the merozoite surface. Thus, we have identified cDNA clones to three different P. falciparum antigens that may be important in protective immunity.

Animals↗
PubMed1985

A role for carbohydrate moieties in the immune response to malaria.

Treatment of antigen prepared from asexual blood stages of the human malarial parasite Plasmodium falciparum with a mixture of glycosidases resulted in a reduction in the ability of the antigen to bind antibodies from immune human and monkey sera in an ELISA assay. Some of the epitopes in the parasite material were heat stable, protease resistant, and sensitive to glycosidases. Proteins of Mr 110,000 and 65,000 from parasitized RBC were shown to have reduced antigenicity in Western blots after glycosidase treatment. The carbohydrate side chains of parasite glycoproteins therefore make a contribution to the total antigenicity of the parasite.

Adult↗
PubMed1985

Recognition of a Mr 56K glycoprotein on the surface of Plasmodium falciparum merozoites by mouse monoclonal antibodies.

Hybridomas were prepared from mice repeatedly injected with disrupted Plasmodium falciparum (FVO isolate) schizonts and merozoites. Antibodies secreted by two of these hybridomas were shown by immunoelectron microscopy to bind to the surface of merozoites from the FVO isolate. These monoclonal antibodies (McAb) reacted with the FVO and Geneva isolates by an indirect fluorescence antibody test (IFAT) and immunoprecipitated a protein of relative molecular weight (Mr) 56K from both isolates. The 56K protein could be labeled with [35S] methionine and [3H]glucosamine. Glycosidase treatment of the affinity-purified polypeptide proved that the [3H]glucosamine had been incorporated into sugar side chains and that this protein (called gp56) was glycosylated. The anti-gp56 McAb did not react by IFAT or immunoprecipitation with four isolates (Honduras I, Indochina I, Tanzania I, and Kenya) that lack gp56 but contain major glycoproteins of Mr 50K. Antibodies from an Aotus monkey immune to the FVO isolate immunoprecipitated gp56 from both the FVO and Geneva isolates, but did not immunoprecipitate the 50K glycoproteins from the other four isolates. Extraction experiments conducted with the nonionic detergent Triton X-114 indicate that some of the gp56 molecules are hydrophilic and that the others are either hydrophobic or interact with hydrophobic molecules. These results, together with the electron microscopic data, suggest that the hydrophilic gp56 is a component of the extracellular matrix and that the hydrophobic gp56 may be associated with the plasma membrane of the merozoite.

Animals↗
PubMed1984

Reversal of knob formation on Plasmodium falciparum-infected erythrocytes.

The human malarial parasite Plasmodium falciparum can produce surface protrusions (knobs) on infected erythrocytes; however, long-term culturing of the parasite results in the appearance of knobless cells. In this study it was found that a knob-producing clone lost the ability to produce knobs in vitro. Furthermore, a clone not producing knobs derived from the knob-producing clone regained the capacity to produce knobby cells in vitro. Certain parasite proteins were associated with the knobby phenotype but not with the knobless type. These results indicate that the parasites change in vitro in a spontaneous and reversible manner independent of immunological selection.

Animals↗
PubMed1984

Complement activation by the surface of Plasmodium falciparum infected erythrocytes.

The surface of trophozoite-stage Plasmodium falciparum infected erythrocytes will, in the presence of immune human or owl monkey serum, activate the classical complement pathway. This was demonstrated with a sensitive, enzyme-linked immunosorbent assay which detects the complex, C1s-C1 inhibitor, which is only generated when the classical pathway is activated. A second enzyme-linked immunosorbent assay, as well as Covaspheres coated with affinity-purified anti-C3, showed that immune activation of the classical pathway by infected erythrocytes resulted in the accumulation of significant amounts of C3b on the erythrocyte surface. During the development of the parasite to the trophozoite stage, the erythrocyte membrane is also transformed from a non-activator into a surface capable of activating complement by the alternative pathway. Erythrocytes infected with trophozoite-stage parasites directly activated the alternative complement pathway. This activation led to the specific binding of an average of 15,000 C3b molecules per infected cell. Alternative pathway activation was augmented by anti-parasite antibody. Such conditions mediated the accumulation of an average of 36,000 C3b molecules per infected erythrocyte. The amounts of C3b on the infected erythrocyte surface did not lead to cellular lysis. They are, however, likely to have a major impact on the total in vivo response to this parasite.

Cells, Cultured↗
PubMed1984

Synthesis of merozoite proteins and glycoproteins during the schizogony of Plasmodium falciparum.

We have investigated the protein and glycoprotein content of Plasmodium falciparum merozoites by metabolically labeling cultures of schizont-stage parasites with [35S]methionine or with [3H]glucosamine followed by incubation in nonradioactive medium to allow the schizonts to mature into merozoites, infect new erythrocytes, and develop into ring-stage parasites. The ring stages were separated from schizonts by sedimentation through Percoll. Labeled proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by fluorography. Using [35S]methionine, four major proteins (p) with apparent relative molecular weights (Mr) = 202k , 136k , 82k , and 46k and two proteins of intermediate labeling (Mr = 185k and 142k ) were observed in the schizont-labeled ring-stage parasites. Because corresponding proteins were also observed in the schizont stage, we conclude that they had been present in the invading merozoite. In contrast, prominent proteins which were generally labeled during the ring stage and some major schizont-stage proteins were virtually absent in the schizont-labeled ring-stage. By labeling the parasite proteins with [3H]glucosamine followed by separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, five major glycoproteins (gp) of apparent Mr = 185k , 88k , 56k , 46k , and 34k were identified. Their presence in both the schizont and the schizont-labeled ring stage demonstrated that the merozoite contains glycoproteins. Immune owl monkey serum recognized all five glycoproteins. A comparison of proteins by two-dimensional gel electrophoresis (isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis) suggested that p185 and gp185 were identical, as were p46 and gp46 .

Animals↗
PubMed1984

Protein and nucleic acid synthesis during synchronized growth of Plasmodium falciparum.

Plasmodium falciparum, the human malarial parasite, was synchronized for asexual growth and pulse labeled to determine when RNA, protein, and DNA synthesis occurred. RNA was synthesized during two periods in the 48-h developmental cycle, protein synthesis occurred throughout the cycle, and most DNA was synthesized just before nuclear division (schizogony).

Animals↗
PubMed1984

Proteins responsible for a punctate fluorescence pattern in Plasmodium falciparum merozoites.

Mouse monoclonal antibodies (McAbs) have been used to characterize the proteins of the asexual erythrocytic cycle of Plasmodium falciparum. Three different McAbs react with antigens of the schizont and extracellular merozoite to give a punctate fluorescence pattern. In many cases, such areas of fluorescence were composed of two adjacent, fluorescent bodies; these were distinct from the nuclei. In contrast, McAbs which bound to the ring-stage parasite were not localized, but were diffusely distributed within or around the ring-stage parasite. These McAbs immunoprecipitated five prominent, 35S-methionine-labeled schizont proteins (p) of Mr 82K, 70K, 67K, 39K, and 37K. Only p82, p39, and p37 were immunoprecipitated from schizont-labeled ring-stage parasites; thus, it appears that p70 and p67 are modified, degraded, or secreted some time between intracellular merozoite maturation and erythrocyte invasion.

Animals↗
PubMed1984

In vitro inhibition of intracellular growth of Plasmodium falciparum by immune sera.

Beginning at the ring stage, synchronized cultures of Plasmodium falciparum were grown in suspension for 22-32 hours. Intracellular growth was assayed by measuring cellular uptake and incorporation into protein of 35S-methionine. Low concentrations (2%) of serum from immune humans and Aotus monkeys were found to inhibit the uptake of the 35S-methionine. The amount of inhibition for a given serum was often inversely related to its indirect fluorescent antibody test titer. Inhibition occurred during the trophozoite stage and was not obtained with a clone lacking the erythrocyte modifications referred to as knobs. Thus, a sensitive new assay is described which allows detection of factors in immune primate sera which can affect maturation of P. falciparum within the erythrocyte. These serum factors are likely to be antibodies which react with antigens expressed at the trophozoite stage on the surface of K+-infected erythrocytes.

Animals↗
PubMed1983

Plasmodium falciparum: comparison of in vitro growth of knobby and knobless isolates.

Variants (K-) of three strains of Plasmodium falciparum which do not produce the erythrocyte surface alterations that have been called knobs have been compared with their wildtype knobby (K+) parents. The K- variants achieve higher parasitemias, incorporate radiolabeled isoleucine more rapidly, and produce a higher percentage of multiply-infected cells than do their K+ parents. Nevertheless, immune owl monkey sera cause approximately the same percentage inhibition of growth of both K+ and K- organisms when included in the growth medium at a 1% concentration.

Animals↗
PubMed1982

Translation in vitro of RNA from the human malarial parasite Plasmodium falciparum.

RNA has been isolated from the human malarial parasite, Plasmodium falciparum, and translated in vitro using two systems: rabbit-reticulocyte lysate and Xenopus laevis oocytes. Polypeptides are synthesized that are characteristic of the parasite isolate and the developmental stage from which the RNA was purified. Many of these polypeptides are precipitated by antibody from an immune monkey. Analysis of the primary structure of these polypeptides should give the information necessary to produce a vaccine against this parasite.

Animals↗
PubMed1982

Plasmodium falciparum merozoites: isolation by density gradient centrifugation using Percoll and antigenic analysis.

Merozoites of Plasmodium falciparum were isolated and immunocytochemically analyzed. Mature parasites from knobby (K+) and knobless (K-) strains were incubated for 4 to 5 hr in RPMI 1640 with 10% serum and 10% RBC extract. About 12 to 14% of the merozoites released were recovered by density gradient centrifugation using Percoll. From 1 to 3 X 10(9) merozoites were obtained per collection. The merozoite preparations were contaminated with 10% residual bodies, about 0.1% infected and uninfected erythrocytes, about 0.1% RBC-free trophozoites and schizonts, and numerous small (less than 0.5 microns) membrane vesicles. Merozoites from the K+ and K- strains were morphologically and, by an indirect, ferritin-labeled antibody assay using serum from immune Aotus, antigenically indistinguishable. Although the residual body coats reacted with the immune Aotus serum, the membrane vesicles, some of which were seen to be blebbing from merozoites, did not react with this serum or a serum against erythrocytes. This paper describes a procedure that can be used to obtain large numbers of merozoites with little contamination by host erythrocytes.

Centrifugation, Density Gradient↗
PubMed1981

Reaction of immune sera with components of the human malarial parasite, Plasmodium falciparum.

Human and monkey sera from individuals exposed to Plasmodium falciparum were characterized by indirect immunofluorescence, in vitro parasite growth inhibition, and immunoprecipitation of 125I-labeled parasite antigens followed by analytical sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In general there was a good correlation between fluorescence titer and the ability of a serum to inhibit parasite growth in vitro. Exceptions were found, however. Some variance was seen in the ability of a given serum to inhibit different strains of the parasite. The significance of this is unknown. The proteins bound by human sera with high and low in vitro inhibitory capacities were compared by SDS-PAGE. The human sera which did not inhibit parasite growth in vitro well differed from those which did by failing to efficiently bind certain parasite components having molecular weights in the range of 200,000, 70,000-85,000, and 45,000.

Animals↗
PubMed1979

Antigenicity of the infected-erythrocyte and merozoite surfaces in Falciparum malaria.

The antigenicity of altered structures induced by Plasmodium falciparum in the membranes of infected Aotus monkey and human erythrocytes was examined. Antisera were obtained from monkeys made immune to malaria. Bound antibodies were shown to be localized on the knob protrusions of infected erythrocytes of both human and monkey origin and from both in vitro and in vivo infections. Therefore, P. falciparum infection has produced similar antigenic changes in the erythrocyte surfaces of both man and monkey. Uninfected erythrocytes and all knobless-infected erythrocytes bound no antibody from immune sera. Strains of P. falciparum from widely different geographic areas that were cultured in vitro in human erythrocytes induced structures (knobs) which have common antigenicity. Merozoites were agglutinated by cross-linking of their cell coats when incubated with immune sera. The binding of ferritin-labeled antibody was heavy on the coats of both homologous and heterologous strains of the parasite, indicating that the merozoite surfaces of these strains share common antigens.

Animals↗
PubMed1979

Inhibition of the in vitro growth of Plasmodium falciparum. I. The effects of immune serum and purified immunoglobulin from owl monkeys.

Sera from Aotus sp. monkeys (karyotypes II, III, and IV) which were immune to Plasmodium falciparum have been used to inhibit the in vitro growth of this human malaria parasite. Culture conditions used for the assays allowed 50- to 100-fold increases in the number of A+ erythrocytes infected in a 96-hr period in control cultures. Although normal monkey serum did not support growth as well as normal human serum, mixtures of normal monkey and human serum were found that did. Compared to such controls, as little as 3.5% immune monkey serum was found to cause approximately 56% inhibition in 4 days (2 replicative cycles). Purified globulin from immune monkeys inhibited 40% at 2 mg/ml and 75% at 7 mg/ml after a single replicative cycle. These data suggest that serum antibody is likely to play a major role in providing Aotus monkeys with protective immunity to P. falciparum.

Animals↗
PubMed1979

Isolation of stages of the human parasite Plasmodium falciparum from culture and from animal blood.

Procedures for isolation of various forms of the asexual erythrocytic stages of the human parasite Plasmodium falciparum are outlined. The procedures employ the plasma expander Physiogel, which is composed of a chemically modified, partially hydrolysed gelatin dissolved in Ringer's lactate. Based on the observation that parasitized cells which are easily separable by this technique differ appreciably at the ultrastructural level, a mechanism by which separation occurs is proposed.

Animals↗