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Biomedical subjects

L H Miller

Publications and source records attributed to L H Miller.

At least 181 records · Page 10Linked to original sources
PubMed1986

Localization of protective 143/140 kDa antigens of Plasmodium knowlesi by the use of antibodies and ultracryomicrotomy.

Immune sera from mice immunized with the 143/140 kDa protein have been shown to partially block erythrocyte invasion by P. knowlesi merozoites. Therefore, immunoelectron microscopy utilizing ultracryomicrotomy, antibody to 143/140 kDa protein, and protein A gold particles were used to determine the precise localization of this protein in malarial parasites. Gold particles were not seen associated with young trophozoites but appeared in the parasite cytoplasm as the parasites grew to multi-nucleate schizonts. In presegmenter-schizonts, gold particles were associated with the well-developed endoplasmic reticulum, the parasite plasma membrane, and the parasitophorous vacuole membrane. The surface of merozoites was covered with gold particles. Maurer's clefts, which appeared in Plasmodium infected erythrocytes, were also associated with gold particles. These observations suggest that 143/140 kDa protective malarial proteins may be synthesized in the endoplasmic reticulum of P. knowlesi schizonts before being transported to the surface of the schizonts and merozoites. Shedding of the merozoite surface coat may be responsible for the presence of the 143/140 kDa proteins in the parasitophorous vacuole and Maurer's clefts.

Animals↗
PubMed1985

Sequence of the immunodominant epitope for the surface protein on sporozoites of Plasmodium vivax.

Plasmodium vivax is one of the four malaria parasites that cause disease in humans. The structure of the immunodominant repeating peptide of the circumsporozoite (CS) protein of P. vivax was determined. A fragment of P. vivax DNA that encodes this tandemly repeating epitope was isolated by use of an oligonucleotide probe whose sequence is thought to be conserved in CS protein genes. DNA sequence analysis of the P. vivax clone indicates that the CS repeat is nine amino acids in length (Gly-Asp-Arg-Ala-Asp-Gly-Gln-Pro-Ala). The structure of the repeating region was confirmed with synthetic peptides and monoclonal antibodies directed against P. vivax sporozoites. This information should allow synthesis of a vaccine for P. vivax that is similar to the one being tested for P. falciparum.

Amino Acid Sequence↗
PubMed1985

Expression of Plasmodium falciparum circumsporozoite proteins in Escherichia coli for potential use in a human malaria vaccine.

The circumsporozoite (CS) protein of the human malaria parasite Plasmodium falciparum may be the most promising target for the development of a malaria vaccine. In this study, proteins composed of 16, 32, or 48 tandem copies of a tetrapeptide repeating sequence found in the CS protein were efficiently expressed in the bacterium Escherichia coli. When injected into mice, these recombinant products resulted in the production of high titers of antibodies that reacted with the authentic CS protein on live sporozoites and blocked sporozoite invasion of human hepatoma cells in vitro. These CS protein derivatives are therefore candidates for a human malaria vaccine.

Animals↗
PubMed1985

Wright (a + b-) human erythrocytes and Plasmodium falciparum malaria.

We find Wr(a + b-) erythrocytes of donor M. Fr., which appear to carry a rare glycophorin A variant, to be fully susceptible to invasion by nine isolates of Plasmodium falciparum. Thus we fail to confirm the previous publication on the refractoriness of these erythrocytes. In addition the serum of donor M. Fr., which is known to contain anti-Wrb directed against an epitope located on glycophorin A in close proximity to the erythrocyte membrane, was not found to inhibit P. falciparum invasion of blood group O Rh- red blood cells. Despite this, different lines of evidence still indicate that glycophorin A is one of the receptors for erythrocyte invasion by P. falciparum. The Wrb epitope, however, does not appear to represent a distinct receptor site, which is in contrast to previous suggestions.

ABO Blood-Group System↗
PubMed1985

An in vitro assay for sequestration: binding of Plasmodium falciparum-infected erythrocytes to formalin-fixed endothelial cells and amelanotic melanoma cells.

Erythrocytes infected with Plasmodium falciparum bind specifically to cultured endothelial cells and to a line of amelanotic melanoma cells. We have fixed endothelial cells and amelanotic melanoma cells in various ways and determined whether the fixed cells were still able to bind infected erythrocytes. Only cells fixed with 1.0-2.5% formalin in phosphate-buffered saline continued to bind infected erythrocytes as well as unfixed cells. The mechanism of binding to fixed and unfixed cells appeared to be identical for the following reasons. First, erythrocytes infected by parasite strains that bound to unfixed cells also bound to fixed cells while those that did not bind to unfixed cells did not bind to fixed cells. Second, immune serum that inhibited binding to unfixed cells also inhibited binding to fixed cells. Third, electron microscopy showed that knobs were the points of attachment between infected erythrocytes and both fixed and unfixed melanoma cells. Fixed cells gave reproducible results over at least 2 months. Thus, we have developed a simplified, reproducible assay for measuring binding of P. falciparum-infected erythrocytes to target cells.

Animals↗
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

Structural alteration of the membrane of erythrocytes infected with Babesia bovis.

Babesia bovis, causative agent of cattle babesiosis, induces characteristic alterations on the membrane of infected erythrocytes. Elliptical protrusions measuring about 320 nm in long axis and about 160 nm in short axis appear on the membrane of infected erythrocytes, both in vitro and in vivo. Freeze-fracture demonstrated alignment of intramembrane particles (IMP) along the long axis of both the P and E faces of the protrusions. The number of IMP on the endoplasmic face increases, but the number of IMP on the protoplasmic face of the protrusions is not statistically altered from that of uninfected erythrocytes. In vitro, there are more protrusions per erythrocyte infected with the virulent form (low passage form) of B. bovis than with the avirulent form (high passage form). This suggests that the number of protrusions which appear on the membrane of infected erythrocytes may have a direct relationship to the virulence of the parasites. These protrusions may be attached to the capillary endothelial cells, which causes fatal cerebral babesiosis.

Animals↗
PubMed1985

Immunization of monkeys with a 140 kilodalton merozoite surface protein of Plasmodium knowlesi malaria: appearance of alternate forms of this protein.

The merozoite is the invasive stage of the malaria parasite which is released by rupture of the schizont-infected erythrocyte. A monoclonal antibody against a 140 kilodalton (kDa) merozoite surface antigen of Plasmodium knowlesi was used to characterize and to purify this antigen. It was shown by pulse-chase metabolic labeling of mature schizonts that the 140 kDa merozoite antigen was the processed product of a 143 kDa schizont component, and that processing occurred at the time of erythrocyte rupture. Antiserum, prepared by immunizing a rabbit with the 143/140 kDa antigen purified by immunoaffinity chromatography with the monoclonal antibody, strongly inhibited invasion of erythrocytes in vitro; Fab fragments prepared from purified rabbit IgG were inactive at blocking invasion, suggesting that agglutination of merozoites was the mechanism of invasion inhibition. The purified 143/140 kDa antigen was used in Freund's adjuvant to immunize four rhesus monkeys. Two of the immunized animals developed fulminating infections on challenge with 10(4) schizonts, as did the three control animals. The remaining two immunized animals controlled their infections and developed chronic low-grade parasitemias. The animals which were partially protected were those that had developed anti-143/140 kDa antibodies capable of blocking invasion in vitro. Parasites were isolated from the chronic stage of infection (V5 population) and were compared with the original parasite population used for challenge (P population). Inhibition of invasion, immunofluorescence, and immunoprecipitation with anti-143/140 kDa monoclonal antibody, with immune rabbit, and with monkey sera showed that the 143/140 kDa surface antigen had been replaced by multiple cross-reacting alternate antigenic forms of the molecule in the V population. Thus, specific immune response directed against a purified merozoite surface antigen resulted in the replacement of this antigen by variant or mutant forms.

Animals↗
PubMed1984

Perspectives for malaria vaccination.

The need for vaccines to relieve the current global resurgence of malaria is apparent. Immunity is specific for each species of human malaria and for each stage in the life cycle. Once protective immunogens have been identified for one species, the homologous molecules in other species may lead to protection. The usefulness of a particular immunogen will be determined, in part, by its antigenic diversity in the population and the potential for boosting during natural infection. Successful immunization with malarial antigens may require adjuvants to induce effective, long-lived immunity. If different vaccines become available against each stage in the life cycle, then the composition of a particular vaccine may be tailored for different objectives: protection for short periods (for example, during epidemics and for tourists), decrease in disease and death, and malaria eradication.

Animals↗
PubMed1984

Target antigens in malaria transmission blocking immunity.

Malaria transmission blocking immunity has been found to operate against two distinct phases of development of malaria parasites in the mosquito midgut: (i) against the extracellular gametes and newly fertilized zygotes shortly after ingestion by a mosquito of parasitized blood and (ii) against the zygotes during their subsequent development into ookinetes. Immunity is antibody-mediated and stage-specific. A set of three proteins, synthesized in the gametocytes, expressed on the surface of the gametes and newly fertilized zygotes and subsequently shed during later transformation of the zygotes, has been identified as the target antigens of anti-gamete fertilization blocking antibodies. A single protein, synthesized and expressed on the zygote surface during its development to ookinetes, has been identified as the target of antibodies which block the development of the fertilized parasites in the mosquito. Immunization of human populations against gamete or zygote antigens, while not directly protecting an immunized individual from inflection, would reduce the transfer of malaria within the population. Such immunity, in addition to reducing the overall rate of malaria transmission, would, if combined with a vaccine against the asexual (disease-causing) stages, reduce the chance of selection of parasites that are resistant to the asexual vaccine by preventing their entry into the mosquito population.

Animals↗
PubMed1984

Evolutionary relatedness of Plasmodium species as determined by the structure of DNA.

Malaria parasites can be grouped evolutionarily by analysis of DNA composition and genome arrangement. Those that vary widely with regard to host range, morphology, and biological characteristics fit into only a small number of distinctive groups. The DNA of the human parasite Plasmodium falciparum fits into a group that includes rodent and avian malarias and is unlike the DNA of other primate malaria parasites. The DNA of Plasmodium vivax, which is also a human parasite, fits into a distinctly different group that includes Plasmodium cynomolgi, a parasite of monkeys. The evolutionary lines suggested here appear to be consistent with similarities seen among malaria parasites with regard to gene sequence.

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

Identification of an erythrocyte component carrying the Duffy blood group Fya antigen.

The erythrocyte component carrying the Duffy blood group antigen Fya has been identified as a 35- to 43-kilodalton protein. The protein is degraded by proteases, chymotrypsin, and Pronase, which destroy its antigenicity on intact erythrocytes. Its unusual property of aggregating on being boiled in 5 percent sodium dodecyl sulfate with 5 percent 2-mercaptoethanol distinguishes it from other erythrocyte membrane proteins described to date.

Blood Group Antigens↗
PubMed1984

Processing of a major parasite surface glycoprotein during the ultimate stages of differentiation in Plasmodium knowlesi.

A monoclonal antibody (13C11) was used to investigate the processing of a Plasmodium knowlesi plasma membrane protein during the late stages of schizogony. 13C11 bound to the surface of merozoites, blocked invasion of erythrocytes and immunoprecipitated a 230 kDa glycoprotein from metabolically labelled schizonts. This protein was a major parasite surface component inserted into the membrane of immature schizonts as shown through the study of saponin-freed schizonts which bound 13C11 to their surface (indirect immunofluorescence and immunoelectron microscopy); in addition, the 230 kDa protein on saponin-freed schizonts was susceptible to trypsin treatment. Cleavage of the protein in pulse-chase experiments was followed by immunoprecipitation with 13C11. As schizogony proceeded, the 230 kDa protein was cleaved to 200, 145 and 110 kDa polypeptides. However, this cleavage did not reflect processing but occurred in vitro during detergent extraction and was due to a proteolytic activity which appeared in the parasite during the later stages of schizogony. As schizonts reached maturity and infected erythrocytes lysed, the 230 kDa protein was processed to 75, 57, 50 kDa and 43 kDa polypeptides which were the surface labelled components on purified merozoites immunoprecipitated by 13C11.

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

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

Monoclonal antibody characterization of Plasmodium falciparum antigens.

We characterized a set of eight monoclonal antibodies produced against Plasmodium falciparum. In an indirect fluorescent antibody assay the antibodies produced small dots of fluorescence in schizonts and individual merozoites. This merozoite-associated dot reactivity occurred with 21 different strains of P. falciparum, but there was no reactivity with other human, nonhuman primate, or rodent Plasmodium species. Three of the monoclonal antibodies precipitated proteins of Mr 145,000, 135,000, and 104,000. Five of the monoclonal antibodies precipitated proteins of Mr 78,000, 63,000, 42,000, and 40,000.

Antibodies, Monoclonal↗