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Query: UMLS:C0024530 (malaria)
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Monolayers of human erythrocytes (E) infected with Plasmodium falciparum were briefly fixed with 1% glutaraldehyde and air dried. They were then exposed to sera from patients with P. falciparum malaria or from donors immune to this parasite and tested in an indirect immunofluorescence assay (IFA). Parasites in infected E were made visible by counterstaining with ethidium bromide. Immunofluorescence (IF) was restricted to the surface of infected E. No antibody binding was detected unless the E were dried, suggesting that the relevant antigens were not available on the outer layers of the E surface. Staining over large parts of the E surface was seen already when the merozoite penetrated noninfected cells and was strong in E containing early stages of the parasite (rings, trophozoites). It was weak or absent from E containing schizonts. Antibodies in sera from different parts of Africa, Colombia, or Sweden reacted similarly with E infected with a Tanzanian P. falciparum strain kept in culture for many years and with parasitized E freshly drawn from African, Swedish, or Colombian patients. All sera from residents of a holoendemic area (Liberia) were IFA positive. In contrast, some sera from Colombian or Swedish patients with primary infection gave negative results. The results of the IFA and of an enzyme-linked immunosorbent assay in which fixed and dried E were the targets were well-correlated, suggesting that the same antibodies were detected by these assays. The antigens involved in the IFA were susceptible to pronase but not to trypsin or neuraminidase. E surface IF was inhibited by lysates of infected E, merozoite extracts, or soluble antigens present in P. falciparum culture supernatants but not by lysates of normal E or ghost extracts. The inhibitory antigens were heat stable (100 degrees C, 5 min). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting of either antigen-enriched preparations from culture supernatants or merozoite extracts showed that antibodies eluted from monolayers of infected E reacted consistently with a predominant polypeptide of Mr 155,000 and two to four minor polypeptides of lower molecular weights. Metabolic labeling of the parasites with 75Se-methionine indicated that these antigens were parasite derived. We conclude that the antigens involved in these reactions are released from bursting schizonts or merozoites and are deposited in the E membrane in the course of invasion.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Antibodies in malarial sera to parasite antigens in the membrane of erythrocytes infected with early asexual stages of Plasmodium falciparum. 637 12

Protective immune responses against the asexual stages of the human malaria parasite, Plasmodium falciparum, are most probably directed against exposed antigenic determinants on the surface of the free merozoite or the infected red blood cell, and therefore antigens in these locations are candidates for testing as components of a defined molecular vaccine. To facilitate the search for such antigens, we recently developed a method for the expression of P. falciparum proteins in Escherichia coli as fused polypeptides. Many clones producing antigens were detected by screening with immune human sera. We show here that antibodies against the fused polypeptide expressed by one such clone react with a P. falciparum protein that is synthesized late in schizogony and is later present on the surface of the ring-infected erythrocyte. The protein is composed of repeating subunits of 8, 4 and 3 amino acids and is present in all isolates of P. falciparum examined.
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PMID:Immune sera recognize on erythrocytes Plasmodium falciparum antigen composed of repeated amino acid sequences. 638 25

A library of cDNA clones expressing antigens of the asexual blood-stages of Plasmodium falciparum (isolate FCQ27/PNG) was constructed in the bacteriophage vector gamma gt11-Amp3. Clones expressing P. falciparum antigens (as polypeptides fused to beta-galactosidase) were selected by their reactivity in an in situ colony immunoassay with affinity-purified malaria antibodies. A detailed analysis of 78 antigen-positive clones selected from approximately 10,000 recombinant clones has shown them to correspond to many different parasite antigens. cDNA hybridization studies on this array of 78 antigen-positive clones have so far identified 18 families of sibling clones with 22 clones as yet unassigned, the majority of which may represent additional unique sequences. Only about 20% of the clones synthesized abundant amounts of the malaria antigen/beta-galactosidase fused polypeptide but each multi-member family except one was represented by at least one clone producing a fused polypeptide in abundance. Antisera have been raised against cloned malaria antigens by immunizing mice and rabbits with bacterial lysates and purified fused polypeptides, respectively. These antisera have been used to characterize the antigens in P. falciparum that correspond to the various antigen-positive clones. The variety of distinct antigens recognized by these antisera confirms that the clone library contains coding sequences for many different antigens.
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PMID:Plasmodium falciparum complementary DNA clones expressed in Escherichia coli encode many distinct antigens. 639 47

Two rat monoclonal antibodies (both IgG2a isotype and having closely related specificities) and a pool of rhesus immune IgG, all of which inhibit Plasmodium knowlesi merozoite invasion of rhesus erythrocytes, have been studied before and after proteolytic digestion. The F(ab')2 and Fab fragments of both rat monoclonal antibodies show considerably enhanced inhibition of merozoite invasion as compared with the intact IgG. Inhibition by monovalent fragments indicates that these antibodies are not dependent upon merozoite agglutination and may act by blocking merozoite attachment to the specific red cell receptor. The fact that the inhibitory activities of F(ab')2 and Fab are equally enhanced on a weight basis, as compared with IgG, suggests that the removal of Fc may reduce electrostatic repulsion between antibody and merozoite surface, both of which are negatively charged at neutral pH. By contrast, papain digestion of polyclonal IgG derived from an immunised rhesus pool markedly reduces its inhibitory activity. This suggests that much of the inhibition mediated by polyclonal IgG results from merozoite agglutination and that the specificity of the rat inhibitory monoclonal antibodies is poorly represented in the immune pool. The P. knowlesi antigen reactive with the inhibitory monoclonal antibodies is known to be synthesized as a minor 66 kDa polypeptide during the last 1.5 h. of schizont development and is processed to smaller products (44 and 42 kDa) present on the merozoite surface. The present results suggest that this antigen may have particular interest as a vaccine against P. knowlesi malaria.
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PMID:The Fab fragments of monoclonal IgG to a merozoite surface antigen inhibit Plasmodium knowlesi invasion of erythrocytes. 651 92

The histidine-rich protein (HRP) of the avian malaria parasite Plasmodium lophurae contains 70% histidine. It is found in dense cytoplasmic granules and during the erythrocytic cycle it accumulates to represent 10% of the dry weight of the parasite. In the present work the HRP mRNA was studied by in vitro translation and by the use of a polyhistidine oligonucleotide probe. The HRP mRNA contains 2,000-2,100 nucleotides encoding a protein with an apparent molecular weight of 50,000. In addition a HRP of molecular weight 35,000-40,000 is also produced in vitro, probably as a result of proteolytic cleavage of the molecular weight 50,000 polypeptide which corresponds to in vivo labeled and purified HRP. The HRP represents a much larger proportion of the in vitro products synthesized in the homologous cell-free system compared to the rabbit reticulocyte system, and it reflects more closely the pattern of protein synthesis seen in vivo. In addition, HRP mRNA is more abundant in polysomes isolated from young parasites than in polysomes from mature schizonts. These results indicate that the HRP accumulates as a result of amplified translation of its mRNA at certain stages of its erythrocytic cycle.
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PMID:In vitro translation and characterization of a unique histidine-rich protein mRNA in the avian malaria parasite Plasmodium lophurae. 657 48

Sera from patients with Plasmodium falciparum, P. vivax or P. ovale malaria were selected according to their high levels of antibodies against human erythrocyte membranes as measured in a microELISA. The specificity of the anti-erythrocyte antibodies in these sera and two normal sera was investigated by means of an immunoblotting technique in combination with SDS-polyacrylamide gel electrophoresis. All the patients' sera as well as the control sera contained antibodies against several erythrocyte polypeptides. As compared with normal sera, most malaria sera showed elevated levels of antibodies against polypeptides of 80K, 70K, 40K and 28K molecular weights. Two sera reacted strongly against a polypeptide with an electrophoretic mobility similar to the alpha subunit of spectrin. One serum showed strong reaction and several other sera, including normal sera, showed weak reaction against a 45K molecular weight polypeptide corresponding to actin. No pervading differences were seen in the pattern of specificities of the anti-erythrocyte ghost antibodies between sera from patients with P. falciparum, P. vivax or P. ovale infections.
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PMID:Studies on the specificity of anti-erythrocyte antibodies in the serum of patients with malaria. 665 62

Stage-specific protein synthesis by the erythrocytic forms of the malaria parasite Plasmodium falciparum was investigated by pulse labeling synchronous parasite cultures with [35S]methionine at 6-h intervals during a complete 48-h developmental cycle. About 40 labeled parasite proteins could be immunoprecipitated with human immune serum, and most of these were associated with the schizont stage of development. In particular, one schizont protein was a 195,000-mol wt species against which a murine monoclonal antibody was produced. This monoclonal antibody, 89.1 reacted with the parasite membrane in schizonts and also with the surface of free merozoites in the indirect immunofluorescence test. In addition to the 195,000-mol wt protein, antibody 89.1 immunoprecipitated a series of lower-molecular weight polypeptides from extracts of labeled asynchronous P. falciparum parasite cultures. These were shown to be related to the 195,000-mol wt protein by peptide mapping. Pulse-chase labeling of synchronized cultures, and immunoprecipitation with antibody 89.1, showed that specific processing of the 195,000-mol wt polypeptide to the lower-molecular-weight products in concomitant with schizont maturation and merozoite release. It is suggested that this P. falciparum protein may be analogous to a similarly processed 230,000-mol wt protective antigen of the rodent malaria parasite, P. yoelii.
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PMID:Biosynthesis and processing of a Plasmodium falciparum schizont antigen recognized by immune serum and a monoclonal antibody. 675 28

Twenty monoclonal antibodies have been prepared to the erythrocytes from CBA/Ca mice infected with the rodent malaria Plasmodium chabaudi. By immunofluorescence, 15 of these antibodies recognized parasite antigens expressed only during the development of mature trophozoites to schizonts and merozoites, 2 recognized parasite antigens that were expressed throughout most of the intraerythrocytic cycle, and 3 recognized the membranes of all infected and uninfected erythrocytes. By immunoprecipitation of [35S]methionine-labeled, parasitized erythrocytes, parasite antigens recognized by all of the antiparasite antibodies were characterized. Eleven precipitated a 250,000-dalton parasite polypeptide which was synthesized and expressed late in the intraerythrocytic cell cycle and which appeared to be the major coat protein of the merozoites. In passive protection experiments, transfer of hyperimmune serum before infection with the parasite resulted in a delay in the rise of parasitemia, reduction in peak parasitemias, and a delay in the clearance of the parasitemia. Two monoclonal antibodies to the 250,000-dalton polypeptide had a similar but not as marked effect on parasitemia when given as a single dose before infection. When mixed and administered throughout the course of infection, their effects were greater. They had no influence on the course of Plasmodium berghei KSP11 parasitemia. Monoclonal antibodies to other parasite antigens and normal erythrocyte antigens failed to have a significant and reproducible effect on P. chabaudi parasitemia. The results suggest that this 250,000-dalton malaria parasite antigen may be important in the induction and expression of antibody-mediated immunity to malaria.
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PMID:Monoclonal antibodies that protect in vivo against Plasmodium chabaudi recognize a 250,000-dalton parasite polypeptide. 714

The immunosuppressive drug cyclosporin A (CsA) inhibits the growth of malaria parasites in vitro and in vivo. Cyclosporin A exerts its immunosuppressive effect in T lymphocytes by binding to cyclophilin (CyP), a peptidylprolyl cis-trans isomerase (PPIase). It is believed that the cyclosporin/cyclophilin complex inhibits a Ca(2+)-activated protein phosphatase, calcineurin, involved in T-cell activation. A cDNA encoding a cyclophilin of the human malaria parasite Plasmodium falciparum has been isolated as a step in the elucidation of the mechanism of antimalarial action of CsA. This cDNA, termed PfCyP, encodes a protein of 195 amino acids which has highest similarity with the Candida albicans (73.1%) and the Drosophila melanogaster (73.1%) cytoplasmic cyclophilins. A Northern blot reveals an approximately 900-bp nucleotide transcript that is consistent with the predicted size of the encoded polypeptide. The predicted PfCyP protein has a putative endoplasmic-reticulum-directed signal sequence at its N-terminus and two potential N-linked glycosylation sites. Expression of PfCyP RNA in an in vitro translation/translocation system reveals that the PfCyP protein is translocated across microsomes, that the signal peptide is cleaved and that the PfCyP protein is glycosylated at two sites. The PfCyP cDNA open reading frame coding for the predicted mature protein has been expressed in Escherichia coli. The purified recombinant protein is an active PPIase (kcat/Km = 2.3 x 10(6) s-1 M-1); this enzymic activity is inhibited by CsA (IC50 = 10 nM). The PfCyP protein has thus the same sensitivity to CsA as the PPIase activity associated with P. falciparum extracts [Bell, A. et al. (1994) Biochem. Pharmacol. 48, 495-503] suggesting that PfCyP may be responsible for the PPIase activity in those extracts. If different cyclophilins exist in P. falciparum, we conclude that either the PfCyP protein is the major cyclophilin detected in the parasite or that there are other cyclophilins with similar susceptibilities to CsA.
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PMID:Molecular and biochemical characterization of a Plasmodium falciparum cyclophilin containing a cleavable signal sequence. 758 14

The need for an alternative methodology to assess disease activity in the case of malaria led us to evaluate the usefulness of studying the humoral immune response to establish the diagnosis of past or recent malaria. For this purpose, we analyzed sera from 439 individuals living in endemic areas of the Amazon region (Ariquemes, Rondonia). Individuals were classified according to the number and the date of past crises. The enzyme-linked immunosorbent assay (ELISA) was performed to evaluate the IgG/IgM ratio so as to discriminate acute or recent malaria from past infections against crude and defined (SPF70) Plasmodium falciparum asexual blood-stage antigens. We also analyzed the humoral immune response against components presented in crude P. falciparum antigen by the immunoblot technique. Use of the IgG/IgM ratio values did not allow us to differentiate acute from past infections. However, when we analyzed the humoral immune response to parasite components, we were capable of identifying a polypeptide with a molecular weight ranging up to 40 kDa, which was recognized by all parasitized polyinfected individuals studied but not by individuals with negative thick blood smears. In view of these data, we conclude that the 40-kDa polypeptide may represent a powerful tool in the diagnosis of acute malaria, mainly for screening blood donors in endemic areas.
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PMID:Malaria diagnosis: identification of an anti-40-kDa polypeptide antibody response associated with active or recent infection and study of the IgG/IgM ratio of antibodies to blood-stage Plasmodium falciparum antigens. 762 88


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