Falciparum Lysate Research Articles

Inter‐Test Comparison Between Filter Paper Absorbed Blood Eluate and Serum for Malaria Serology by Enzyme Immunoassay: An Operational Feasibility

Antimalarial IgG and IgM were detected by enzyme immunoassay in finger‐stick blood samples collected in capillary tubes and also spotted onto Whatman filter paper. Assay was performed in 92 blood samples obtained from 53 falciparum malaria patients, representing 23 fever cases (malaria negative) and 16 healthy individuals. A simple indirect ELISA was done using Plasmodium falciparum lysate and MSP119 peptide as antigens. Total IgG and IgM contents were also estimated in individual sera and filter paper eluate by single radial immunodiffusion (SRID). Assay results of both serum and filter paper eluates were compared. The sensitivity and specificity of the assays for IgG measurement were comparable between serum and filter paper eluates (P < 0.001), whereas, in case of IgM, detection level was poor in filter paper eluates as observed by ELISA and SRID. The filter paper eluates may serve the purpose of antigen‐specific IgG detection in seroepidemiological surveys.

Spectrophotometric determination of de novo hemozoin/β-hematin formation in an in vitro assay

Formation of hemozoin in the malaria parasite, due to its unique nature, is an attractive molecular target. Several laboratories have been trying to unravel the molecular mechanism of hemozoin biosynthesis within the parasite digestive vacuoles. Use of different assay protocols for in vitro β-hematin (synthetic identical to hemozoin) formation by these laboratories has led to inconsistent and often contradictory findings. Much of the difficulty may be attributed to oligomeric heme aggregates, which may be indistinguishable in some detection approaches if adequate separation of β-hemtin is not achieved. Therefore, there is an urgent need for a widely accepted protocol for in vitro β-hematin formation. We describe here a spectrophotometric assay for in vitro β-hematin formation. The assay has been validated with the Plasmodium falciparum lysate, the parasite lipid extracts, and some commercially available fatty acids, which are known to initiate/catalyze β-hematin formation in vitro. The necessity for multiple wash steps for accurate quantification of de novo hemozoin/β-hematin formation was verified experimentally. It was necessary to wash the pellet, which contains β-hematin and heme aggregates, sequentially with Tris/SDS buffer and alkaline bicarbonate solution for complete removal of monomeric heme and heme aggregates and accurate quantification of β-hematin formed during the assay. The pellets and side products in the supernatant were characterized by infrared spectroscopy. No β-hematin formation occurred in the absence of a catalytic/initiating factor. Based on these findings, a filtration-based assay that uses 96-well microplates, and which has important application in in vitro screening and identification of novel inhibitors of hemozoin formation as potential blood schizontocidal antimalarials, has been developed.

Synthesis of chloroplast galactolipids in apicomplexan parasites.

Monogalactosyldiacylglycerol and digalactosyldiacylglycerol are major chloroplast lipids of algae and land plants and are synthesized within the plastid envelope. Here we report that in Toxoplasma gondii and Plasmodium falciparum lysates, radiolabeled UDP-galactose is incorporated into monogalactosylcerebrosides, monogalactosyldiacylglycerol, and digalactosyldiacylglycerol due to distinct enzymological activities. Furthermore, DGDG is immunologically detected in apicomplexans.

An IRP-like protein from Plasmodium falciparum binds to a mammalian iron-responsive element

AbstractThis study cloned and sequenced the complementary DNA (cDNA) encoding of a putative malarial iron responsive element-binding protein (PfIRPa) and confirmed its identity to the previously identified iron-regulatory protein (IRP)–like cDNA from Plasmodium falciparum. Sequence alignment showed that the plasmodial sequence has 47% identity with human IRP1. Hemoglobin-free lysates obtained from erythrocyte-stage P falciparum contain a protein that binds a consensus mammalian iron-responsive element (IRE), indicating that a protein(s) with iron-regulatory activity was present in the lysates. IRE-binding activity was found to be iron regulated in the electrophoretic mobility shift assays. Western blot analysis showed a 2-fold increase in the level of PfIRPa in the desferrioxamine-treated cultures versus control or iron-supplemented cells. Malarial IRP was detected by anti-PfIRPa antibody in the IRE-protein complex fromP falciparum lysates. Immunofluorescence studies confirmed the presence of PfIRPa in the infected red blood cells. These findings demonstrate that erythrocyte P falciparum contains an iron-regulated IRP that binds a mammalian consensus IRE sequence, raising the possibility that the malaria parasite expresses transcripts that contain IREs and are iron-dependently regulated.

The effects of interleukin-15 on human γδ T cell responses to Plasmodium falciparum in vitro

We observed that the γ δ T cell subset expands when human peripheral blood mononuclear cells (PBMC) from malaria-naive donors are cultured with Plasmodium falciparum lysate in the presence of IL-2 or IL-15, cytokines that utilize two common IL-2 receptor subunits. IL-15 induced the expansion of the γ δ T cell subset at all levels tested, whereas IL-2 was not stimulatory at high levels. Flow cytometric analysis of apoptosis using the TUNEL assay indicated that the percentage and absolute number of γ δ T cells undergoing apoptosis were greater in cultures stimulated with antigen and IL-2 than in cultures stimulated with either antigen and IL-15 or control erythrocyte lysate and IL-2. The ability of IL-15 to enhance γ δ T cell function was also assessed; the results suggest that IL-15 can function with IL-2 to enhance the capacity of γ δ T cells to inhibit parasite replication. Together these data indicate that IL-2 and IL-15, which both bind to IL-2R β and IL-2R γc, enhance γ δ T cell function, but they appear to have different effects on proliferation and survival.

Serum antibody immunoglobulin G of mice convalescent from Plasmodium yoelii infection inhibits growth of Plasmodium falciparum in vitro: blood stage antigens of P. falciparum involved in interspecies cross-reactive inhibition of parasite growth.

We demonstrated that antibodies in the serum of BALB/c mice convalescent from Plasmodium yoelii infection inhibit the in vitro growth of Plasmodium falciparum. Blood stage P. falciparum antigens that cross-react with the convalescent-phase mouse serum antibodies were identified and partially characterized. Convalescent-phase mouse serum immunoglobulin G (IgG) reacted with P. falciparum lysates at up to a 1:15,000 dilution of the immune sera and bound to P. falciparum-parasitized erythrocytes at up to a 1:5,000 dilution of the sera. The cross-reactive moieties of antigens in parasite lysates were resistant to oxidation by periodate but sensitive to trypsinization. About 15 polypeptides (M(r)s of 15,000 to 110,000) of P. falciparum blood stages were recognized by the convalescent-phase mouse anti-P. yoelii sera; many of these antigens were metabolically 35S labeled and specifically immunoprecipitated. Also, virtually all of the cross-reactive antigens were recognized by human malaria-immune sera. The anti-P. yoelii serum antibodies bound, with high affinity, to at least five of the cross-reactive antigens (M(r)s of 107,000, 84,000, 53,000, 36,000, and 30,000). By phase separation in Triton X-114, eight interspecies cross-reactive antigens (M(r)s of 84,000, 76,000, 51,000, 31,000, 29,000, 28,000, 23,000, and 22,000) were found to be integral membrane proteins. Convalescent-phase mouse serum IgG strongly inhibited the differentiation of P. falciparum from schizonts to rings; 75 micrograms of IgG per ml caused 80% inhibition of release of merozoites and their invasion into erythrocytes. On the other hand, the anti-P. yoelii serum antibodies also inhibited intracellular development of P. falciparum from rings to schizonts; 25 micrograms of IgG per ml caused 50% inhibition. Inhibition of P. falciparum growth by anti-P. yoelii serum IgG suggests that some of the interspecies cross-reactive antigens contain important conserved epitopes and induce protective antibodies against P. falciparum.

Molecular analysis of Plasmodium falciparum hexokinase

Hexokinase, a key glycolytic enzyme, is involved in the initial phosphorylation reaction of imported glucose and specific blocking of this activity may therefore arrest the development of malaria parasites. We describe here the cloning of a single copy hexokinase gene of Plasmodium falciparum (PfHK) from cDNA or genomic DNA libraries. The deduced amino acid sequence of PfHK has 26% identity with human hexokinase I and its predicted molecular mass assigns it as an invertebrate type isoenzyme of hexokinase. A single 1.5-kb exon is translated from a 3-kb mRNA in asexual stages of the parasite. In contrast to aldolase and GPI, the gene for this glycolytic enzyme is located on chromosome 8. Poly- and monoclonal antibodies against recombinant PfHK support our cloning results at the protein level as they detect a protein of the predicted size and isoelectric point by Western blotting in parasite protein samples. Moreover, polyclonal rabbit 1gG against recombinant PfHK partially inhibits the hexokinase activity of a P. falciparum lysate which provides direct proof that the gene cloned encodes hexokinase of the parasite.

Preferential expansion of V gamma 9 V delta 2 T cells following stimulation of peripheral blood lymphocytes with extracts of Plasmodium falciparum.

A Plasmodium falciparum schizont lysate has been previously described as being a powerful inducer of proliferation for human peripheral T lymphocytes. In this report we study the phenotype of cycling T cells from unexposed donors and examine how the P. falciparum lysate compares with the conventional T cell mitogen phytohemagglutinin (PHA), a known superantigen staphylococcal enterotoxin B (SEB), and a classical antigen pure protein derivative (PPD). We show that for this lymphoproliferative activity interaction with the MHC class II molecule is required and that in the presence of P. falciparum the great majority of the cycling cells at day 6 are gamma delta T cells, all of them bearing V gamma 9 V delta 2. Our results suggest that P. falciparum induces a T cell proliferative response that resembles a response of human peripheral blood gamma delta T cells to superantigen. This observation is in agreement with the elevated level of peripheral gamma delta lymphocytes observed during and after malaria acute infection.

Antibodies to Human T Lymphotropic Virus Type I in a Population from the Philippines: Evidence for Cross-Reactivity with Plasmodium falciparum

Residents of two areas in the Philippines were tested for human T lymphotropic virus type I (HTLV-I): Napsan, a site endemic for malaria, and a nonmalarious site, Bacolod. By ELISA, 30% of the 1743 Napsan residents were positive. Western blot immunoreactivity with two or more HTLV-I proteins was present in 81% of the ELISA positives; however, there was no reactivity with the env proteins. The frequency of this indeterminate immunoreactivity increased with the malaria antibody titer. None of the 200 Bacolod sera were Western blot immunoreactive. In competitive serologic assays, an HTLV-I lysate blocked the HTLV-I immunoreactivity of the Napsan sera but did not reduce malaria antibody. A Plasmodium falciparum lysate blocked both the HTLV-I and malaria immunoreactivity of the Napsan sera. These results show that indeterminate HTLV-I immunoreactivity can be caused by P. falciparum antibody. Serosurveys conducted in malaria endemic areas should consider this possibility.