Concentration of Plasmodium ovale- and Plasmodium vivax-infected erythrocytes from nonhuman primate blood using Percoll gradients.

Plasmodium vivax and Plasmodium ovale schizont-infected erythrocytes were separated from peripheral blood by centrifugation using discontinuous Percoll (colloidal silica) gradients. Infected Aotus monkey or chimpanzee blood was diluted and placed on a discontinuous gradient containing 30%, 40%, 45%, and 50% Percoll (v/v in media) layers before centrifugation at 1,450 X g. Parasitized erythrocytes were concentrated to greater than 95% schizont-infected cells in two bands that contained an average of one leukocyte per 500 infected cells. Mononuclear cells and trophozoites were isolated in another band and noninfected red cells, ring-infected cells, and granulocytes were pelleted to the bottom. The yield of parasitized erythrocytes ranged from 50% to close to 100% of the estimated number of infected cells in the original whole blood. Use of this Percoll procedure results in a high yield of concentrated parasitized erythrocytes and separation of these cells from host white blood cells.

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.

Identification of malaria species by ELISA in sporozoite and oocyst infected Anopheles from western Kenya.

Enzyme-linked immunosorbent assays (ELISAs) for the circumsporozoite (CS) antigens of Plasmodium falciparum, P. malariae, and P. ovale were used to identify species of sporozoite and oocyst infections detected by dissection in Anopheles gambiae s.1. and An. funestus collected in western Kenya. ELISAs identified 92.5% of 1,113 salivary gland infections; Plasmodium species infections included 79.4% P. falciparum, 3.2% P. malariae, 1.7% P. ovale, and 2 or more Plasmodium species were detected in 15.7% of the Anopheles in which the species of parasite was identified. Identification was more likely with greater numbers of sporozoites observed in dissections, increasing from 65% ELISA positivity in mosquitoes with 1-10 sporozoites in their salivary glands to 96% in mosquitoes with over 1,000 sporozoites. ELISAs detected CS antigen in 66% of 294 Anopheles that by dissection had oocysts but uninfected salivary glands. Of 112 Anopheles with a single species of Plasmodium detected in the salivary glands, 29 (25.9%) had 1 or more additional species detected in the midgut, indicating a high potential for multiple infections. Similar proportions of Plasmodium species were found in An. gambiae s.1. and An. funestus.

Assessment of a synthetic DNA probe for Plasmodium falciparum in African blood specimens.

Synthetic DNA oligomers homologous to 21-base long repetitive sequences of Plasmodium falciparum DNA were labeled with 32P using T4 kinase, and were hybridized with purified DNA and with processed blood samples from Africa. The sequence PFR1, its antiparallel oligomer PFR1R, and PFR1 covalently attached to biotin hybridized similarly to P. falciparum DNA. One-microliter aliquots of blood from Zaire spotted on prewet nylon filters and hybridized with PFR1 gave detectable autoradiogram signals from samples with parasitemias as low as 1,000 parasites/mm3. Blood lysis and protein digestion followed by alkylation allowed dot-blot processing of larger aliquots of blood. After hybridization with PFR 1 and autoradiography, 26 samples were scored positive visually, compared with 34 scored positive by microscopy. The effective sensitivity for processed 10-microliter samples was about 500 parasites/mm3. Signals from hybridized probes were quantitated by liquid scintillation counting and densitometry, and were proportional to the amounts of purified P. falciparum DNA applied to the filter. Autoradiogram signals also were roughly proportional (correlation coefficient, r = 0.77) to the number of parasites/mm3 of blood from field samples as determined by microscopic examination.

T cell-accessory cell interactions that initiate allospecific cytotoxic T lymphocyte responses: existence of both Ia-restricted and Ia-unrestricted cellular interaction pathways.

The specificity of the T-accessory cell interactions that initiate primary allospecific cytotoxic T lymphocyte (CTL) responses were found to be surprisingly diverse and of three distinct major histocompatibility complex (MHC) specificities, involving responder T cell recognition of: a) self-Ia accessory cell determinants, b) allo-Ia accessory cell determinants, or c) allo-K/D accessory cell determinants. Any one of these T-accessory cell interactions was sufficient to initiate allospecific CTL responses. It was observed that when accessory cells did not express foreign class I MHC determinants, primary allospecific CTL responses were invariably initiated by Ia-restricted T-accessory cell interactions. In contrast, it was observed that when accessory cells did express foreign class I MHC determinants, primary allospecific CTL responses could be initiated by Ia-independent T-accessory cell interactions that were specific for allogeneic, but not self, K/D determinants and that did not involve recognition of polymorphic Ia determinants. The MHC specificities of the T-accessory cell interactions that initiate primary allospecific and primary trinitrophenyl (TNP)-self CTL responses were also compared. It was observed that primary allospecific and primary TNP-self CTL responses could be initiated by self-Ia-restricted T-accessory cell interactions, and that in both responses the Ia determinants that the responding T cells recognized as self-specificities on the accessory cell surface were those that their precursors had encountered on radiation-resistant thymic elements in their differentiation environment. In contrast to the initiation of primary TNP-self CTL responses that required the activation by accessory cells of Ia-restricted T helper (TH) cells, allospecific CTL responses could also be initiated by class I-restricted T cells specific for accessory cell K/D determinants. Interestingly, such class I-restricted T cells present in primary responder cell populations were triggered only by recognition of allogeneic, but not self, K/D accessory cell determinants, even when the accessory cells were modified with TNP. Thus, the present study demonstrates that primary allospecific CTL responses, but not TNP-self CTL responses, are initiated by Ia-restricted or Ia-independent cellular interaction pathways. These results raise the possibility that unprimed class I-restricted TH cells that mediate the Ia-independent cellular interaction pathway may predominantly express an allospecific, but not a self + X-specific, receptor repertoire. Possible mechanisms by which these distinct T-accessory cell interactions initiate primary allospecific CTL responses are discuss

Self recognition of accessory cell Ia determinants is required for the in vitro generation of hapten-specific cytotoxic T lymphocyte responses.

The present study has addressed the involvement of Ia determinants in the in vitro generation of antigen-specific cytotoxic T lymphocyte (CTL) responses. It demonstrated that the in vitro generation of TNP-specific CTL responses strictly requires responder T cell recognition of self-Ia determinants expressed by accessory cells, and that this recognition could be specifically inhibited by monoclonal anti-Ia-antibodies. The generation of TNP-specific CTL responses was unaffected by the presence of anti-I-A antibodies or the absence of accessory cells when cultures were performed in the presence of an exogenous source of T helper cell factors, Con A SN. Thus, these results indicate that T helper cell recognition of self-Ia determinants expressed by accessory cells is required for the generation of TNP-specific CTL responses. These results preclude the possibility that accessory cells perform only immunologically nonspecific roles in the generation of hapten-specific CTL, but instead demonstrate that accessory cells function in such responses as Ia-bearing antigen-presenting cells for the activation of self-Ia-specific T cells.

Stage-specific and species-specific antigens of Plasmodium vivax and Plasmodium ovale defined by monoclonal antibodies.

Monoclonal antibodies (MAbs) were produced against the asexual blood stages of Plasmodium vivax and Plasmodium ovale and used to define antigens of plasmodial parasites in an indirect fluorescent antibody assay. The anti-P. vivax MAbs produced two distinct patterns in the indirect fluorescent antibody assay. Four patterns were found with the anti-P. ovale MAbs. Species-specific epitopes were defined for P. vivax and P. ovale; epitopes shared among all four species of human malaria parasites were also defined. Some of the anti-P. vivax MAbs reacted only with mature stages, and others reacted with all asexual stages. No asexual blood-stage specificity could be found with the anti-P. ovale antibodies. Five of the anti-P. vivax MAbs and three of the anti-P. ovale MAbs also reacted with sporozoites.

Differential helper and effector responses of Lyt-2+ T cells to H-2Kb mutant (Kbm) determinants and the appearance of thymic influence on anti-Kbm CTL responsiveness.

The goal of this study was to assess and compare the allorecognition requirements for eliciting Lyt-2+ helper and effector functions from primary T cell populations. By using interleukin 2 (IL 2) secretion as a measure of T helper (Th) function, and cytolytic T lymphocyte (CTL) generation as a measure of effector function, this study compared the responses of Lyt-2+ T cells from wild-type B6 mice against a series of H-2Kb mutant determinants. Although all Kbm determinants stimulated B6 Lyt-2+ T cells to become cytolytic effector cells, the various Kbm determinants differed dramatically in their ability to stimulate Lyt-2+ T cells to function as IL 2-secreting helper cells. For example, in contrast to Kbm1 determinants that stimulated both helper and effector functions, Kbm6 determinants only stimulated B6 Lyt-2+ T cells to become cytolytic and failed to stimulate them to secrete IL 2. The distinct functional responses of Lyt-2+ T cells to Kbm6 determinants was documented by precursor frequency determinations, and was not due to an inability of the Kbm6 molecule to stimulate Lyt-2+ Th cells to secrete IL 2. Rather, it was the specific recognition and response of Lyt-2+ T cells to novel mutant epitopes on the Kbm6 molecule that was defective, such that anti-Kbm6 Lyt-2+ T cells only functioned as CTL effectors and did not function as IL 2-secreting Th cells. The failure of Lyt-2+ anti-Kbm6 T cells to function as IL 2-secreting Th cells was a characteristic of all Lyt-2+ T cell populations examined in which the response to novel mutant epitopes could be distinguished from the response to other epitopes expressed on the Kbm6 molecule. The absence of significant numbers of anti-Kbm6 Th cells in Lyt-2+ T cell populations was examined for its functional consequences on anti-Kbm6 CTL responsiveness. It was found that primary anti-Kbm6 CTL responses could be readily generated in vitro, but unlike responses to most class I alloantigens that can be mediated by Lyt-2+ Th cells, anti-Kbm6 CTL responses were strictly dependent upon self-Ia-restricted L3T4+ Th cells. Because the restriction specificity of L3T4+ Th cells is determined by the thymus, in which their precursors had differentiated, anti-Kbm6 CTL responsiveness, unlike responsiveness to most class I alloantigens, was significantly influenced by the Ia phenotype of the thymus in which the responder cells had differentiated.(ABSTRACT TRUNCATED AT 400 WORDS)

Ultrastructure of the erythrocytic stages of Plasmodium malariae.

This report describes the fine structure of the erythrocytic stages of Plasmodium malariae. Erythrocytic parasites from a naturally acquired human infection and an experimentally infected chimpanzee were morphologically indistinguishable and structurally similar to other primate malarias. New findings included observations of highly structured arrays of merozoite surface coat proteins in the cytoplasm of early schizonts and on the surface of budding merozoites and the presence of knobs in the membranes of Maurer's clefts. Morphological evidence is presented suggesting that proteins are transported between the erythrocyte surface and intracellular parasites via two routes: one associated with Maurer's clefts for transport of membrane-associated knob material and a second associated with caveolae in the host cell membrane for the import or export of host- or parasite-derived substances through the erythrocyte cytoplasm.

Immunization of Aotus monkeys with recombinant proteins of an erythrocyte surface antigen of Plasmodium falciparum.

Recent studies have identified and characterized a ring-infected erythrocyte surface antigen (RESA) of the human malaria parasite Plasmodium falciparum with a relative molecular mass (Mr) of approximately 155,000 (refs 1-7). RESA is localized in the micronemes of merozoites and also the membrane of red cells infected with ring-stage parasites. It is thought to be released through the apical pore from the rhoptry at the time of merozoite invasion. Because antibodies directed against this antigen strongly inhibit parasite growth in vitro, RESA may be useful in developing a vaccine against this parasite Here we describe an immunization trial using Aotus monkeys and Escherichia coli-derived fused polypeptides corresponding to various regions of the RESA molecule. Some monkeys in all test groups, but not in the control group, were protected against overwhelming infection. Strikingly, protection correlated with antibody responses to either of two different repetitive sequences in RESA.