Identification of three stage-specific proteinases of Plasmodium falciparum.

We have identified and characterized three stage-specific proteinases of Plasmodium falciparum that are active at neutral pH. We analyzed ring-, trophozoite-, schizont-, and merozoite-stage parasites by gelatin substrate PAGE and characterized the identified proteinases with class-specific proteinase inhibitors. No proteinase activity was detected with rings. Trophozoites had a 28 kD proteinase that was inhibited by inhibitors of cysteine proteinases. Mature schizonts had a 35-40 kD proteinase that also was inhibited by cysteine proteinase inhibitors. Merozoite fractions had a 75 kD proteinase that was inhibited by serine proteinase inhibitors. The stage-specific activity of these proteinases and the correlation between the effects of proteinase inhibitors on the isolated enzymes with the effects of the inhibitors on whole parasites suggest potential critical functions for these proteinases in the life cycle of malaria parasites.

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.

Knob-independent cytoadherence of Plasmodium falciparum to the leukocyte differentiation antigen CD36.

The survival of Plasmodium falciparum-infected erythrocytes is enhanced by the sequestration of mature trophozoites and schizonts from the peripheral circulation. Cytoadherence of infected erythrocytes in vivo is associated with the presence of knobs on the erythrocyte surface, but we and others have shown recently that cytoadherence to C32 melanoma cells may occur in vitro in the absence of knobs. We show here that a knobless clone of P. falciparum adheres to the leukocyte differentiation antigen, CD36, suggesting that binding to CD36 is independent of the presence of knobs on the surface of the infected erythrocyte. This clone showed little cytoadherence to immobilized thrombospondin or to endothelial cells expressing the intercellular adhesion molecule 1. Furthermore, an Mr approximately 300-kD trypsin-sensitive protein doublet was immunoprecipitated from knobless trophozoite-infected erythrocytes. Finding a P. falciparum erythrocyte membrane protein 1 (PfEMP1)-like molecule on these infected erythrocytes is consistent with a role for PfEMP1 in cytoadherence to CD36 and C32 melanoma cells.

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.

Secretion of a malarial histidine-rich protein (Pf HRP II) from Plasmodium falciparum-infected erythrocytes.

Plasmodium falciparum-infected erythrocytes (IRBCs) synthesize several histidine-rich proteins (HRPs) that accumulate high levels of [3H]histidine but very low levels of amino acids such as [3H]isoleucine or [35S]methionine. We prepared a monoclonal antibody which reacts specifically with one of these HRPs (Pf HRP II) and studied the location and synthesis of this protein during the parasite's intracellular growth. With the knob-positive Malayan Camp strain of P. falciparum, the monoclonal antibody identified a multiplet of protein bands with major species at Mr 72,000 and 69,000. Pf HRP II synthesis began with immature parasites (rings) and continued through the trophozoite stage. The Mr 72,000 band of Pf HRP II, but not the faster moving bands of the multiplet, was recovered as a water-soluble protein from the culture supernatant of intact IRBCs. Approximately 50% of the total [3H]histidine radioactivity incorporated into the Mr 72,000 band was extracellular between 2 and 24 h of culture. Immunofluorescence and cryothin-section immunoelectron microscopy localized Pf HRP II to several cell compartments including the parasite cytoplasm, as concentrated "packets" in the host erythrocyte cytoplasm and at the IRBC membrane. Our results provide evidence for an intracellular route of transport for a secreted malarial protein from the parasite through several membranes and the host cell cytoplasm.

A malarial cysteine proteinase is necessary for hemoglobin degradation by Plasmodium falciparum.

To obtain free amino acids for protein synthesis, trophozoite stage malaria parasites feed on the cytoplasm of host erythrocytes and degrade hemoglobin within an acid food vacuole. The food vacuole appears to be analogous to the secondary lysosomes of mammalian cells. To determine the enzymatic mechanism of hemoglobin degradation, we incubated trophozoite-infected erythrocytes with peptide inhibitors of different classes of proteinases. Leupeptin and L-transepoxy-succinyl-leucyl-amido-(4-guanidino)-butane (E-64), two peptide inhibitors of cysteine proteinases, inhibited the proteolysis of globin and caused the accumulation of undegraded erythrocyte cytoplasm in parasite food vacuoles, suggesting that a food vacuole cysteine proteinase is necessary for hemoglobin degradation. Proteinase assays of trophozoites demonstrated cysteine proteinase activity with a pH optimum similar to that of the food vacuole and the substrate specificity of lysosomal cathepsin L. We also identified an Mr 28,000 proteinase that was trophozoite stage-specific and was inhibited by leupeptin and E-64. We conclude that the Mr 28,000 cysteine proteinase has a critical, perhaps rate-limiting, role in hemoglobin degradation within the food vacuole of Plasmodium falciparum. Specific inhibitors of this enzyme might provide new means of antimalarial chemotherapy.

Plasmodium falciparum malaria. An amelanotic melanoma cell line bears receptors for the knob ligand on infected erythrocytes.

Erythrocytes infected with Plasmodium falciparum trophozoites and schizonts are not seen in the peripheral circulation because they attach to venular endothelium via knoblike structures on the infected erythrocyte membrane. We have recently shown that erythrocytes containing P. falciparum trophozoites and schizonts likewise attach to cultured human venous endothelial cells via knobs. In search of a more practical target cell for large scale binding studies designed to characterize and isolate the knob ligand, we tested various normal cells and continuous cell lines for their ability to bind P. falciparum-infected erythrocytes. Of the 18 cell types tested, binding of infected erythrocytes was observed to a human amelanotic melanoma cell line and amnion epithelial cells as well as to human aortic and umbilical vein endothelial cells. 96-100% of amelanotic melanoma cells bound 17+/-4 (+/-1 SEM) infected erythrocytes per positive cell, whereas fewer endothelial cells (4-59%) and amnion epithelial cells (8-19%) were capable of binding 12+/-5 and 4+/-1 infected erythrocytes per positive cell, respectively. Further studies designed to compare the mechanism of binding to the amelanotic melanoma cell line and endothelial cells showed the following results. First, that adhesion of infected erythrocytes to these two cell types was parasite stage-specific in that only erythrocytes containing late ring forms, trophozoites, and schizonts bound. Erythrocytes containing early ring forms, which do not attach to venular endothelium in vivo, did not bind to either cell type. Second, erythrocytes infected with trophozoites and schizonts of P. vivax or a knobless strain of P. falciparum, both of which continue to circulate in vivo, did not bind to either target cell type. Third, transmission electron microscopy showed that infected erythrocytes attached to the amelanotic melanoma cells via knobs. We conclude that cultured human endothelial cells and an amelanotic melanoma cell line share common determinants on their surface and that the mechanism of binding to these two different cell types is similar. The amelanotic melanoma cell line offers a useful substitute for endothelial cells in binding studies requiring large numbers of target cells.

Malignant lymphomas of follicular center cell origin in man. I. Immunologic studies.

Lymphomas with histologic features indicating a follicular center cell (FCC) origin were analyzed from 26 patients of a group of 45 consecutive non-Hodgkin's lymphoma patinets whose tumors were studied for B- and T-cell characteristics. They were compared with benign, reactive lymphoid tissue from 14 patients. Cell suspensions from biopsy material, blood, or bone marrow were examined for surface Ig and for rosette formation with sheep erythrocytes (E rosettes). Of the 26 patients with FCC lymphomas, 22 had 40% or more Ig-bearing cells; all patients with FCC lymphoma tissues had 25% or less E rosette-forming cells. Cells from most FCC lymphomas of the cleaved type had surfac IgM; those from several FCC lymphomas had both IgM and IgD. Cells from lymphomas of noncleaved cell type had surface IgG or IgA. Light-chain analysis showed that cells from FCC lymphomas bore a predominant light-chain type, which indicated their monoclonal nature. Neoplastic cells from several FCC lymphomas synthesized the surface Ig which they bore. Reactive tissues usually contained fewer Ig-bearing and more E rosette-forming cells than FCC lymphomas; the Ig-bearing cells, with one exception, had a polyclonal distribution. Correlation of histologic and immunologic observations indicates that most lymphomas identified as FCC in origin by light micorscopic criteria mark as B cells with the use of immunologic techniques and that FCC lymphomas are the most common type of non-Hodgkin's lymphoma.

Malignant lymphomas of follicular center cell origin in man. II. Ultrastructural and cytochemical studies.

Tissues from malignant lymphomas with both nodular and diffuse growth patterns, thought by light microscopy to be composed of cells of follicular center cell (FCC) origin, Were examined by electron microscopy; the tumor cells were similar to lymphoid cells found in reactive follicular centers. Tumor cells from neoplasms thought to be composed of cleaved FCC often had more pronounced nuclear folding than did cleaved FCC of reactive follicles, whereas cells in tumors of noncleaved FCC type were indistinguishable from their presumed counterparts in reactive follicles. Large cell noeplasms, previously classified as "histiocytic" lymphomas were composed of cells with ultrastructural characteristics of transformed lymphocytes; they showed neither ultrastructural nor cytochemical features of mononuclear phagocytes. These findings support the concept that a major group of lymphomas arises from lymphocytes of follicular centers.

Rectal leishmaniasis in a patient with acquired immunodeficiency syndrome.

A severe rectal lesion due to Leishmania infection is described in an American-born homosexual man with the acquired immunodeficiency syndrome. The infection, which may have been venereally transmitted, responded to treatment with amphotericin B. There was no evidence of visceral leishmaniasis. The contribution of the patient's immunodeficiency to the development of the atypical cutaneous leishmanial lesion is unclear. The case may foretell increasing problems with protozoan infections in AIDS as the epidemic spreads to areas with endemic protozoan diseases.

Plasmodium falciparum: inhibitors of lysosomal cysteine proteinases inhibit a trophozoite proteinase and block parasite development.

Trophozoites of Plasmodium falciparum obtain free amino acids for protein synthesis by degrading host erythrocyte hemoglobin in an acidic food vacuole. We previously reported that leupeptin and L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane (E-64), two inhibitors of the cysteine class of proteinases, blocked hemoglobin degradation in the trophozoite food vacuole, and we identified a 28-kDa trophozoite cysteine proteinase as a potential food vacuole hemoglobinase. We now report that the biochemical properties of the trophozoite cysteine proteinase closely resembled those of the lysosomal cysteine proteinases cathepsin B and cathepsin L. The trophozoite proteinase had a pH optimum of 5.5-6.0, near that of both lysosomal proteinases, and it was efficiently inhibited by highly specific diazomethylketone and fluoromethylketone inhibitors of cathepsin B and cathepsin L. The trophozoite proteinase preferred peptide substrates with arginine adjacent to hydrophobic amino acids, as does cathepsin L. Micromolar concentrations of the fluoromethylketone inhibitor Z-Phe-Ala-Ch2F blocked the degradation of hemoglobin in the trophozoite food vacuole and prevented parasite multiplication. In previous studies much higher concentrations of the inhibitor were not toxic for mice. Our results provide additional evidence that the 28-kDa trophozoite proteinase is a food vacuole hemoglobinase and suggest that specific inhibitors of the enzyme may have potential as antimalarial drugs.

A comparison of knobby (K+) and knobless (K-) parasites from two strains of Plasmodium falciparum.

Erythrocytes infected with Plasmodium falciparum develop knob-like protrusions on their membranes. Knobby (K+) parasites of the FCR-3 (Gambian) strain have been shown to possess a histidine-labelled protein of apparent molecular weight 80 000 which is absent from knobless (K-) variants of the same strain. Here we report similar findings with K+ and K- parasites of another strain, the Malayan Camp strain, and also with cloned K+ and K- parasites of the FCR-3 strain. A histidine-labelled protein unique to the two K+ parasites was identified as a broad band with an apparent molecular weight of 89 000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The presence of this protein in both K+ Malayan Camp parasites and K+ FCR-3 (Gambian) parasites and its absence from K- parasites of both strains is consistent with this protein being a major component of knobs.

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.

Inhibitory effects of histidine analogues on growth and protein synthesis by Plasmodium falciparum in vitro.

The human malaria parasite Plasmodium falciparum synthesizes several proteins that are unusually rich in histidine. We therefore screened histidine analogues for their capacity to inhibit in vitro parasite growth. Analogues were added to cultures of ring-stage parasites, and parasite morphological development was assessed by light microscopy after a 22-hr culture. Inhibition of morphological development was identified as the appearance of condensed or pycnotic parasites rather than mature trophozoites. Inhibition of parasite protein synthesis was assessed by radioactivity counting of [3H] isoleucine incorporated into acid-insoluble products and by sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography of [3H]histidine-labeled malarial proteins. 2-F-L-Histidine and 2-I-D, L-histidine exerted the most pronounced inhibitory effects, the fluoro-analogue being the more effective of the two. At a 0.125 mM concentration, both compounds inhibited parasite growth and 2-F-L-histidine also inhibited protein synthesis. At a 1.0 mM concentration, 2-azido-L-histidine, alpha-methyl-L-histidine and WR 177589A also inhibited P. falciparum growth and protein synthesis. Twenty other histidine analogues, including 5-F-L-histidine and 5-I-L-histidine, showed little or no effect under these conditions. The inhibitory histidine analogues may be of interest for antimalarial chemotherapy if they should prove to have greater effect on P. falciparum protein synthesis than on host protein synthesis.

Rosetting: a new cytoadherence property of malaria-infected erythrocytes.

Plasmodium fragile infection of the toque monkey is a natural host-parasite association in which parasite sequestration occurs as during P. falciparum infection of humans. We have studied parasite sequestration of P. fragile and demonstrated the existence of a new property of cytoadherence of infected erythrocytes, "rosetting," which is defined as the agglutination of uninfected erythrocytes around parasitized erythrocytes. Rosetting in vitro and sequestration in vivo appear simultaneously as the parasite matures. The spleen plays a role in modulating cytoadherence; both sequestration and rosetting, which occur with cloned parasites from spleen-intact animals, are markedly reduced in splenectomized animals infected with parasites derived from the same clone. Sequestration and rosetting can be reversed by immune serum. Protease treatment of infected blood abolishes rosetting; however, if treatment is performed at an early stage of schizogony, rosetting reappears if parasites are allowed to further develop in the absence of protease. These results indicate that with P. fragile in its natural primate host, rosetting and sequestration are related to the presence on the infected erythrocyte surface of a parasite-derived antigenic component, the expression of which is modulated by the spleen.

Characterization of a Cryptosporidium parvum sporozoite glycoprotein.

Polyclonal and monoclonal antibodies directed against Cryptosporidium oocysts or sporozoites were developed to identify and characterize sporozoite pellicle and apical complex antigens. A very large glycoprotein of Cryptosporidium sporozoites was identified by three monoclonal antibodies that also reacted with intracellular merozoites. The glycoprotein was also identified by polyclonal antibodies that were affinity-purified on nitrocellulose-bound recombinant proteins expressed by four lambda gtll genomic clones.

Plasmodium falciparum: D260, an intraerythrocytic parasite protein, is a member of the glutamic acid dipeptide-repeat family of proteins.

Members of a serologically cross-reacting family of proteins including Ag332 and Pf11.1, megadalton proteins of schizont-infected red blood cells, and gametocytes, respectively, and Pf155-RESA, a 155-kDa protein of ring-infected red blood cells, have been reported to share amino acid repeat sequences. These repeats are rich in glutamic acid dipeptides postulated to be involved in generating serologic cross-reactivity. We report the identification and characterization of another member of this cross-reacting family, a 260-kDa glutamic acid-rich intraerythrocytic protein. Human antibodies affinity purified on the 260-kDa region of Western boots of trophozoite proteins of Plasmodium falciparum were used to screen a trophozoite-stage lambda gt11 cDNA library. A 1.8-kb clone was identified and human antibodies were affinity purified on the expressing clone. Using this affinity-purified antibody and the 1.8-kb clone, the corresponding protein, its gene, and its chromosomal location were investigated. The 260-kDa corresponding protein serologically cross-reacts with Pf155-RESA, but is the product of a different gene. The 260-kDa protein is Triton X-100 soluble and is variable in molecular weight in different isolates. Immunoprecipitation of [35S]methionine-labeled infected red blood cells indicates that the protein is synthesized throughout the intraerythrocytic cycle but is most prominent in schizonts. The protein, as has been shown previously, is not immunoprecipitated from 125I surface-labeled infected red blood cells and is thus not PfEMP1, the antigen associated with cytoadherence. Indirect fluorescent antibody studies using fixed infected red blood cells suggest that the protein is localized to the periphery of the intraerythrocytic parasite.

Cytoadherence of Plasmodium falciparum-infected erythrocytes to human melanoma cell lines correlates with surface OKM5 antigen.

OKM5 antigen and thrombospondin are currently under investigation as potential receptors on the surface of human monocytes, endothelial cells, and melanomas responsible for the cytoadherence of Plasmodium falciparum-infected erythrocytes. We have studies the binding capacity of six human melanoma cell lines and related this property to the cytoplasmic and surface expression of the OKM5 antigen and thrombospondin by using indirect immunofluorescence assays on methanol-fixed and nonfixed melanomas. The presence of OKM5 antigen was detectable only in the melanoma lines which bound P. falciparum-infected erythrocytes. Thrombospondin was present in the cytoplasm of all the melanoma lines but was not detectable on the surface of any cells. Our work demonstrates a direct correlation between surface OKM5 antigen and cytoadherence in vitro. While our results do not exclude thrombospondin as a mediator of cytoadherence to endothelial cells in vivo, they showed no correlation between the presence of thrombospondin and the ability of melanoma cell lines to cytoadhere in vitro.

Ultrastructural study of Reed-Sternberg cells. Comparison with transformed lymphocytes and histiocytes.

The ultrastructural features of Reed-Sternberg cells from 17 patients with Hodgkin's disease were compared with those of histiocytes and transformed lymphocytes in both benign and malignant conditions. Transformed lymphocytes and Reed-Sternberg cells appeared to have similar features, including large nuclei with dispersed chromatin, large nucleoli, and great numbers of cytoplasmic polyribosomes. Histiocytes contained abundant cytoplasmic lysosomal granules and microfilaments. These results are indicative of the origin of Reed-Sternberg cells from lymphocytes.

Identification and initial characterization of five Cryptosporidium parvum sporozoite antigen genes.

Cryptosporidium parvum, an Apicomplexan parasite of gastrointestinal epithelial cells, causes severe disease in persons with AIDS and is a common cause of self-limited diarrhea in children, animal handlers, and residents of developing countries. No approved therapy exists; in research studies, however, hyperimmune bovine colostrum raised to Cryptosporidium oocysts and sporozoites has eradicated disease or decreased parasite burden in some AIDS patients. Although the protective antigens recognized by bovine hyperimmune colostrum have not been defined, protective antigens of other Apicomplexan parasites frequently have been associated with two unique structures of invasive forms, the trilaminar pellicle and the apical complex. In order to identify immunogenic Cryptosporidium proteins that may be protective antigens for use as recombinant immunogens in passive and/or active immunotherapy, we screened two genomic DNA expression libraries with polyspecific anti-Cryptosporidium antibodies. We used an approach to cloning apical complex and pellicle protein antigens that succeeded despite the lack of large numbers of organisms that would be necessitated for conventional biochemical approaches requiring organelle or membrane purification. We report here the molecular cloning of five C. parvum genes and the characterization of the cognate sporozoite proteins having molecular masses of greater than 500, 68/95, 45, 23, and 15/35 kDa. The light microscopic immunofluorescence pattern of antibodies recognizing these protein antigens suggest that they are located in the pellicle or apical complex of Cryptosporidium sporozoites.