Retrieving parasite specific liver stage gene products in Plasmodium yoelii infected livers using differential display.

Differential display (DD) has been routinely used to identify genes whose expression pattern is altered by changes in the cellular environment and/or at different stages of development. Most reports utilizing DD contain conventional DD primers that have high guanine and cytosine content and would not be expected to be optimal for Plasmodium which has approximately 30-40% G+C. In an attempt to accommodate the high adenine and thymidine rich genome of Plasmodium yoelii, we utilized PCR primers containing 40, 50 and 60% G+C and modified the existing DD technique. Thus 40% G+C appeared to be the most suitable to amplify Plasmodium genome. Gene specific primers were generated from the sequences of selected DD bands amplified using the 40% G+C primers and were used to verify that the DD clones were of parasite origin by PCR and sequence alignment. Additional data on five of the selected DD clones, designated P2T1L5, P2T1L6, P2T6L11, P2T7L12 and P2T7L13, suggested that all are expressed during the P. yoelii liver stage infection. Interestingly, P2T1L5 is also expressed during the sporozoite stage of the life cycle and both P2T1L6 and P2T6L11 are present as blood stage antigens. The results of this study suggest that DD incorporating primers with low G+C content allows the identification of P. yoelii messages from infected mouse livers.

Purification and characterization of the 3'-nucleotidase/nuclease from promastigotes of Leishmania donovani.

The surface membrane-associated 3'-nucleotidase/nuclease (3'-N'ase) of Leishmania donovani has been purified from detergent extracted promastigotes by anion and cation exchange, lectin affinity and gel filtration chromatography. SDS-PAGE analysis of the purified enzyme preparation revealed a 43-kDa polypeptide as well as faster migrating bands. These bands co-migrated, following both one- and two-dimensional electrophoretic analyses, with enzyme activity as determined by an in situ 3'-nucleotidase gel activity assay. It is suggested that the lower molecular weight species arise during purification as a result of proteolytic cleavage of the intact 43-kDa enzyme. The 3'-N'ase exhibited a pI of 5.4, as revealed by 2-dimensional gel electrophoresis. The glycoprotein nature of the 3'-N'ase was suggested by its binding to concanavalin A and by its electrophoretic shift following incubation with N-glycanaseR. In nucleotidase and nuclease assays, the 3'-N'ase was most active with 3'-AMP and poly(A), respectively. Both nucleotidase and nuclease activities exhibited broad pH optima with peaks at 8.5 and 7.5, respectively. At pH 8.5 nucleotidase activity was inhibited by EDTA, Zn2+ and thiols, but was insensitive to tartrate, molybdate and fluoride ions, commonly used inhibitors of phosphatases. The properties of the leishmanial 3'-N'ase was similar to the 3'-N'ase purified from purine-starved Crithidia luciliae, a related trypanosomatid protozoan, and to group of nucleases from fungi and germinating plant seedlings.

Rapid detection of Leishmania amastigotes in fluid aspirates and biopsies of human tissues.

When using a genus-specific monoclonal antibody (83-J3D2) as the primary reagent in an indirect immunofluorescent antibody assay (IFA), intracellular amastigotes of Leishmania were easily identified in 9 of 9 biopsies and in 11 of 12 needle aspirates taken from human lesions. In contrast, only 5 of the biopsies and 4 of the aspirates yielded promastigotes upon culture in vitro. Similarly, all but 2 of the aspirates and one-half of the biopsies were reported as negative for parasites when stained with Wright's and hematoxylin-eosin, respectively. Serum antibody titers, ranging from 1:8 to 1:128, corroborated the results of the amastigote detection assays when histopathology and isolation were negative. These findings support the practicality of using the genus-specific monoclonal IFA in those field situations where it becomes necessary to differentiate leishmaniasis from other skin infections.

Subcellular and taxonomic specificity of monoclonal antibodies to New World Leishmania.

Murine monoclonal antibodies to flagellar, surface membrane and cytoplasmic antigens of New World Leishmania were assessed for their taxonomic specificity in enzyme-linked immunosorbent assays with three genera of the family Trypanosomatidae and three species and seven subspecies of the genus Leishmania. Antibodies exhibiting exclusive reactivity with either the flagellum, flagellar pocket, kinetoplast, or nucleus lacked specificity at all phylogenetic levels and, in fact, recognized epitopes common to cultured mammalian cells. Monoclonals to intracellular antigens were capable of distinguishing Leishmania from Trypanosoma and Endotrypanum. Antibodies reactive at the surface membrane could separate six isolates of L. braziliensis from three isolates of L. mexicana but the differences in antigen expression were frequently quantitative rather than qualitative. Antigenic variability within species and/or subspecies often exceeded that which was observed between species and/or subspecies. At least one monoclonal antibody was specific for a surface antigen peculiar to a subpopulation of promastigotes of an L. braziliensis panamensis isolate.

Serodiagnosis of New World leishmaniasis by using a genus-specific antigen in enzyme linked immunosorbent assays.

A genus-specific monoclonal antibody (83L-5G9), generated against promastigotes of Leishmania braziliensis panamensis (WRAIR-470), has been used as a ligand in immunochromatography columns for the recovery and purification of the genus-specific antigen. When the purified polypeptide, actually a doublet comprised of a 58 kd and a 31 kd moiety, was used as the antigen in enzyme linked immunosorbent assays (ELISA), it was reactive with 36 of 85 sera from Leishmania patients and with 1 of 49 sera from confirmed cases of Chagas' disease. An additional 25 sera from an unexposed population and five specimens from individuals seropositive for Toxoplasma gondii were nonreactive.

In vitro expression and in vivo immunogenicity of Plasmodium falciparum pre-erythrocytic stage DNA vaccines.

DNA vaccine plasmids were constructed that encoded four pre-erythrocytic antigens from the human malaria parasite Plasmodium falciparum: circumsporozoite protein (PfCSP); sporozoite surface protein 2 (PfSSP2); carboxyl terminus of liver stage antigen 1 (PfLSA-1 c-term); and, exported protein 1 (PfExp-1). Antigen expression was evaluated in vitro by immunoblot analysis of tissue culture cells following transient transfection with each plasmid. Clearly detectable levels of expression depended upon, or were markedly enhanced by, fusion of the antigen encoding sequences in-frame with the initiation complex and peptide leader sequence of human tissue plasminogen activator protein. Mice injected with these plasmids produced antigen specific antibody and cytotoxic T lymphocyte responses. However, the magnitudes of the responses were not always predicted by the in vitro expression assay. The results of this study provided the basis for further testing of these plasmids in primates and the formulation of multi-component pre-erythrocytic DNA vaccines for efficacy testing in human volunteers.

Murine typhus: updated roles of multiple urban components and a second typhuslike rickettsia.

Studies using serologic and polymerase chain reaction-(PCR) facilitated analysis of field samples from southern Texas indicate the presence of Rickettsia typhi and ELB agent infected cat fleas, Ctenocephalides felis (Bouché), and the first observation of ELB infected vertebrates (opossums). The ELB agent is a recently described typhus-like rickettsia that is not distinguished from R. typhi or R. prowazekii by currently available serologic reagents. Restriction digests of PCR products from 399 fleas revealed an ELB agent infection rate of 3.8% and a R. typhi infection rate of 0.8%. Three of nine tested opossums (Didelphis virginiana) were shown to harbor ELB agent infections. No R. typhi infected rats, Rattus norvegicus, or rat-fleas, Xenopsylla cheopis Rothschild, were detected among surveyed samples. The persistence of this murine typhus disease focus appears to be better accounted for by the presence of infected cat fleas, opossums, and other non-rat hosts found in close association with human populations. Involvement of the ELB agent in the biology of murine typhus is suggested by its prevalence among suspected vectors and reservoir hosts.

Detection of Plasmodium yoelii stage mRNA in BALB/c mice.

Relatively little information is available concerning the expression of parasite genes during the liver stage of Plasmodium infection, mostly because of low-level infection of host hepatocytes and the lack of purification techniques for the liver stage parasites. We have determined the optimal dosage of Plasmodium yoelii sporozoite inoculum and routes of inoculation, which are intravenous tail vein and the intrahepatic portal circulation. To determine which route was optimal, BALB/c mice were inoculated via 1 of these routes, and parasitemia was detected by reverse transcription polymerase chain reaction (RT-PCR) detecting both murine beta-actin and P. yoelii-specific 28S ribosomal RNA in the liver samples. Murine beta-actin was detected after 15 cycles of PCR, and its expression levels did not differ between treatment groups. However, P. yoelii-specific 28S ribosomal RNA gene product was detected after 15 cycles of PCR in animals inoculated via the tail vein but was not detected until 25 cycles in animals inoculated via the intrahepatic portal circulation. Experiments were then performed to identify the smallest inoculum required to initiate a liver stage infection that would yield sufficient parasite RNA for analysis. Inoculation with different doses of sporozoite inocula was followed by RT-PCR on the livers of the inoculated animals. The P. yoelii-specific 28S ribosomal RNA gene product was first detected in both treatment groups after 15 cycles, suggesting that both doses of sporozoite inocula provided relatively the same level of liver infection rate. We also have analyzed infected mouse liver for parasite-specific mRNA, which was detectable as early as 24 hr postinfection.

Enhanced mucosal priming by cholera toxin and procholeragenoid with a lipoidal amine adjuvant (avridine) delivered in liposomes.

The mucosal adjuvant activity of avridine, a synthetic lipoidal amine [N,N-dioctadecyl-N',N'-(2-hydroxymethyl) propanediamine, previously designated CP-20,961), was studied in rats immunized intraintestinally with cholera toxin or procholeragenoid. Avridine was most efficient as an adjuvant when incorporated into liposomes; liposomes that lacked avridine had no adjuvant effect. Coadministration of avridine-containing liposomes with enteric priming doses of cholera toxin or procholeragenoid enhanced the efficiency of priming for secondary mucosal anti-cholera toxin responses, i.e., the establishment of memory, five- to sevenfold. Avridine-containing liposomes had no significant effect, however, on either the primary mucosal anti-cholera toxin response, when given with the primary dose of antigen, or on the secondary response, when given with the booster dose to previously primed animals. Little or no adjuvant effect occurred when avridine-containing liposomes were given concurrently with antigen, but at a separate mucosal site or parenterally, or at the site of enteric immunization, but 1 day earlier or later. These results support the notion that adjuvants may be developed which enhance the mucosal immunogenicity of locally applied antigens and suggest that liposomes may be effective vehicles for delivery of such adjuvants.

Leishmania donovani: regulated changes in the level of expression of the surface 3'-nucleotidase/nuclease.

Leishmania donovani promastigotes have previously been shown to possess a surface membrane bound 3'-nucleotidase/nuclease (3'-N'ase) capable of hydrolyzing both nucleic acids and 3'-ribonucleotides. The specific activity of the 3'-N'ase was increased following transfer of the parasites to fresh, nutrient-replete media or to media lacking purines and/or inorganic phosphate (Pi). In nutrient-replete media, the enzyme activity was transiently elevated during the lag and early logarithmic phases of the growth curve; enzyme activity fell as the cells continued into late log and stationary phases. Purine- and Pi-starved cells exhibited significantly greater levels of 3'-N'ase activity than nutrient-replete cells. These levels remained elevated as long as the organisms were maintained in the deficient media. Nutrient-replete and purine-starved 125I surface-labeled parasites displayed differences in electrophoretic patterns. Upon purine starvation, incorporation of radiolabel was increased in proteins which migrated with apparent molecular weights of 70, 43, and 40 kDa. Comigration, in both one- and two-dimensional systems, of 3'-N'ase activity with the radiolabeled 43-kDa band demonstrated that this band was the catalytically active protein. Peptide mapping of the 70-, 43-, and 40-kDa proteins failed to demonstrate similarities in peptide sequence consistent with either a degradation or a precursor/product relationship. Treatment of the 43- and 40-kDa peptides with N-Glycanase indicated that they were differentially glycosylated. The cumulative results of these studies indicated that L. donovani can respond to altered culture conditions by the differential expression of surface proteins. In particular, the differential expression of the protein responsible for 3'-N'ase activity is consistent with the role of this enzyme in purine acquisition.

Oral immunization of dogs with purified cholera toxin, crude cholera toxin, or B subunit: evidence for synergistic protection by antitoxic and antibacterial mechanisms.

The immunogenicity and safety of purified cholera toxin (CT), its B subunit, and a crude culture filtrate of toxigenic Vibrio cholerae (CrT) were compared in dogs immunized orally and challenged with virulent V. cholerae. CT and CrT caused marked protection in two- or three-dose regimens. Protection due to CT occurred only with doses that caused transient, sometimes severe, diarrhea in most dogs; this protection was proportional to the peak antitoxin response in jejunal mucosa and lasted at least 15 weeks. In contrast, minimum protective doses of CrT contained much less cholera toxin, caused very mild diarrhea in only 21% of the dogs, and evoked protection that was greater than predicted from the modest jejunal antitoxin response. B subunit caused smaller jejunal antitoxin responses than did similar doses of CT and was poorly protective, the 50% protective dose being >40-fold greater than that of CT. Two observations indicated that protection due to CrT involved synergy between antibacterial and antitoxic immune responses. First, the 50% protective dose of CrT was 24-fold and >36-fold smaller than the 50% protective doses of its CT and non-CT antigenic components, respectively, when tested separately. Second, protection was greater in CrT-immunized dogs than in CT-immunized dogs for a given mucosal antitoxin response. Low doses of CrT evoked serotype-specific protection, indicating that the serotype-specific O somatic antigen contributed significatly to antibacterial protection. These results suggest that a simple, effective, nonliving oral vaccine for cholera based on combined antibacterial and antitoxic immunity can probably be achieved. However, further studies are needed to determine how a protective antitoxic response can be evoked without causing diarrhea during immunization.

Host responses to Plasmodium yoelii hepatic stages: a paradigm in host-parasite interaction.

The liver stage of malaria, caused by the genus Plasmodium, is clinically silent, but immunologically significant. Ample evidence exists for an effective CD8(+) T cell response to this stage as well as the involvement of gammadeltaT cells and NK1.1(int) cells in immunized animal models. In contrast, there is little information concerning responses in a naive host. Here we report that several host gene expressions in the liver, spleen, and kidney of BALB/c mice are altered during the liver stage of Plasmodium yoelii infection. Really interesting new gene 3 (Ring3), semaphorin subclass 4 member G, glutamylcysteine synthetase, and p45 NF erythroid 2 were all up-regulated 24 h after infection with P. yoelii. Semaphorin subclass 4 member G expression was elevated in the kidney, whereas Ring3 was elevated in both spleen and kidney. The expression of TNF-alpha (TNF-alpha and IFN-gamma) were down-regulated in all three tissues tested except in infected spleen where IFN-gamma was elevated. P. yoelii-related host gene changes were compared with those in Toxoplasma gondii-infected livers. Ring3 expression increased 5-fold over control values, whereas expression of the other transcripts remained unchanged. TNF-alpha and IFN-gamma expressions were increased in the Toxoplasma-infected livers. The uniform increase of Ring3 expression in both Plasmodium- and Toxoplasma-infected livers suggests an innate immune response against parasitic infections, whereas the other gene expression changes are consistent with Plasmodium parasite-specific responses. Taken together, these changes suggest the immune responses to P. yoelii infection are both parasite and organ specific.

Mercury exposure and murine response to Plasmodium yoelii infection and immunization.

Malaria has re-emerged in Amazonia over the past two decades. Many factors have been proposed for this, among them changes in population distribution, failures of vector control and pharmacologic management, and local as well as global environmental changes. Among the latter factors, we have studied the potential role of increasing exposures to the immunotoxic metal mercury, which is widely used in Amazonia for artisanal extraction of alluvial gold deposits. We report here that Hg impairs host resistance to malaria infection at exo-erythrocytic stages. Hg exposed mice have higher parasitemia following infection with sporozoites, but not after transfusion of infected red cells. In mice inoculated with irradiated sporozoites, Hg blocks acquisition of immunity. In addition Hg affects immunologic parameters that are known to be involved in host response to malaria infection. These results have potential implications for the incidence and prevalence of malaria among populations exposed to mercury from artisanal goldmining and consumption of contaminated fish regions with high rates of malaria and other infectious diseases.

Identification and recovery of Leishmania antigen displayed on the surface membrane of mouse peritoneal macrophages infected in vitro.

A murine monoclonal antibody (83L-2D3/a) to a dominant surface antigen of Leishmania braziliensis panamensis (WRAIR-470) recognized a determinant expressed on the surface membrane of mouse peritoneal macrophages infected in vitro. This determinant, also demonstrable on the surface membrane of intracellular amastigotes, was not displayed by the macrophage until at least 6 hr post-infection. This delay in expression and the obvious negativity of all uninfected macrophages inherent to infected cultures implied that the leishmania determinant had an intracellular origin. Furthermore, expression was dependent upon maintenance of macrophage function. When the parasite burden became overwhelming, additional antigen processing ceased, and that which had accumulated was either shed into the medium or was internalized. Immunochemical analyses revealed that the 83L-2D3/a reactive epitope was part of a 15,000 dalton molecule, which in all likelihood represents a breakdown product of a major surface glycoconjugate that had been degraded in the phagolysosome.

Characterization and quantitation of membrane antigens of New World Leishmania species by using monoclonal antibodies in Western blot and flow microfluorometric assays.

Membrane-specific monoclonal antibodies generated against promastigotes of New World Leishmania species were used in Western blot, ELISA, and flow microfluorometric assays to characterize their antigen specificity and to determine the external surface distribution of the reactive epitopes. Three major membrane antigens of molecular weight 72 KD, 55 KD, and 42 KD were identified as well as a dominant antigen that migrated as a broad band on SDS-PAGE, corresponding to a molecular weight of 10-15 KD. By dot-ELISA this antigen was also found to be excreted by promastigotes into their culture medium. One minor membrane antigen of 25 KD and a triplet component of 66, 58, and 56 KD were also identified. While assays performed on air-dried promastigotes revealed the almost ubiquitous presence of the 72 KD and 55 KD antigens, indirect immunofluorescent staining of live promastigotes followed by flow cytometric analysis revealed that these antigens had no external exposure. Antibodies binding the 55 KD component were also reactive toward purified mammalian tubulin. The remaining antigens had a variable distribution on the eight isolates utilized, and these quantitative differences could be used to distinguish isolates of the Leishmania mexicana complex from those belonging to the Leishmania braziliensis complex.

Flow cytometric analysis of the effects exerted by monoclonal antibodies on binding and uptake of Leishmania mexicana subsp. mexicana promastigotes by murine peritoneal macrophages.

A flow cytometric assay was developed to quantitate the binding kinetics of Leishmania mexicana subsp. mexicana promastigotes to murine peritoneal macrophages and to determine if selected membrane-specific monoclonal antibodies would exert an effect on the binding process. A total of three monoclonal antibodies, all reactive with a similar 42-kilodalton surface membrane component by Western blot analysis, enhanced parasite-macrophage binding at levels greater than 45%. An additional three monoclonal antibodies that identified low-molecular-weight antigens of the promastigote (15 to 20 kilodaltons) had no effect on the binding process. Of these antibodies, however, one did appear to inhibit the internalization of the parasites after it attached to the macrophage membrane.

Enhancement by lipid A of mucosal immunogenicity of liposome-associated cholera toxin.

Two methods that might enhance the mucosal immunogenicity of a protein antigen, cholera toxin (CT), were studied in rats: association of CT with liposomes, and coadministration of CT with lipid A. Enteric priming by CT was not enhanced when the antigen was trapped within liposomes or bound to their surface via GM1 ganglioside, nor was it improved when CT was mixed with lipid A or with liposomes containing lipid A. However, lipid A did enhance priming by liposome-associated CT when the lipid A was incorporated into CT-bearing liposomes. It is concluded that lipid A can act as an adjuvant for a local IgA response to a mucosally applied antigen, at least when lipid A and the antigen are associated on a liposome carrier.

Detection of polymerase chain reaction-amplified malarial DNA in infected blood and individual mosquitoes.

Chelex treatment of Plasmodium falciparum and P. berghei infected tissues, in lieu of organic extraction, was followed directly by polymerase chain reaction amplification of primed circumsporozoite gene sequences. The amplified DNA products were detected in stained gels and hybridization blots of extracts from individual infected mosquitoes and dissected mosquito tissues as well as small volumes of infected blood. Parasite development, within the mosquito midgut and salivary gland, was also monitored as a function of time post infectious blood meal. The temporal presence of amplifiable circumsporozoite gene sequences in the infected mosquito midgut lumen, midgut endothelium, and salivary glands corresponded directly to the visual identification of ookinetes, oocysts, and salivary gland sporozoites, respectively.