The identification and characterization of antigens that elicit human T cell responses is an important step toward understanding of Leishmania major infection and ultimately in the development of a vaccine. Micropreparative SDS-PAGE followed by electrotransfer to a PVDF membrane and elution of proteins from the PVDF, was used to separate 2 novel proteins from L. major promastigotes, which can induce antibodies of the IgG2a isotype in mice and also are recognized by antisera of recovered human cutaneous leishmaniasis subjects. Fractionation of the crude extract of L. major revealed that all detectable proteins of interest were present within the soluble Leishmania antigens (SLA). Quantitation of these proteins showed that their expression in promastigotes is relatively very low. Considering the molecular weight, immunoreactivity, chromatographic and electrophoretic behavior in reducing and non-reducing conditions, these proteins are probably 2 isoforms of a single protein. A digest of these proteins was resolved on Tricine-SDS-PAGE and immunoreactive fragments were identified by human sera. Two immunoreactive fragments (36.4 and 34.8 kDa) were only generated by endoproteinase Glu-C treatment. These immunoreactive fragments or their parent molecules may be ideal candidates for incorporation in a cocktail vaccine against cutaneous leishmaniasis.
Animals ; Antigens, Protozoan/*chemistry/*isolation & purification ; Blotting, Western ; Chromatography, High Pressure Liquid ; Electrophoresis, Polyacrylamide Gel/methods ; Humans ; Leishmania major/growth & development/*immunology ; Protein Isoforms/chemistry/isolation & purification

Plasmodium vivax, a protozoan malaria parasite of humans, represents a major public health concern in the Republic of Korea (= South Korea). However, little is known about the genetic properties and population structures of the P. vivax isolates circulating in South Korea. This article reviews known polymorphic genetic markers in South Korean isolates of P. vivax and briefly summarizes the current issues surrounding the gene and population structures of this parasite. The critical genetic characteristics of major antigens of the parasite, such as circumsporozoite protein (CSP), merozoite surface protein 1 (MSP-1) and MSP-3, Duffy binding protein (DBP), apical membrane antigen 1 (AMA-1), and GAM-1, are also discussed.
Amino Acid Sequence ; Animals ; Antigens, Protozoan/chemistry/*genetics ; Base Sequence ; Humans ; Malaria, Vivax/*parasitology ; Molecular Sequence Data ; Plasmodium vivax/chemistry/*genetics/isolation & purification ; *Polymorphism, Genetic ; Protozoan Proteins/chemistry/*genetics ; Republic of Korea ; Sequence Alignment

Identification of expressed protein profiles and antigenic determination are some of the most challenging aspects of proteomics. Two-dimensional gel electrophoresis (2-DE) combined with immunoblot analysis were employed to study the N. caninum proteome. Protein sample preparation was carried out by first conducting sonication, followed by adding lysis buffer containing 7M urea plus 2M thiourea to the purified tachyzoites in order to complete disruption. A total of 335 differentially expressed protein spots were detected using pH 4-7 IPG strip (7 cm) that were run in a 56 kVh isoelectric focusing (IEF) system. Of the spots analyzed, 64 were identified as antigenic spots on immunoblot profile. Major antigenic spots appeared at 65 kDa (pI 5.2-5.3), 51 kDa (pI 5.5), 38 kDa (pI 5.1), 33 kDa (pI 4.4), 29 kDa (pI 5.6) and 15.5 kDa (pI 5.0) were observed to be significantly distinct compared to the rest of the antigenic spots. The results indicate that combination of 2-DE and immunoblotting methods were thought as very useful tools in defining both proteins and antigens of N. caninum tachyzoites. Additionally, present 2-DE profiles may be valuable in further proteomic approaches and study of the pathogen.
Animals ; Antibody Formation ; Antigens, Protozoan/*analysis/isolation&purification ; Electrophoresis, Gel, Two-Dimensional/methods ; Electrophoresis, Polyacrylamide Gel ; Epitopes/analysis ; Image Processing, Computer-Assisted ; Immunoblotting/methods ; Isoelectric Focusing ; Neospora/*chemistry/immunology ; Proteome/analysis ; Proteomics ; Protozoan Proteins/*analysis/isolation&purification

In order to develop tools for an early serodiagnosis of Plasmodium falciparum infection, we evaluated the usefulness of P. falciparum liver stage antigen-3 (LSA-3) as a serodiagnostic antigen. A portion of LSA-3 gene was cloned, and its recombinant protein (rLSA-3) was expressed in Escherichia coli and purified by column chromatography. The purified rLSA-3 and 120 test blood/serum samples collected from inhabitants in malaria-endemic areas of Mandalay, Myanmar were used for this study. In microscopic examinations of blood samples, P. falciparum positive rate was 39.1% (47/120) in thin smear trials, and 33.3% (40/120) in thick smear trials. Although the positive rate associated with the rLSA-3 (30.8%) was lower than that of the blood stage antigens (70.8%), rLSA-3 based enzyme-linked immunosorbent assay could detect 12 seropositive cases (10.0%), in which blood stage antigens were not detected. These results indicate that the LSA-3 is a useful antigen for an early serodiagnosis of P. falciparum infection.
Recombinant Proteins/biosynthesis/genetics/*immunology ; Plasmodium vivax/isolation & purification ; Plasmodium falciparum/*immunology ; Molecular Sequence Data ; Malaria, Falciparum/blood/*diagnosis ; Humans ; Genes, Protozoan/genetics/immunology ; Fluorescent Antibody Technique, Direct/methods ; Escherichia coli/genetics ; Enzyme-Linked Immunosorbent Assay/methods ; Early Diagnosis ; DNA, Protozoan/chemistry ; DNA Primers/chemistry ; Cloning, Molecular/methods ; Base Sequence ; Antigens, Protozoan/biosynthesis/chemistry/genetics/*immunology ; Animals ; Amino Acid Sequence

Toxoplasma gondii is a persistent protozoan parasite capable of infecting almost any warm-blooded vertebrates. SAG1 (p30) is the prototypic member of a superfamily of surface antigens called SRS (SAG1-related sequence). It constitutes the most abundant and predominant antigen. In this paper the primary structure of mature SAG1 gene of an Indonesian T. gondii isolate is described and sequence comparison is made with published sequence data of 7 other strains or isolates. Sequence comparison indicated that SAG1 is highly conserved through evolution and despite parasite spreading world-wide. Sequences may be divided into two major families, independent of the strain/isolate geographic origin. Variations were mainly localized at the C-terminal half or domain 2 and some clustered in restricted areas. Sequence comparison allowed us to define the Indonesian isolate as genuine virulent RH strain. A phylogenetic tree of Toxoplasma strains/isolates was constructed based on SAG1.
Amino Acid Sequence ; Animals ; Antigens, Protozoan/chemistry/*genetics ; Base Sequence ; Cloning, Molecular ; DNA, Protozoan/chemistry/genetics ; Goat Diseases/parasitology ; Goats ; Indonesia ; Molecular Sequence Data ; Phylogeny ; Polymerase Chain Reaction ; Protozoan Proteins/chemistry/*genetics ; Sequence Alignment ; Sequence Analysis, DNA ; Toxoplasma/*genetics/*immunology/isolation&purification ; Toxoplasmosis/parasitology ; Zoonoses/parasitology

The present study was designed to investigate polymorphism in Duffy binding protein (DBP) gene of Plasmodium vivax isolates of Korea. Thirty samples were obtained from P. vivax patients in Yonchon-gun, Kyonggi-do in 1998. The PCR products of the samples were subjected to sequencing and hybridization analyses of the regions II and IV of P. vivax DBP gene. Two genotypes, SK-1 and SK-2, were identified on the basis of amino acid substitution and deletion. The genotype of 10 isolates was SK-1 and that of 20 isolates was SK-2. Most of the predicted amino acids in the region II of DBP gene were conserved between the Korean isolates and Belem strain except for 4-5 amino acid substitutions. In the region IV of DBP, a 6-bp insert that was shown in the Sal-1 allele type was found in SK-1, and a 27-bp insert that was shown in the Papua New Guinea allele type was found in SK-2. In conclusion, the present findings suggest that two genotypes of P. vivax coexist in the endemic area of Korea.
Amino Acid Sequence ; Animals ; *Antigens, Protozoan ; Base Sequence ; Carrier Proteins/*analysis/chemistry/*genetics ; DNA, Protozoan/genetics ; Genotype ; Human ; Korea ; Malaria, Vivax/parasitology ; Molecular Sequence Data ; Plasmodium vivax/*genetics/isolation & purification ; Polymerase Chain Reaction ; *Polymorphism (Genetics) ; *Protozoan Proteins ; Receptors, Cell Surface/*analysis/chemistry/*genetics ; Support, Non-U.S. Gov't

The protective effect of the Synadenium carinatum latex lectin (ScLL), and the possibility of using it as an adjuvant in murine model of vaccination against American cutaneous leishmaniasis, were evaluated. BALB/c mice were immunized with the lectin ScLL (10, 50, 100 microgram/animal) separately or in association with the soluble Leishmania amazonensis antigen (SLA). After a challenge infection with 10(6) promastigotes, the injury progression was monitored weekly by measuring the footpad swelling for 10 weeks. ScLL appeared to be capable of conferring partial protection to the animals, being most evident when ScLL was used in concentrations of 50 and 100 microgram/animal. Also the parasite load in the interior of macrophages showed significant reduction (61.7%) when compared to the control group. With regard to the cellular response, ScLL 50 and 100 microgram/animal stimulated the delayed-type hypersensitivity (DTH) reaction significantly (P < 0.05) higher than SLA or SLA plus ScLL 10 weeks after the challenge infection. The detection of high levels of IgG2a and the expression of mRNA cytokines, such as IFN-gamma, IL-12, and TNF-alpha (Th1 profiles), corroborated the protective role of this lectin against cutaneous leishmaniasis. This is the first report of the ScLL effect on leishmaniasis and shows a promising role for ScLL to be explored in other experimental models for treatment of leishmaniasis.
*Adjuvants, Immunologic ; Animals ; Antibodies, Protozoan/immunology ; Antibody Formation ; Antigens, Protozoan/immunology ; Cytokines/genetics/immunology ; Euphorbiaceae/*chemistry ; Hypersensitivity, Delayed/immunology ; Immunization ; Immunoglobulin G/immunology ; Latex/chemistry ; Leishmania/immunology ; Leishmaniasis, Cutaneous/*immunology/pathology ; Mice ; Mice, Inbred BALB C ; Nitric Oxide Synthase Type II/genetics/immunology ; Plant Lectins/*immunology/isolation & purification ; Protozoan Vaccines/immunology/pharmacology ; Skin/pathology

Cancer is the main cause of death in developed countries. However, in underdeveloped countries infections and parasitic diseases are the main causes of death. There are raising scientific evidences indicating that parasitic infections induce antitumor activity against certain types of cancers. In this study, the effects of Toxoplasma gondii and Toxocara canis egg antigens in comparison with Bacillus Calmette Guerin (BCG) (known to have anticancer distinctive) on WEHI-164 fibosarcoma transplanted to BALB/c mice was investigated. Groups of 6 male BALB/c mice injected with T. gondii antigen, BCG, or T. canis egg antigen as case groups and alum alone as control groups. All mice were then challenged with WEHI-164 fibrosarcoma cells. The mice were examined for growth of the solid tumor and the tumor sizes were measured every other day up to 4 wk. The mean tumor area in T. gondii, BCG, or alum alone injected mice in 4 different days of measurements was 25 mm2, 23 mm2, and 186 mm2 respectively. Also the mean tumor area in T. canis injected mice in 4 different days was 25.5 mm2 compared to the control group (alum treated) which was 155 mm2. T. gondii parasites and T. canis egg antigens induced inhibition of the tumor growth in the fibrosarcoma mouse model. We need further study to clarify the mechanisms of anti-cancer effects.
Animals ; Antigens, Helminth/isolation & purification/pharmacology/*therapeutic use ; Antigens, Protozoan/isolation & purification/pharmacology/*therapeutic use ; Antineoplastic Agents/isolation & purification/pharmacology/*therapeutic use ; Chemoprevention/*methods ; Female ; Fibrosarcoma/pathology/*prevention & control ; Male ; Mice ; Mice, Inbred BALB C ; Mycobacterium bovis ; Toxocara canis/*chemistry ; Toxoplasma/*chemistry