Giardia lamblia: RNA translation products

The in vitro translation products of two different human isolates of Giardia lamblia, WB 2x and GS/E, were compared in order to determine common protein constituents and to identify proteins recognized by the infected host. Multiple polypeptides ranging from 20 to 185 kDa were synthesized using a rabbit reticulocyte cell-free translation system and although most were identical some differences were noted. GS/E compared to WB 2x showed different polypeptides of 23.5, 24.5, 26.5, 27.5, 32.5, 33.5, and 41 kDa. Some of these polypeptides were antigenic and were immunoprecipitated with anti-isolate antiserum from experimentally infected humans and gerbils. The sera of humans experimentally infected with isolate GS/M recognized a 24-kDa polypeptide from WB 2x and 23.5- and 24.5-kDa polypeptides from GS/E in vitro translation products. Sera from WB 2x- and GS/E-infected gerbils recognized 74- and 24-kDa polypeptides present in WB 2x translation products and 23.5-, 24.5-, 32.5-, 33.5-, and 74-kDa polypeptides when GS/E in vitro translation products were used. These studies identified both unique and common antigens in two different Giardia isolates and they may be of use in the serologic diagnosis of giardiasis and characterization of Giardia isolates.     

Immunoblotting reactivity of human sera from various sources against purified epstein-barr virus

The immunoblotting technique was used to analyse polypeptides of purified Epstein-Barr virus reacting with antibodies present in sera from clinically healthy individuals, from patients with infectious mononucleosis (IM) or AIDS, and from renal transplant recipients with molecular sizes in the range of 40–290 kDa were detected.The 47- and 160-kDa nucleocapsid polypeptides, as well as the 72-, 74-, 140-, 220- and 290-kDa membrane polypeptides were the major viral proteins detected in the sera. Sera from clinically healthy individuals contained antibodies directed against all EBV membrane and nucleocapsid antigens. Sera from renal transplant recipients, from patients with IM and from patients with AIDS failed to react with certain nucleocapsid and membrane antigens; in particular, sera from AIDS patients and renal transplant recipients did not react with the 220-kDa polypeptide, one of the major membrane antigens, while sera from subjects with IM and from healthy individuals did.A high proportion of sera from patients with IM (38% vs 5% of clinically healthy individuals and 0–5% of the AIDS patients and renal transplant recipients) reacted with a 42-kDa polypeptide, suggesting its possible role in acute EBV infection.     

Entamoeba histolytica: purification and characterization of ornithine decarboxylase

Ornithine decarboxylase, a rate-limiting enzyme in polyamine biosynthesis in eukaryotes, was stabilized and purified from trophozoites of the parasite protozoan E. histolytica. Analytical electrophoresis revealed the presence in the purified preparations of a major polypeptide of 45 kDa and barely detectable amounts of two other proteins of 70 and 120 kDa. Both the 45 and 70 kDa polypeptides were recognized by a mouse anti-ODC monoclonal antibody. The major polypeptide exhibited amino terminal sequence homology in the range of 40-73% with ODCs from other organisms. The immunoreactive polypeptide of 70 kDa was not identified. The molecular masses of 216 and 45 kDa determined for the native enzyme by gel filtration and for the major polypeptide by SDS-PAGE, respectively, suggest that the amoeba ODC is a homopentamer. Dialysis against hydroxylamine rendered the enzyme activity fully dependent on pyridoxal 5'-phosphate (PLP). As expected for an oligomeric enzyme, ODC activity exhibited sigmoidal kinetics when it was measured as a function of increasing concentrations of L-ornithine and PLP yielding S(0.5) values of 0.45 and 0.18 mM, respectively. Purified ODC was inhibited by 1,3-diaminopropane and 2,4-diamino-2-butanone but was largely insensitive to inhibition by alpha-difluoromethylornithine (DFMO), indicating that the enzyme may not be a suitable target for this anti-parasitic drug. Other features of the amoeba ODC were common with the enzyme from prokaryotes and eucaryotes.     

Antigenic specificity of human antibody to chlamydia in trachoma and lymphogranuloma venereum

An understanding of the molecular basis of the humoral immune response to chlamydial infections in man requires the identification of target antigens to which antibodies are directed. The antigenic specificity of antibody from patients with lymphogranuloma venereum (LGV) or trachoma was therefore assessed by Western blotting. Surface polypeptides were first identified using purified chlamydial outer membrane complex as antigen. Antibodies in sera from patients with LGV but not from control negative sera reacted with a wide range of chlamydial surface polypeptides with molecular masses of 19, 29, 41, 58, 63 and 65 kDa. The major component of the antibody response detected by both immunoblotting and immunoprecipitation assay was directed against the major outer membrane protein (MOMP). Antibody to MOMP was species-specific on Western blotting, whereas antibody to several other polypeptides recognized common immunodeterminants on polypeptides of C. psittaci Cal-10 of equivalent molecular mass. Immunologically C. psittaci Cal-10 was more closely related to LGV strains of C. trachomatis than a guinea pig inclusion conjunctivitis strain of C. psittaci. Trachoma sera collected from a village in southern Iran showed predominantly type-specific antibody on micro-immunofluorescence to serotype A or B trachoma agents. These sera showed a weak immune response to MOMP, a pronounced response to a polypeptide of 36 kDa and much less widespread reactivity with other chlamydial polypeptides. The lack of an immune response to SDS-stable immunodeterminants on MOMP might contribute to the susceptibility of trachoma patients to repeated cycles of ocular infection with chlamydiae.     

Schistosoma mansoni: identification and characterization of schistosomula polypeptides

Schistosomula proteins separated by a two-dimensional (NEPHGE) gel system identify 94 major silver-stained polypeptides. When compared to polypeptides similarly separated from cercariae and adult worms; cercariae share the same polypeptides as schistosomula, adult worms share ca. 60% of the polypeptides. A group of five schistosomula polypeptides 15-31 kDa (apparent pI 8.2-8.9) was not found in adult worm extracts. To identify which polypeptides were immunogens, Western blots of the NEPHGE gels were probed with sera either from humans with chronic schistosomiasis or from mice vaccinated with irradiated cercariae. For characterization studies, polyclonal antibodies were made against the five schistosomula-specific and selected immunogenic polypeptides by immunizing mice with silver-stained spots removed from NEPHGE gels. We show that the polyclonal serum against a polypeptide of 12.5 kDa and an apparent pI of 6.70 mediated complement and eosinophil-dependent killing of schistosomula in an in vitro assay. Epitopes recognized by antibody against the 12.5-kDa polypeptide show a diffuse distribution and are found on flame cells of the excretory system of the schistosomula.     

Antibody profiles by EITB and ELISA of cattle and sheep infected with Fasciola hepatica

In evaluating potential mechanisms of immunity in fascioliasis we compared the time-course analysis of the antibody responses between a resistant (cattle) and a susceptible model (sheep). Sera from sheep and cows experimentally infected with F. hepatica were reacted with both somatic (FhWWE) and excretory-secretory (ES) antigens in order to evaluate the antibody repertoires in the 2 different hosts. Analysis of these sera by ELISA showed a significant increase in antibody levels by 2 wk in most infected cattle using both somatic and ES antigens, whereas with most infected sheep antibodies are not clearly detected until week 4. By EITB, both infected sheep and cows recognize major somatic polypeptides in a molecular weight range of 30-38 kDa by 8 wk. Cattle recognized 3 additional major antigens of 56, 64, and 69 kDa as early as 6 wk. Various polypeptides of 20-25 kDa are prominently detected by most sheep and very faintly, if at all, by the cow sera. The sera from both sheep and cows also identify ES polypeptides of 20-28 kDa. The patterns of polypeptides recognized by sheep infected with S. mansoni and challenged with F. hepatica in EITB are almost identical to those with a simple F. hepatica primary infection. No significant differences were detected in the antibody kinetics in ELISA between these 2 groups. Differences and similarities between these models could eventually help determine which antibodies may be predictive of resistance or susceptibility in fascioliasis.     

Trypanosoma cruzi: identification of specific epimastigote antigens by human immune sera

Soluble antigens from epimastigotes of Trypanosoma cruzi were analyzed by western blot in terms of their reactivity with sera from patients with Chagas' disease. In addition, sera from patients with visceral (AVL) and tegumental leishmaniasis (ATL) were also tested in order to identify cross-reactivities with Trypanosoma cruzi antigens. Twenty eight polypeptides with molecular weights ranging from 14 kDa to 113 kDa were identified with sera from Chagas' disease patients. An extensive cross-reactivity was observed when sera from human visceral leishmaniasis were used, while only a slight cross-reaction was observed with sera from tegumental leishmaniasis. On the other hand, 10 polypeptides specifically reacting with sera from Chagas' disease patients were identified. Among them, the antigens with molecular weights of 46 kDa and 25 kDa reacted with all sera tested and may be good candidates for specific immunodiagnosis of Chagas' disease.     

Evaluation of immunodiagnostic antigens in the excretory-secretory products of Fasciola hepatica

The metabolic antigens of F. hepatica have been shown to be a source of potential immunodiagnostic antigens. We have fractionated F. hepatica excretory-secretory (ES) antigens by conventional gel filtration and HPLC, analyzed these fractions in PAGE, and evaluated their immunogenicity by ELISA with sera from experimentally infected rabbits to identify potential serodiagnostic antigens for fascioliasis. A fraction enriched in high molecular weight components of ca. 150-160 kDa was found to be very reactive with sera from early fascioliasis. This fraction was successfully adapted to the DOT-ELISA, where titers up to 1:16,000 still appeared visually as positive. Both acute and chronic fascioliasis sera also recognized, in the enzyme-linked immunoelectrotransfer blot technique (EITB), prominent 25-30-kDa polypeptides that have previously been shown to be recognized by infected rabbits, cows, and sheep. We have therefore employed conventional gel filtration and HPLC gel exclusion chromatography as a 1-step procedure to obtain fractions enriched in antigens recognized in early fascioliasis. In addition, these antigens have been successfully applied to a sensitive, visual immunodiagnostic technique that can be easily employed in field studies.     

Entamoeba histolytica: transferrin binding proteins

Entamoeba histolytica trophozoites depend on iron for their growth; thus, they must use some host iron-containing molecules to fulfill this requirement. In this work we report that amoebas are able to utilize human holo-Tf as iron source and to recognize it through transferrin binding proteins. By use of an anti-human transferrin antiserum in an immunoblotting assay, two main polypeptides with apparent molecular masses of 70 and 140 kDa were found in total extract of trophozoites cultured in vitro. However, when a monoclonal anti-human transferrin receptor antibody was used, only one band with molecular mass of 140 kDa was observed. Both the human transferrin and the monoclonal antibody recognized a protein on the amoebic surface, demonstrated by confocal microscopy. Furthermore, the complex transferrin-transferrin binding protein was internalized by an endocytic process and probably dissociated inside the cell. This mechanism could be one manner in which E. histolytica acquires iron from the human host transferrin.     

Shared and non-shared antigens from three different extracts of the metacestode of Echinococcus granulosus

Hydatid cyst fluid (HCF), somatic antigens (S-Ag) and excretory-secretory products (ES-Ag) of Echinococcus granulosus protoscoleces are used as the main antigenic sources for immunodiagnosis of human and dog echinococcosis. In order to determine their non-shared as well as their shared antigenic components, these extracts were studied by ELISA-inhibition and immunoblot-inhibition. Assays were carried out using homologous rabbit polyclonal antisera, human sera from individuals with surgically confirmed hydatidosis, and sera from dogs naturally infected with E. granulosus. High levels of cross-reactivity were observed for all antigenic extracts, but especially for ES-Ag and S-Ag. Canine antibodies evidenced lesser avidity for their specific antigens than antibodies from human origin. The major antigenic components shared by HCF, S-Ag, and ES-Ag have apparent molecular masses of 4-6, 20-24, 52, 80, and 100-104 kDa, including doublets of 41/45, 54/57, and 65/68 kDa. Non-shared polypeptides of each antigenic extract of E. granulosus were identified, having apparent masses of 108 and 78 kDa for HCF, of 124, 94, 83, and 75 kDa for S-Ag, and of 89, 66, 42, 39, 37, and 35 kDa for ES-Ag.     

Characterization of membrane antigens on human cytomegalovirus-infected fibroblasts recognized by human antibodies.

The antigens on the surface of human cytomegalovirus (HCMV)-infected fibroblasts which are recognized by human HCMV antibody-positive sera were characterized. Three HCMV-induced polypeptides, with apparent molecular masses of 53 to 63, 94, and 94 to 120 kilodaltons, were precipitated from 125I-surface-labeled cell extracts with different sera obtained from healthy individuals. Renal transplant recipients who were suffering from active HCMV infections recognized the same set of antigens. By the use of monoclonal antibodies, these antigens were identified as polypeptides belonging to the gcI and gcIII families of HCMV glycoproteins.     

Schistosoma mansoni polypeptides immunogenic in mice vaccinated with radiation-attenuated cercariae

We compared the humoral immune response of mice protected against Schistosoma mansoni by vaccination with radiation-attenuated cercariae to that of patently infected mice, and we identified antigens that elicit a greater, or unique, immune response in the vaccinated mice. These comparisons were based upon radioimmunoprecipitations and immunodepletion of [35S]methionine-labeled schistosomular and adult worm polypeptides, followed by one- and two-dimensional polyacrylamide gel analyses. The humoral responses of patently infected mice and of mice vaccinated once were remarkably similar and were directed against schistosome glycoproteins ranging in molecular size from greater than 300 to less than 10 kDa. Exposing mice to a second vaccination resulted in a marked change in the immune response, to one predominantly directed toward high molecular size glycoproteins. Sequential immunodepletion techniques identified five schistosomular and seven adult worm antigens that showed a greater or unique immunogenicity in vaccinated mice as compared with patently infected mice. These adult worm antigens were purified by preparative sequential immunoaffinity chromatography and used to prepare a polyclonal antiserum, anti-irradiated vaccine. This antiserum bound to the surface of live newly transformed and lung-stage schistosomula, as assessed by immunofluorescence assays, and was reactive with a number of 125I-labeled schistosomular surface polypeptides, including a doublet of 150 kDa that was also recognized by sera of vaccinated mice but not by sera of patently infected mice.     

Molecular and immunochemical analysis of Treponema pallidum

Abstract Molecular analysis of polypeptides and antigens of Treponema pallidum has been used increasingly during the past 5 years in investigation of the immunology, pathogenicity and molecular biology of this organism. Failure to culture the organism has severely limited our knowledge of its constituent polypeptides and antigens, but many profiles of these unknown constituents, revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting techniques have been published. In order to compare meaningfully the results obtained by different groups, we have identified a standard pattern of prominent 'landmark' polypeptides in such gel profiles and where possible have assigned functional identities to them. A preliminary nomenclature for the prominent polypeptides of T. pallidum is proposed. These are: P1, 80 kDa; P2, 60 kDa; P3, 47 kDa, an outer membrane-associated polypeptide; P4, 40 kDa; P5, 37 kDa, the major polypeptide of the axial filament; P6, 34 kDa; and P7, 31.5 kDa.     

Precursors of molecules related to mammalian opioid peptides in brain of a marine worm.

Total mRNA were extracted from brain of Nereis diversicolor (Annelida, Polychaeta) and were translated in vitro or in ovo. The newly synthesized polypeptides were analyzed through electrophoresis of immunoprecipitated products or the Western blotting technique using polyclonal antibodies raised against mammalian dynorphin 1-17 and mammalian alpha-neo-endorphin. Among the products translated in vitro, only one class of polypeptide of 70 kDa was recognized by anti-dynorphin 1-17 antibodies. Furthermore, some in ovo translated products as well as proteins extracted from brain of worms showed identical immunoreactivity. These polypeptides, 60-70 kDa, reacted with anti-dynorphin 1-17 and anti alpha-neo-endorphin antibodies. These results suggest the existence of epitopes common to in ovo and in vitro translated products, to polypeptides extracted from the brain and to some mammalian opioid peptides of the prodynorphin family. We postulate the presence, in the brain of N. diversicolor, of precursors of peptides related to mammalian dynorphin 1-17 and alpha-neo-endorphin. Data reported in this investigation do not allow us to propose or even postulate the presence, in the brain of the worm, of one precursor molecule common to polypeptides related to mammalian dynorphin 1-17 and alpha-neo-endorphin. Furthermore, the Nereis precursor molecules exhibit a clear-cut difference in molecular mass with the mammalian prodynorphin: 70 kDa versus 30 kDa.     

The antibody response in Lyme disease.

We determined the antibody response against the Ixodes dammini spirochete in Lyme disease patients by indirect immunofluorescence and an enzyme-linked immunosorbent assay (ELISA). The specific IgM response became maximal three to six weeks after disease onset, and then declined, although titers sometimes remained elevated during later disease. Specific IgM levels correlated directly with total serum IgM. The specific IgG response, often delayed initially, was nearly always present during neuritis and arthritis, and frequently remained elevated after months of remission. Although results obtained by indirect immunofluorescence and the ELISA were similar, the ELISA was more sensitive and specific. Cross-reactive antibodies from patients with other spirochetal infections were blocked by absorption of sera with Borrelia hermsii, but titers of Lyme disease sera were also decreased. To further characterize the specificity of the humoral immune response against the I. dammini spirochete, 35S-methionine-labeled spirochetal antigens were identified by immunoprecipitation with sera from Lyme arthritis patients. These polypeptides had molecular weights of 62, 60, 47, 37, 22, 18, and 15 kDa, and were not recognized by control sera. We conclude that the ELISA, without absorption, is the best method to assay the humoral immune response in Lyme disease, and we have identified methionine-containing spirochetal polypeptides that may be important in Lyme arthritis.     

Analysis of antigenic specificities of Paragonimus westermani developmental stages using immunoblot technique

Serodiagnosis of parasitic infections is widely used, since parasites or their eggs are not always detected by ordinary methods. The sensitive tests such as ELISA are highly dependent on the purity of antigens used. To solve this problem, many workers have tried to find species-specific components of antigens. The present study was performed to determine the antigenic profile of crude saline extracts of 3, 5, 8 and 12-week old P. westermani worms, which were collected from experimentally infected cats, based on SDS-PAGE and immunoblot technique. The results were as follows: 1. The SDS-PAGE showed at least 30 protein bands ranging from 229 kDa to 10 kDa molecular weight. The protein components of P. westermani changed chronologically during its developmental period. The 229 kDa band was recognized only in 12-week old worms (SEP12). 2. Analysis by ELISA showed a significant increase in antibody levels at 3 weeks in infected cats using crude saline extract antigens (SEP3, SEP5, SEP8, SEP12). 3. By EITB using SEP3 and SEP5, infected cats recognized major protein bands with molecular weight of 60, 35, 28, 25 or 21 kDa at 3-12 weeks of infection, and 3 additional antigens, 19, 13 and 10 kDa, were detected at 8-12 weeks of infections. 4. Using SEP8, 5 antigens, 91, 85, 31, 25 and 21 kDa, were consistently detected by all infected sera tested. In addition, 3 antigens of 19, 13 and 10 kDa were detected at 8-12 weeks of infection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunoblotting analysis of anti-rickettsial antibodies produced in patients of Tsutsugamushi disease

The reactivity of sera from patients of Tsutsugamushi disease (scrub typhus) with the antigenic polypeptides of Rickettsia tsutsugamushi was analyzed by the immunoblotting method. The reactivity varied greatly among the sera of individual patients tested. IgG and IgM antibodies of most patients reacted with the 54-56 kilodalton (54-56K) polypeptide located on the rickettsial surface, suggesting that this polypeptide is a predominant antigen in the infection. Other polypeptides of 60K, 50-52K, 46-47K, 35K, and 21-25K were reactive with some but not all sera. From the reactivity of these polypeptides, it was suggested that the 54-56K polypeptide is both strain-specific and group-specific, the 60K polypeptide is group-specific, and the 35K and 21-25K polypeptides are subgroup-specific. IgG antibodies seem to be more cross-reactive with polypeptides of multiple strains than IgM antibodies and have a tendency of increased cross-reactivity that was observed in the sera obtained at the later stage of illness.     

Demonstration of the humoral immune response of horses to Babesia caballi by western blotting.

Babesia caballi-infected or normal equine erythrocytes were solubilized in sodium dodecyl sulfate (SDS) buffer and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Antigens were allowed to react with sera from horses experimentally or field-infected with B. caballi and with sera from non-infected horses. Major babesial antigens recognized by immune sera had apparent mol. wts of 141, 112, 70, 50, 48, 34, and 30 kDa. The polypeptides at 50 and 48 kDa were recognized earliest and throughout infection, but also weakly by 3/100 equine sera tested negative and 1/33 sera tested false positive by the complement fixation test (CFT) and immunofluorescence antibody test (IFAT). Thus, further characterization and purification of B. caballi antigens are required to identify target antigens for an improved enzyme immuno assay. Until such an assay is available, Western blotting can provide a specific tool for the diagnosis of B. caballi infections, particularly in cases of contradicting CFT and IFAT results.     

Analysis of excretory-secretory and somatic antigens of Gastrothylax crumenifer

The excretory/secretory (ES) metabolic products released by Gastrothylax crumenifer (Trematoda: Digenea) during in vitro incubations and the somatic extract of the adult parasite were analysed using polyacrylamide gel electrophoresis (PAGE). Immunogenicity of ES and somatic extracts were evaluated by immunoblotting and ELISA using sera raised against ES and somatic antigens in rabbits. The electropherograms of ES and somatic extracts have been resolved into 38 and 41 polypeptides, respectively. The apparent molecular weights of these polypeptides range from < 29 to > 205 kDa. A total of 14 polypeptides were found to be common to both of the samples. The analysis of immunoblot results revealed 22 and 27 antigenic polypeptides in ES and somatic extracts respectively. Only 11 and 13 antigenic polypeptides were found specific to ES and tissue extract respectively. The molecular weights of these specific polypeptides were calculated to be < 14.4, 16, 20, 25, 33, 42, 119, 125 and > 205 kDa for metabolic products and < 14.4, 25, 30, 35, 78, 84 and > 205 kDa for the tissue extracts, respectively. Analysis of ELISA results revealed that a dilution of up to 1:3200 of the test sera could react with the ES product. Further, when the ES antigens were allowed to react with antisomatic extracts in hyperimmune sera the titre of IgG increased up to a dilution of 1:12800. The potential importance of these antigens in the immunodiagnosis of amphistomiasis is discussed.     

Characterization of transforming growth factors produced by the insulin-independent teratoma-derived cell line 1246-3A

The 1246-3A cell line is an insulin-independent variant derived from the adipogenic cell line 1246. Data presented in this paper indicate that the 1246-3A cell line releases in its culture medium two types of transforming growth factors, TGF-alpha- and TGF-beta-like polypeptides, and a growth inhibitor. TGF-alpha like polypeptide eluted from Biogel P60 column into two fractions with an apparent molecular weight of 50 kDa and 13 kDa. These high-molecular-weight TGF-alpha-like factors competed with 125I-EGF for binding to epidermal growth factor (EGF) receptors and were specifically immunoprecipitated by incubation with antirat TGF-alpha antibody, not by incubation with anti-EGF antibody. Both fractions promoted anchorage-independent growth of normal rat kidney NRK cells in the absence of EGF and stimulated DNA synthesis in quiescent Balb/c-3T3 cells in a fashion similar to EGF and synthetic TGF-alpha. In addition to secreting TGF-alpha-like polypeptides, 1246-3A cells produce TGF-beta. This polypeptide, eluted from Biogel P60 chromatography with an apparent molecular weight of 25 kDa, promoted anchorage-independent growth of NRK cells in the presence of EGF and was growth inhibitory for Chinese hamster lung fibroblasts CCL 39 cells. Interestingly, another growth inhibitory activity was detected in Biogel P60 fractions and eluted with an apparent molecular weight of between 9.5-11 kDa. This fraction was different from TGF-beta and TGF-alpha as determined by specific radioreceptor competition assays. TGF-alpha and TGF-beta-like polypeptides could represent autocrine growth stimulators for the insulin-independent 1246-3A cells and act in synergy with insulin-related factor (IRF) for an optimal stimulation of 1246-3A cell proliferation in serum-free medium.