RESUMEN

We have previously reported on the cloning of the 14-3-3 protein of Schistosoma mansoni. Here, we evaluate the potential use of this protein as a vaccine candidate against infection by S. mansoni. Sm14-3-3 was expressed and purified either as a free protein or as a fusion protein to SjGST or MBP. Sera from mice infected with S. mansoni recognized both SjGST and 14-3-3, indicating that antibodies against these two proteins are induced in the course of the natural infection. Furthermore, mice immunized with either 14-3-3, GST or 14-3-3-GST, reacted with cercaria lysate. A cellular immune response was also detected, particularly in mice immunized with 14-3-3-GST. With respect to the effect on biological functions, antibodies to 14-3-3 and 14-3-3-GST caused 23-32% complement-mediated cytotoxcity of S. mansoni schistosomula compared to only 10-11% induced by either normal mouse serum, or GST alone. In challenge infection with S. mansoni, immunization with 14-3-3, either as a fusion protein or as a free protein, led to protection ranging from 25-46%, as determined by reduction of adult worm burden, while SjGST alone elicited only 0-8% protection and MBP alone did not elicit any protection.

Asunto(s)

Antígenos Helmínticos/administración & dosificación , Proteínas del Helminto/administración & dosificación , Schistosoma mansoni , Esquistosomiasis/prevención & control , Vacunas/administración & dosificación , Proteínas 14-3-3 , Animales , Anticuerpos Antihelmínticos/sangre , Antígenos Helmínticos/inmunología , Glutatión Transferasa/inmunología , Proteínas del Helminto/inmunología , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes/administración & dosificación , Schistosoma mansoni/inmunología , Schistosoma mansoni/aislamiento & purificación , Esquistosomiasis/inmunología , Esquistosomiasis/parasitología , Tirosina 3-Monooxigenasa/administración & dosificación , Tirosina 3-Monooxigenasa/inmunología , Vacunación , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología

RESUMEN

Synthetic recombinant vaccines are constructs in which a synthetic oligonucleotide coding for a protective epitope is inserted into an adequate gene for expression of the epitope. We report the results obtained using recombinant flagella of Salmonella vaccine strain expressing epitopes of influenza virus or of the parasite Schistosoma mansoni. In the case of influenza virus, three conserved epitopes of the haemagglutinin and the nucleoprotein of the virus inducing B- and T-cell immune response, were expressed and the flagella were used for intranasal immunization without any adjuvant. Both humoral and cellular immune responses specific to the virus induced in mice cross-strain long-term protection against challenge infection. Aged mice were also able to resist infection. For the design of a human influenza vaccine, epitopes recognized by the HLAs prevalent in Caucasian populations were used, and the resulting vaccine was evaluated in human/mouse radiation chimaera in which human PBMC are functionally engrafted. The vaccinated mice demonstrated efficient clearance of the virus after challenge and resistance to lethal infection. In the case of the parasitic disease schistosomiasis, a 14-residue peptide denoted 9B peptide 1 was expressed in the flagella. Intranasal vaccination of mice with this construct, without the use of adjuvant, resulted in 40% protection against challenge infection.

Asunto(s)

Antivirales/química , Péptidos/química , Schistosoma mansoni/inmunología , Vacunas Sintéticas/química , Animales , Antivirales/farmacología , Virus de la Influenza A/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Infecciones por Orthomyxoviridae/prevención & control , Péptidos/farmacología , Salmonella/inmunología , Infecciones por Salmonella/prevención & control , Esquistosomiasis/prevención & control , Vacunas Sintéticas/farmacología

RESUMEN

The 14-3-3 protein is a key player in signal transduction processes in various species. We have previously cloned and expressed the 14-3-3 of Schistosoma mansoni. Using the purified protein we have now raised antibodies against it. A highly specific, affinity-purified antibody preparation was employed for the localization of the 14-3-3 protein in the parasite, by both immunohistochemistry and immunoelectron microscopy. The results demonstrate wide distribution of this protein. It was observed in the female excretory system, the nephridia as well as in the genital systems of both sexes, namely in the vitelline gland of female and in the testis of the male. It is also present in the parenchyma and muscle of both male and female worms. Immunoelectron microscopy demonstrated the presence of immunogold-labelled protein in the tegument, subtegument, muscle, parenchyma and in the female reproductive system, in both the cytoplasm and nucleus of vitelline cells, and oocytes. The possible role of the 14-3-3 protein in the genital organs is discussed.

Asunto(s)

Schistosoma mansoni/metabolismo , Tirosina 3-Monooxigenasa/biosíntesis , Proteínas 14-3-3 , Animales , Anticuerpos Antihelmínticos/biosíntesis , Western Blotting , ADN de Helmintos/genética , Femenino , Regulación de la Expresión Génica , Inmunohistoquímica , Masculino , Microscopía Inmunoelectrónica , Reacción en Cadena de la Polimerasa , Conejos , Schistosoma mansoni/genética , Schistosoma mansoni/ultraestructura , Transducción de Señal/fisiología , Tirosina 3-Monooxigenasa/metabolismo

RESUMEN

A panel of four mimotopes of the epitope recognized by the highly protective monoclonal antibody against Schistosoma mansoni (152-66-9B) was obtained by screening a solid-phase 8mer random peptide library. Three of the four mimotopes (p28, p29 and p30) were efficiently recognized in an in vitro radioimmunoassay by the monoclonal antibody and by sera from infected mice and one (p30) induced in vitro proliferation of primed lymphocytes. When the mimotopes were conjugated to bovine serum albumin (BSA) and the conjugates used to immunize C57BL/6J mice, only the p30-BSA-induced antibodies which were effective at complement-mediated killing of schistosomula. The level of complement-mediated killing obtained with the anti-p30 antibodies was comparable to that seen with serum from mice immunized with the protective 9B-antigen. Furthermore, following challenge infection of mimotope-BSA-immunized mice, a greater than 40% reduction in worm burden was observed in p30-BSA-immunized mice, a level comparable to that seen following immunization with the intact 9B-antigen. These results show that a simple synthetic peptide immunogen comprising an eight-amino acid mimotope of a conformational epitope on the 9B-antigen can induce protective immune responses against S. mansoni that are comparable to those obtained following immunization with the far more complex intact antigen. This mimotope may well represent a potential component of a synthetic peptide vaccine against S. mansoni. The inclusion of other B-cell- and T-cell-stimulating synthetic epitopes in such a vaccine, together with a more appropriate carrier, adjuvant and delivery systems may well result in a level of protection even greater than that seen with the single mimotope.

Asunto(s)

Imitación Molecular/inmunología , Schistosoma mansoni/inmunología , Esquistosomiasis mansoni/prevención & control , Vacunas Sintéticas/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antihelmínticos/biosíntesis , Anticuerpos Monoclonales/inmunología , División Celular/inmunología , Proteínas del Sistema Complemento/inmunología , Citotoxicidad Inmunológica , Inmunización , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Biblioteca de Péptidos , Vacunas Sintéticas/química

RESUMEN

In the cercarial and schistosomal stages of the life cycle of the trematode Schistosoma mansoni, acetylcholinesterase occurs as two principal molecular forms (both globular), present in approximately equal amounts, with sedimentation coefficients of 6.5 S and 8 S. The 6.5 S form is solubilized by bacterial phosphatidylinositol-specific phospholipase C from intact schistosomula. It is thus located on the outer surface of the schistosomal tegument and is most probably analogous to the glycosylphosphatidylinositol-anchored G(2) form of acetylcholinesterase found in the electric organ of Torpedo, on the surface of mammalian erythrocytes, and elsewhere. Both forms are fully solubilized by the non-ionic detergent Triton X-100. Upon passing such a detergent extract over a heparin-Sepharose column, only the 8 S form was retained on the column. The bound acetylcholinesterase could be progressively eluted by increasing the salt concentration, with approx. 0.5-0.6 M NaCl being needed for complete elution. Selective inhibition experiments carried out on live parasites using the covalent acetylcholinesterase inhibitor echothiophate (phospholine), which does not penetrate the tegument, selectively inhibited the 6.5 S form, but not the 8 S form, suggesting an internal location for the latter. Monoclonal antibodies raised against S. mansoni acetylcholinesterase also distinguished between the two forms. Thus monoclonal antibody SA7 bound the 6.5 S form selectively, whereas SA57 recognized the 8 S form. The selective binding of the 8 S form to heparin suggests that, within the parasite, this form may be associated with the extracellular matrix of the musculature.

Asunto(s)

Acetilcolinesterasa/metabolismo , Heparina/metabolismo , Schistosoma mansoni/enzimología , Animales , Anticuerpos Monoclonales/metabolismo , Centrifugación por Gradiente de Densidad , Cromatografía en Gel , Isoenzimas/metabolismo , Lactonas/farmacología , Octoxinol/farmacología , Compuestos Organofosforados/farmacología , Unión Proteica , Sefarosa/análogos & derivados , Solubilidad

RESUMEN

Schistosomiasis is the cause of a chronic debilitating disease which accounts for significant mortality and morbidity every year, especially in tropical and subtropical areas. An epitope derived from the protective surface protein 9B-Ag of Schistosoma mansoni, designated 9B peptide-1, was previously showed to be protective in mice when conjugated to bovine serum albumin and administered subcutaneously in complete Freund's adjuvant. In this work, this protective peptide was expressed in the flagellin of a Salmonella vaccine strain, and the isolated recombinant flagella were used for immunization of mice. Since during the invasion of the parasite into the host the schistosomula migrate first to the lungs, the intranasal route of administration was employed in order to halt the parasite at an early stage of the infection. Such intranasal immunization with this peptide expressed in flagellin, without the addition of adjuvants, resulted in a significant humoral response and also led to protection against challenge infection, manifested as a reduction of the worm burden by an average of 42%.

Asunto(s)

Antígenos Helmínticos/inmunología , Péptidos/inmunología , Schistosoma mansoni/inmunología , Esquistosomiasis mansoni/prevención & control , Vacunas Sintéticas/inmunología , Administración Intranasal , Animales , Anticuerpos Antihelmínticos/sangre , Anticuerpos Antihelmínticos/inmunología , Antígenos Helmínticos/administración & dosificación , Antígenos Helmínticos/genética , Antígenos Helmínticos/metabolismo , Biomphalaria , Modelos Animales de Enfermedad , Flagelina/genética , Flagelina/inmunología , Ratones , Ratones Endogámicos C57BL , Péptidos/administración & dosificación , Péptidos/genética , Péptidos/metabolismo , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Schistosoma mansoni/genética , Esquistosomiasis mansoni/parasitología , Distribución Tisular , Vacunas Conjugadas/administración & dosificación , Vacunas Conjugadas/inmunología , Vacunas Sintéticas/administración & dosificación

RESUMEN

We have previously characterized a stage specific, partially protective protein denoted 9B-antigen. This antigen is of 450 kDa in its native form but upon SDS-PAGE in reducing conditions it exhibits two subunits of 30 kDa and 45 kDa. The 9B-antigen is localized at the surface of schistosomula and persists at the surface of lung schistosomula. The 9B-antigen is also localized in internal organs of a vital function in the parasite such as flame cells and cytoplasmic tubes. Infected individuals or mice vaccinated with irradiated cercariae recognize the 9B-antigen. We have previously shown that when injected with complete Freunds adjuvant, the 9B-antigen can induce 40% protection against challenge infection. In this study we have used a more effective delivery system for this antigen. The 9B-antigen was coupled to proteosomes derived from meningoccocal outer membrane proteins. Vaccination of mice with this complex increased the protection level to 60%. Sera from these vaccinated mice induced high levels of complement mediated cytotoxicity of the parasite. Since the proteosomes are approved for human use, these results are promising towards the development of a vaccine against schistosomiasis.

Asunto(s)

Adyuvantes Inmunológicos/administración & dosificación , Antígenos Helmínticos/inmunología , Cisteína Endopeptidasas/inmunología , Complejos Multienzimáticos/inmunología , Schistosoma mansoni/inmunología , Esquistosomiasis mansoni/prevención & control , Vacunas/inmunología , Animales , Antígenos Helmínticos/administración & dosificación , Antígenos Helmínticos/química , Proteínas Bacterianas/administración & dosificación , Proteínas Bacterianas/inmunología , Enfermedad Crónica , Proteínas del Sistema Complemento/inmunología , Cisteína Endopeptidasas/administración & dosificación , Humanos , Sueros Inmunes/química , Sueros Inmunes/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Microscopía Inmunoelectrónica , Complejos Multienzimáticos/administración & dosificación , Complejo de la Endopetidasa Proteasomal , Schistosoma mansoni/ultraestructura , Esquistosomiasis mansoni/inmunología , Esquistosomiasis mansoni/patología , Vacunas/administración & dosificación , Vacunas/química

RESUMEN

Acetylcholinesterase (AChE) is an enzyme broadly distributed in many species, including parasites. It occurs in multiple molecular forms that differ in their quaternary structure and mode of anchoring to the cell surface. This review summarizes biochemical and immunological investigations carried out in our laboratories on AChE of the helmint, Schistosoma mansoni. AChE appears in S. mansoni in two principal molecular forms, both globular, with sedimentation coefficients of approximately 6.5 and 8 S. On the basis of their substrate specificity and sensitivity to inhibitors, both are "true" acetylcholinesterases. Approximately half of the AChE activity of S. mansoni is located on the outer surface of the parasite, attached to the tegumental membrane via a covalently attached glycosylphosphatidylinositol anchor. The remainder is located within the parasite, mainly associated with muscle tissue. Whereas the internal enzyme is most likely involved in termination of neurotransmission at cholinergic synapses, the role of the surface enzyme remains to be established; there are, however, indications that it is involved in signal transduction. The two forms of AChE differ in their heparin-binding properties, only the internal 8 S form of the AChE being retained on a heparin column. The two forms differ also in their immunological specificity, since they are selectively recognized by different monoclonal antibodies. Polyclonal antibodies raised against S. mansoni AChE purified by affinity chromatography are specific for the parasite AChE, reacting with both molecular forms, but do not recognize AChE from other species. They interact with the surface-localized enzyme on the intact organism, and produce almost total complement-dependent killing of the parasite. S. mansoni AChE is thus demonstrated to be a functional protein, involved in multifaceted activities, which can serve as a suitable candidate for diagnostic purposes, vaccine development, and drug design.

Asunto(s)

Acetilcolinesterasa/química , Isoenzimas/química , Schistosoma mansoni/enzimología , Acetilcolinesterasa/metabolismo , Animales , Isoenzimas/metabolismo , Estructura Secundaria de Proteína , Especificidad por Sustrato

RESUMEN

Two synthetic peptides, corresponding to the N-terminal sequence of the 45-kDa subunit of the protective 9B antigen of Schistosoma mansoni and differing in only one amino acid residue, were synthesized. These peptides were recognized by the protective monoclonal antibody 152-66-9B, as well as by sera of mice and humans infected with schistosomiasis. The peptides were coupled to a protein carrier and used for immunization. One of the peptides, 9B-peptide1, induced in mice significant protection against challenge infection, manifested in a 40 to 50% reduction in worm burden.

Asunto(s)

Antígenos Helmínticos/inmunología , Péptidos/inmunología , Schistosoma mansoni/inmunología , Esquistosomiasis mansoni/prevención & control , Vacunas Sintéticas/inmunología , Animales , Anticuerpos Antihelmínticos/inmunología , Anticuerpos Monoclonales/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Proteínas del Sistema Complemento/inmunología , Humanos , Ratones , Ratones Endogámicos C57BL , Péptidos/síntesis química , Esquistosomiasis mansoni/sangre , Esquistosomiasis mansoni/inmunología

RESUMEN

The Na/K-pump or Na+/K(+)-ATPase (EC 3.6.1.37), couples the hydrolysis of ATP to the active transport of Na+ and K+ ions across the plasma membrane of virtually all animal cells. The relationship between activity of the Na+/K(+)-ATPase and the sensitivity of Schistosoma mansoni to immunological attack has been investigated. It has been observed that ouabain, the specific inhibitor of the pump, via a synergistic effect with specific antibody and complement, affects the average membrane potential causing depolarization and death of complement resistant parasites. Thus, apparently, there is association between the inhibition of the Na/K-pump and the lysis and death of the complement-resistant parasite.

Asunto(s)

Proteínas del Sistema Complemento/inmunología , Schistosoma mansoni/inmunología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Proteínas del Sistema Complemento/farmacología , Inhibidores Enzimáticos/farmacología , Cobayas , Inmunidad Innata , Indicadores y Reactivos/metabolismo , Indicadores y Reactivos/farmacología , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Endogámicos DBA , Compuestos Onio/metabolismo , Compuestos Onio/farmacología , Compuestos Organofosforados/metabolismo , Compuestos Organofosforados/farmacología , Ouabaína/farmacología , Schistosoma mansoni/efectos de los fármacos , Schistosoma mansoni/metabolismo

RESUMEN

Monoclonal antibodies (MAbs) were raised in mice against acetylcholinesterase (AChE, EC 3.1.1.7) of the parasite Schistosoma mansoni. Specific tests were used, in which the hybridoma culture supernatants were screened for MAbs capable of recognizing AChE. The MAbs were characterized by their recognition of different stages of the parasite life cycle, by their binding to epitopes of protein or of carbohydrate, and by their capability of blocking AChE activity of the intact parasites. Furthermore, the MAbs were tested for their cross-reaction with AChE derived from various species. One of the MAbs, termed SA31, showed strong cross-reactivity with invertebrate and vertebrate species, indicating some similarity of cross-reaction between schistosome and mammalian AChE. However, most of the schistosome AChE epitopes are not shared with vertebrate AChE. The specific interaction of three other MAbs with intact schistosomula resulted in a marked complement (C)-dependent cytotoxicity. Specific schistosome AChE epitopes might be suitable candidates for drug design and vaccine preparation.

Asunto(s)

Acetilcolinesterasa/inmunología , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/química , Schistosoma mansoni/enzimología , Schistosoma mansoni/inmunología , Acetilcolinesterasa/química , Animales , Anticuerpos Antihelmínticos/biosíntesis , Anticuerpos Antihelmínticos/química , Anticuerpos Antihelmínticos/toxicidad , Anticuerpos Monoclonales/toxicidad , Especificidad de Anticuerpos , Citotoxicidad Celular Dependiente de Anticuerpos , Sitios de Unión de Anticuerpos , Inhibidores de la Colinesterasa/química , Reacciones Cruzadas , Femenino , Ratones , Ratones Endogámicos , Schistosoma mansoni/crecimiento & desarrollo , Especificidad de la Especie

Asunto(s)

Proteínas del Helminto/genética , Proteínas/genética , ARN de Helminto/genética , ARN Mensajero/genética , Schistosoma mansoni/genética , Tirosina 3-Monooxigenasa , Proteínas 14-3-3 , Secuencia de Aminoácidos , Animales , ADN Complementario/genética , ADN de Helmintos/genética , Regulación del Desarrollo de la Expresión Génica , Genes de Helminto , Datos de Secuencia Molecular , Schistosoma mansoni/crecimiento & desarrollo , Homología de Secuencia de Aminoácido

RESUMEN

Acetylcholinesterase (AChE) is present in all stages of the life-cycle of schistosomes and is located in muscle and on the surface of the parasite. Metrifonate is a drug that inhibits AChE. We compared the AChEs from three schistosome species (Schistosoma mansoni, Schistosoma haematobium and Schistosoma bovis) that have different susceptibilities to metrifonate in vivo. Sensitivities to AChE inhibitors were similar. The subunits of AChE were 110 kDa and 76 kDa and the dominant molecular form of AChE was a G2 form in all three species. This was the major form on the tegument while additional molecular forms were associated with the internal tissues. Differences in relative amounts of AChE activity between these species were found in the adults but not in the schistosomula. At the adult stage the major difference between species lay in the relative amounts of AChE activity in their teguments. S. haematobium teguments carried 20 times and S. bovis 6.9 times the activity present on S. mansoni teguments. These quantitative differences associate with the relative sensitivities of these species to metrifonate.

Asunto(s)

Acetilcolinesterasa/análisis , Schistosoma haematobium/efectos de los fármacos , Schistosoma mansoni/efectos de los fármacos , Schistosoma/efectos de los fármacos , Triclorfón/farmacología , Animales , Autorradiografía , Centrifugación por Gradiente de Densidad , Inhibidores de la Colinesterasa/farmacología , Electroforesis en Gel de Poliacrilamida , Pruebas de Precipitina , Schistosoma/enzimología , Schistosoma haematobium/enzimología , Schistosoma mansoni/enzimología , Ultracentrifugación

RESUMEN

Various autoantibodies to nuclear antigens were detected by ELISA in pooled sera of 15 ICR mice 9 weeks after infection with schistosoma. The binding of this serum with acute infection to cercarial extract was inhibited by prior incubation of the sera with DNA, polynucleotides and cercarial extract. Sera from 9 BALB/c mice with experimentally induced lupus, reacted with cercarial extract. No binding was noted with normal mouse sera (NMS). Similarly, sera of SLE patients (N = 113) reacted with the parasite extracts in a higher incidence (14.2%) compared to NHS (3.7%) (P < 0.05). Immunoblot analysis showed common features to SLE sera reacting with two antigenic "groups". The first are antigens that also were recognized by sera of mice infected with schistosomiasis (60, 85, and 94 kDa cercarial proteins). The second group entailed proteins that bound with SLE sera only (10-18 kDa, 29 kDa and 52 kDa). It can be concluded that antinuclear autoantibodies can be found in sera of acute infected mice with schistosomiasis, while SLE sera may react with 3 schistosomal extracts in a higher incidence than NHS (P < 0.05). These results suggest an autoimmune response in schistosomiasis. It is possible that a parasite infection (as bilharzia) may be one of the "trigger" mechanisms for SLE in a subject with the suitable immunogenetic and hormonal background.

Asunto(s)

Anticuerpos Antinucleares/inmunología , Anticuerpos Antihelmínticos/inmunología , Lupus Eritematoso Sistémico/inmunología , Schistosoma mansoni/inmunología , Esquistosomiasis mansoni/inmunología , Animales , Unión Competitiva , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos ICR

RESUMEN

The enzyme acetylcholinesterase (AChE) is present in the trematode Schistosoma mansoni, which infects humans and causes a severe disease called schistosomiasis or Bilharzia. We have purified this enzyme and raised polyclonal antibodies against it. The specificity of these antibodies against the schistosome enzyme was demonstrated by their capacity to precipitate exclusively AChE activity from cercariae extract and to recognize the 8S molecular form of the parasite's AChE. On the other hand, they did not cross-react at all with AChE from human erythrocytes. By employing immunogold electron microscopy, AChE was located on the surface, in the membranal bodies of the tegument and in the muscles of schistosomula. The antibodies raised against the purified AChE of S. mansoni are of protective value, as they led to efficient complement-mediated killing of schistosomula in vitro. It was also demonstrated that antibodies specific towards S. mansoni AChE are present in the sera of mice and of human patients infected with the parasite, suggesting that this enzyme partakes in the immune response towards the parasite during infection. These cumulative data, particularly the schistosomicidal activity of the antibodies and their lack of cross-reactivity with human AChE, are of significance in the consideration of the S. mansoni AChE for vaccination purposes.

Asunto(s)

Acetilcolinesterasa/aislamiento & purificación , Schistosoma mansoni/enzimología , Acetilcolinesterasa/inmunología , Acetilcolinesterasa/metabolismo , Animales , Anticuerpos Antihelmínticos/inmunología , Anticuerpos Antihelmínticos/toxicidad , Biomphalaria , Reacciones Cruzadas , Femenino , Humanos , Ratones , Conejos , Schistosoma mansoni/inmunología , Schistosoma mansoni/ultraestructura , Caracoles , Especificidad de la Especie

RESUMEN

We have previously shown that two ectoenzymes, acetylcholinesterase (AChE) and alkaline phosphatase, are released from the surface and from particulate fractions of the parasite Schistosoma mansoni, by a phosphatidylinositol-specific phospholipase C (PtdIns-PLC) of bacterial origin. Exposure to PtdIns-PLC not only removes large amounts of AChE from the surface of intact, viable Schistosoma in culture, but is accompanied by a concomitant increase in overall levels of AChE in the parasite. The same phenomenon is observed with PtdIns-PLC from two different bacterial sources; Staphylococcus aureus and Bacillus thuringiensis. The increase in AChE levels may be ascribed to de novo synthesis since exposure to PtdIns-PLC, in the presence of the protein-synthesis inhibitor cycloheximide, totally blocked the increase in AChE activity. Furthermore, PtdIns-PLC induced an increased incorporation of [35S]methionine into the AChE immunoprecipitated by a specific anti-AChE serum. This increase is selective for AChE, since total protein synthesis remained almost unchanged after PtdIns-PLC addition, and little or no effect was observed on the enzymatic activity of alkaline phosphatase, which is also glycophosphatidylinositol anchored. Since cleavage of the phosphatidylinositol anchor by PtdIns-PLC should liberate diacylglycerol, which may act as second messenger, we investigated the effect of exogenous diacylglycerols on the synthesis of AChE in S. mansoni. Three different diacylglycerols were tested as possible inducers of AChE activity in the parasite. Both 1-oleoyl-2-acetyl-sn-glycerol and 1,2-dimyristoyl-sn-glycerol were able to increase AChE activity by 35-40% at concentrations of 25 micrograms/ml. A higher concentration of 1,2-dioctanoyl-sn-glycerol (70 micrograms/ml) was needed to produce an equivalent effect. Moreover, addition of phorbol-12-myristate-13-acetate, together with the calcium ionophore A23187, produced a similar increase in AChE activity. Finally, polymixin B, a specific inhibitor of protein kinase C, partially blocked the increase in AChE activity induced by PtdIns-PLC. Our results suggest the involvement of glycophosphatidyl membrane-anchor breakdown products as putative second messengers in the parasite S. mansoni.

Asunto(s)

Acetilcolinesterasa/biosíntesis , Diglicéridos/farmacología , Hidrolasas Diéster Fosfóricas/farmacología , Schistosoma mansoni/enzimología , Animales , Bacillus thuringiensis/enzimología , Biomphalaria , Calcimicina/farmacología , Cicloheximida/farmacología , Diglicéridos/metabolismo , Femenino , Cinética , Metionina/metabolismo , Ratones , Ratones Endogámicos , Fosfatidilinositol Diacilglicerol-Liasa , Fosfoinositido Fosfolipasa C , Polimixina B/farmacología , Staphylococcus aureus/enzimología , Relación Estructura-Actividad , Radioisótopos de Azufre , Acetato de Tetradecanoilforbol/farmacología

RESUMEN

It was previously shown that an antigen preparation termed 9B obtained from Schistosoma mansoni cercarial extracts partially (34%) protects mice from challenge infection with cercariae (R. Tarrab-Hazdai et al., J. Immunol. 135, 2772, 1985). To characterize some of the proteins which comprise this preparation, rabbit antibodies to the 9B antigen preparation were used to screen cDNA libraries of cercariae and adult worms. We isolated and sequenced cDNA clones encoding three proteins: calcium-binding protein, paramyosin, and myosin. The calcium-binding protein was previously shown to be expressed in cercariae but not in sporocysts or adult worms (D. Ram et al., Mol. Biochem. Parasitol. 34, 167, 1989). Northern blots showed the presence of paramyosin and myosin mRNAs in sporocysts and adult worms but not in cercariae. Antibodies to paramyosin detected the protein in sporocysts and adult worms as well as in cercariae. These findings explain, in part, the protective activity of the 9B antigen preparation against challenge infection.

Asunto(s)

Proteínas de Unión al Calcio/genética , Expresión Génica , Miosinas/genética , Schistosoma mansoni/genética , Tropomiosina/genética , Secuencia de Aminoácidos , Animales , Antígenos Helmínticos/genética , Antígenos Helmínticos/inmunología , Secuencia de Bases , Northern Blotting , Southern Blotting , Western Blotting , Proteínas de Unión al Calcio/inmunología , Clonación Molecular , ADN/genética , Datos de Secuencia Molecular , Miosinas/inmunología , ARN Mensajero/análisis , Mapeo Restrictivo , Schistosoma mansoni/inmunología , Tropomiosina/inmunología

RESUMEN

The influence of the I region of the major histocompatibility complex (MHC) on T-dependent immune responses against a purified schistosome antigen (9B antigen) was investigated. H-2 congenic mice expressing both I-A and I-E antigens (I-E+) showed a higher in-vitro proliferation to 9B antigen as compared to the recombinant strains expressing only I-A (I-E-). These two strains of mice differed both qualitatively and quantitatively in the humoral responses elicited by the purified antigen. Furthermore, in-vivo protection experiments showed that mice which do not express I-E molecules can be partially protected against the disease by prior immunization with the 9B antigen in contrast to their I-E expressing counterparts. The possible role of the I-E molecule in the immune responses elicited during Schistosoma mansoni infection is discussed.

Asunto(s)

Antígenos Helmínticos/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Activación de Linfocitos , Schistosoma mansoni/inmunología , Esquistosomiasis mansoni/inmunología , Animales , Anticuerpos Antihelmínticos , División Celular , Relación Dosis-Respuesta Inmunológica , Genes MHC Clase II , Ratones , Ratones Endogámicos , Pruebas de Precipitina , Esquistosomiasis mansoni/genética , Linfocitos T/inmunología , Linfocitos T Colaboradores-Inductores/inmunología

RESUMEN

T-cell lines and clones specific for a partially protective schistosome antigen (9B antigen) were established from mice immunized with such antigen. The H-2 congenic strains B10.A which express both I-A and I-E class II gene products of the major histocompatibility complex (MHC) and B10.A(4R) which only express I-A molecules were used in these studies. The specific T-cell lines recognized the 9B antigen in the context of either A or E molecules, but both class II antigens were necessary for maximal stimulation of the T-cell lines in lymphocyte proliferation assays. T-cell clones were derived from these lines and their MHC restriction was investigated. Both I-A and I-E restricted clones could be isolated. All clones were specific for 9B antigen showing different degrees of cross-reactivity with a total schistosome extract (CA sonicate). A correlation between the fine specificity of the clones and the expression of class II antigens was demonstrated. Clones specific for 9B antigen, or which reacted to the same extent with 9B antigen and CA sonicate, were I-A restricted, whereas clones which proliferated more in the presence of CA sonicate were all I-E restricted. This suggests that I-E restricted clones recognize more cross-reactive epitopes than I-A restricted clones. These antigen-specific T-cell clones should provide a useful tool for examining the role of class II antigens in the modulation of protective immune response during Schistosoma mansoni infection.

Asunto(s)

Anticuerpos Antihelmínticos/biosíntesis , Antígenos de Histocompatibilidad Clase II/inmunología , Schistosoma mansoni/inmunología , Esquistosomiasis mansoni/inmunología , Linfocitos T/inmunología , Animales , Antígenos Helmínticos/inmunología , División Celular , Línea Celular , Células Clonales , Activación de Linfocitos , Ratones , Ratones Endogámicos

RESUMEN

Two enzymes, alkaline phosphatase and acetylcholinesterase (AChE), have been shown previously to be components of the surface of the trematode parasite Schistosoma mansoni. In this study we report that both these enzymes and other serine hydrolases are susceptible to release from the S. mansoni tegumental membrane by a phosphatidylinositol-specific phospholipase C (PIPLC) of bacterial origin. These data suggest that AChE and alkaline phosphatase of S. mansoni, as in higher organisms, are anchored to the membrane via covalently attached phosphatidylinositol. The release of AChE from the vesicular fraction of the parasite with PIPLC occurs in a concentration-dependent manner. Sucrose gradient centrifugation of the PIPLC-released AChE showed a single 8.3 S molecular form, similar to that observed for AChE solubilized by Triton X-100. PIPLC removed large amounts of AChE from the surface of intact schistosomula in culture, with no impairment of the viability of the parasite. In this case, an increase in the overall levels of AChE in the intact parasite was observed after addition of PIPLC.

Asunto(s)

Acetilcolinesterasa/metabolismo , Proteínas de la Membrana/metabolismo , Fosfatidilinositoles/metabolismo , Schistosoma mansoni/enzimología , Fosfolipasas de Tipo C/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Centrifugación por Gradiente de Densidad , Hidrolasas/metabolismo , Serina/metabolismo