RESUMO
The immune response has relevant physiological functions both in the male and female reproductive system, and must be tightly controlled to achieve a successful pregnancy. Several immune factors have been related to infertility, among them humoral and cellular immune responses triggered by sperm antigens. The present study was aimed at evaluating the immune profile induced by DNA immunization against the sperm protease proacrosin in CF1 male mice and its effect upon fertility. Immunized animals exhibited higher anti-proacrosin antibodies levels than controls (indirect ELISA), both in serum (p<0.01) and in seminal vesicle fluid (SVF; p<0.05). IgG2a levels were higher than IgG1 in serum (p<0.01) and similar in SVF. IL-10 and TGF-ß1 mRNA levels were lower in testis (p<0.05), whereas TNF-α and IFN-γ transcript levels were increased in SV tissue (p<0.05). Immunized mice showed a trend toward higher IFN-γ concentration in serum and SVF than controls. Male fertility rate was diminished in immunized mice (p<0.01) and inversely correlated with serum and SVF anti-proacrosin IgG levels (p<0.001). Immunized animals also had fewer pups born than controls (p<0.01). To our knowledge, this is the first report on DNA immunization done in CF1 mice. Injection of proacrosin DNA induces an immune response in the male reproductive tract characterized by high levels of specific antibodies and cytokine changes. These factors may alter the crucial balance of the genital tract microenvironment required for adequate fertilization and pregnancy.
Assuntos
Acrosina/imunologia , Precursores Enzimáticos/imunologia , Infertilidade Masculina/metabolismo , Vacinas de DNA/imunologia , Acrosina/genética , Animais , Coeficiente de Natalidade , Microambiente Celular , Citocinas/metabolismo , Precursores Enzimáticos/genética , Feminino , Imunização Secundária , Imunoglobulina G/sangue , Masculino , Camundongos , Camundongos Endogâmicos , Gravidez , Glândulas Seminais/metabolismoRESUMO
The identification of sperm proteins involved in fertilization has been the subject of numerous investigations. Much interest has been dedicated to naturally occurring antisperm antibodies (ASA) and their impact in fertility. Their presence in men and women has been associated with 2-50% of infertility cases. ASA may impair pre- and post-fertilization steps. Experimental models have been developed using sperm proteins as immunogens to evaluate their involvement in sperm function. Our team has pursued investigations to assess ASA presence in biological fluids from patients consulting for infertility and their effect on fertilization. We found ASA in follicular fluids with ability of inducing the acrosome reaction and blocking sperm-zona pellucida interaction and used them to identify sperm entities involved in these events. We generated and utilized antibodies against proacrosin/acrosin to characterize the sperm protease system. We implemented an ELISA to detect proacrosin/acrosin antibodies in human sera and evaluated their impact upon fertility by developing in vitro assays and a gene immunization model. This review presents a summary of ASA history, etiology, current approaches for detection and effects upon fertility. ASA (naturally occurring, generated by animal immunization and/or of commercial origin) are invaluable tools to understand the molecular basis of fertilization, better diagnose/treat immunoinfertility and develop immunocontraceptive methods.
Assuntos
Anticorpos/análise , Infertilidade Feminina/imunologia , Infertilidade Masculina/imunologia , Espermatozoides/imunologia , Zona Pelúcida/imunologia , Acrosina/genética , Acrosina/imunologia , Reação Acrossômica , Animais , Antígenos/imunologia , Precursores Enzimáticos/genética , Precursores Enzimáticos/imunologia , Feminino , Fertilização/imunologia , Expressão Gênica , Humanos , Masculino , Espermatozoides/metabolismo , Zona Pelúcida/metabolismoRESUMO
PROBLEM: Evaluation of proacrosin/acrosin ability to induce an immune response in male mice after genetic immunization and assessment of animal fertility. METHOD OF STUDY: Mice received 50 µg per animal of a plasmid containing the human proacrosin cDNA (pSF2-Acro) (control: empty plasmid, pSF2). The humoral response was evaluated by ELISA and immunocytochemistry. In vivo fertility was assessed by mating immunized males with control females. The effect of antibodies upon Ca(+2)-ionophore-induced acrosomal exocytosis (AE) and in vitro sperm-zona pellucida (ZP) binding was also studied. RESULTS: pSF2-Acro-immunized mice developed high levels of specific antibodies (P < 0.05) that recognized the sperm acrosomal cap. The number of fertile mice was lower (P = 0.027) in pSF2-Acro-immunized animals than in controls. Litter size was smaller (P < 0.05) in the pSF2-Acro group compared with controls. A negative correlation (P < 0.05) between antibody levels and litter size was found. Antiproacrosin/acrosin antibodies inhibited sperm-ZP binding (P < 0.0001) and Ca(+2)-ionophore-induced AE (P < 0.05). CONCLUSION: DNA immunization against proacrosin elicits an immune response in male mice associated with abnormal sperm functions and reduced fertility.
Assuntos
Acrosina/imunologia , Autoanticorpos/imunologia , Anticoncepção Imunológica , Precursores Enzimáticos/imunologia , Fertilidade/imunologia , Imunização , Vacinas Anticoncepcionais/imunologia , Vacinas de DNA/imunologia , Acrosina/genética , Animais , Precursores Enzimáticos/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Interações Espermatozoide-Óvulo/imunologia , Espermatozoides/imunologia , Vacinas Anticoncepcionais/genética , Vacinas de DNA/genéticaRESUMO
The anti-acrosin monoclonal antibody AcrC5F10 inhibited proacrosin activation, proacrosin-human zona pellucida glycoprotein A (ZPA) binding, and the zona pellucida (ZP)-induced acrosome reaction of the ZP-bound spermatozoa but had no significant effect on sperm-ZP binding. These results suggest that proacrosin-acrosin may play an important role in the ZP-induced acrosome reaction of spermatozoa after primary binding to the ZP.
Assuntos
Acrosina/imunologia , Reação Acrossômica/efeitos dos fármacos , Anticorpos/farmacologia , Precursores Enzimáticos/imunologia , Espermatozoides/efeitos dos fármacos , Zona Pelúcida/fisiologia , Acrosina/metabolismo , Reação Acrossômica/imunologia , Animais , Células CHO , Cricetinae , Cricetulus , Proteínas do Ovo/metabolismo , Proteínas do Ovo/farmacologia , Ativação Enzimática/efeitos dos fármacos , Precursores Enzimáticos/metabolismo , Feminino , Humanos , Masculino , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/farmacologia , Ligação Proteica , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/metabolismo , Espermatozoides/metabolismo , Espermatozoides/fisiologia , Zona Pelúcida/efeitos dos fármacos , Glicoproteínas da Zona PelúcidaRESUMO
Crustacean aquaculture represents a major industry in tropical developing countries. As a result of high culture densities and increasing extension of aquaculture farms, the presence of diseases has also increased, inducing economic losses. Invertebrates, which lack adaptive immune systems, have developed defense systems that respond against antigens on the surface of potential pathogens. The defense mechanisms of crustaceans depend completely on the innate immune system that is activated when pathogen-associated molecular patterns are recognized by soluble or by cell surface host proteins, such as lectins, antimicrobial, clotting, and pattern recognition proteins, which, in turn, activate cellular or humoral effector mechanisms to destroy invading pathogens. This work is aimed at presenting the main characteristics of the crustacean proteins that participate in immune defense by specific recognition of carbohydrate containing molecules, i.e. glycans, glycolipids, glycoproteins, peptidoglycans, or lipopolysaccharides from Gram-negative and Gram-positive bacteria, viruses, or fungi. We review some basic aspects of crustacean effector defense processes, like agglutination, encapsulation, phagocytosis, clottable proteins, and bactericidal activity, induced by these carbohydrate-driven recognition patterns.
Assuntos
Peptídeos Catiônicos Antimicrobianos/imunologia , Catecol Oxidase/imunologia , Crustáceos/imunologia , Precursores Enzimáticos/imunologia , Lectinas/imunologia , Fagocitose/imunologia , Receptores de Reconhecimento de Padrão/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Catecol Oxidase/metabolismo , Crustáceos/metabolismo , Precursores Enzimáticos/metabolismo , Imunidade Inata , Lectinas/metabolismo , Receptores de Reconhecimento de Padrão/metabolismoRESUMO
Proteins stored in insect hemolymph may serve as a source of amino acids and energy for metabolism and development. The expression of the main storage proteins was assessed in bacterial-challenged honey bees using real-time (RT)-PCR and Western blot. After ensuring that the immune system had been activated by measuring the ensuing expression of the innate immune response genes, defensin-1 (def-1) and prophenoloxidase (proPO), we verified the expression of four genes encoding storage proteins. The levels of vitellogenin (vg) mRNA and of the respective protein were significantly lowered in bees injected with bacteria or water only (injury). An equivalent response was observed in orally-infected bees. The levels of apolipophorin II/I (apoLp-II/I) and hexamerin (hex 70a) mRNAs did not significantly change, but levels of Hex 70a protein subunit showed a substantial decay after bacterial challenge or injury. Infection also caused a strong reduction in the levels of apoLp-III transcripts. Our findings are consistent with a down-regulation of the expression and accumulation of storage proteins as a consequence of activation of the immune system, suggesting that this phenomenon represents a strategy to redirect resources to combat injury or infection.
Assuntos
Infecções Bacterianas/imunologia , Abelhas/genética , Hemolinfa/imunologia , Proteínas de Insetos/genética , Animais , Apolipoproteínas/genética , Apolipoproteínas/imunologia , Apolipoproteínas/metabolismo , Infecções Bacterianas/metabolismo , Abelhas/imunologia , Abelhas/metabolismo , Catecol Oxidase/genética , Catecol Oxidase/imunologia , Catecol Oxidase/metabolismo , Defensinas/imunologia , Defensinas/metabolismo , Precursores Enzimáticos/genética , Precursores Enzimáticos/imunologia , Precursores Enzimáticos/metabolismo , Feminino , Regulação da Expressão Gênica , Hemolinfa/metabolismo , Proteínas de Insetos/imunologia , Proteínas de Insetos/metabolismo , RNA/análise , RNA Mensageiro/análise , Especificidade da Espécie , Estresse Fisiológico/genética , Estresse Fisiológico/imunologia , Vitelogeninas/genética , Vitelogeninas/imunologia , Vitelogeninas/metabolismoRESUMO
OBJECTIVE: To detect the presence of antibodies to the proacrosin/acrosin system and to evaluate their effect on the sperm acrosomal protein activities in women consulting for infertility. DESIGN: Retrospective study. SETTING: Basic research laboratory. PATIENT(S): Recombinant proteins derived from human proacrosin (Rec-40, Rec-30, Rec-20, Rec-10) and recombinant human zona pellucida (ZP) glycoprotein A( *) (rec-hZPA). INTERVENTION(S): Development of an ELISA-Acro to test for antiacrosin antibodies using Rec-40 and truncated acrosin proteins as antigens. MAIN OUTCOME MEASURE(S): Evaluation of: 1) the presence of antiacrosin antibodies; 2) the protein regions recognized by the antibodies; 3) the relationship between antiacrosin antibodies and surface antisperm antibodies (ASA) identified by the immunobead binding test (IBT); and 4) the effect of antiacrosin antibodies upon proacrosin/acrosin binding activity to ZPA and acrosin amidase activity. RESULT(S): Antiacrosin antibodies were detected in sera from 34 of 179 women (19%). Detection of ASA by the IBT resulted in a similar incidence (36 of 179, 20%), although only six of them showed correspondence between both assays; five of these six sera were IBT-positive IgGs to the sperm head. Antiacrosin antibodies directed toward different protein regions inhibited proacrosin binding activity to rec-hZPA as well as its activation and acrosin amidase activity in protein sperm extracts. CONCLUSION(S): Antiacrosin antibodies are present in sera of women consulting for infertility in both IBT-positive and IBT-negative samples, and they affect proacrosin/acrosin activities.
Assuntos
Acrosina/imunologia , Autoanticorpos/sangue , Autoanticorpos/farmacologia , Infertilidade Feminina/sangue , Peptídeo Hidrolases/metabolismo , Espermatozoides/efeitos dos fármacos , Acrosina/metabolismo , Adulto , Autoanticorpos/isolamento & purificação , Autoanticorpos/fisiologia , Proteínas do Ovo/imunologia , Proteínas do Ovo/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/imunologia , Precursores Enzimáticos/imunologia , Precursores Enzimáticos/metabolismo , Feminino , Humanos , Infertilidade Feminina/imunologia , Masculino , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Ligação Proteica , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Estudos Retrospectivos , Espermatozoides/enzimologia , Espermatozoides/imunologia , Adulto Jovem , Glicoproteínas da Zona PelúcidaRESUMO
OBJECTIVE: To assess the effect of antiacrosin antibodies upon proacrosin/acrosin activities and animal fertility. DESIGN: Prospective study. SETTING: Basic research laboratory. PATIENT(S): A gene immunization (GI) model was developed; mice were injected with the sequence encoding human proacrosin (h-proacrosin), cloned in an expression vector. INTERVENTION(S): Subcloning of h-proacrosin in a eukaryotic expression vector (promoter, CMV; leader sequence, alpha-1 antitrypsin; pSF2-Acro); GI of female mice with this plasmid. MAIN OUTCOME MEASURE(S): The following parameters were evaluated: [1] adequate conditions for GI protocols, [2] humoral response to GI with pSF2-Acro, [3] protein regions recognized by the antibodies, and [4] effect of antibodies upon proacrosin/acrosin-ZPA binding and amidase activity, and animal fertility. RESULT(S): Conditions of female mice GI with the proacrosin sequence were established (plasmid purification with anion exchange chromatography and 40 microg of pSF2-Acro per dose) to trigger an immune response, reaching maximum levels at week 9 after the first injection. Antibodies produced by GI recognized human and mouse sperm acrosin systems, inhibited human proacrosin/acrosin interaction with recombinant human ZPA and protease activity, and negatively affected mouse IVF and early embryonic development. In addition, mice immunized with SF2-Acro exhibited a significantly lower size of fetuses. CONCLUSION(S): Antiacrosin antibodies developed by using GI inhibit human proacrosin/acrosin activities and impair mouse fertility.
Assuntos
Acrosina/imunologia , Autoanticorpos/fisiologia , Precursores Enzimáticos/genética , Precursores Enzimáticos/imunologia , Fertilidade/imunologia , Infertilidade/etiologia , Acrosina/genética , Acrosina/metabolismo , Animais , Autoanticorpos/sangue , Autoanticorpos/farmacologia , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/imunologia , Precursores Enzimáticos/metabolismo , Feminino , Fertilidade/genética , Fertilização/genética , Fertilização/imunologia , Humanos , Imunização/efeitos adversos , Infertilidade/sangue , Infertilidade/imunologia , Infertilidade/veterinária , Masculino , Camundongos , Camundongos Endogâmicos BALB C , GravidezRESUMO
VTDCE (Vitelin-Degrading Cysteine Endopeptidase) is a peptidase with an active role in Rhipicephalus (Boophilus) microplus embryogenesis. VTDCE is found in the tick's eggs and was shown to be the most active protein in vitellin (VT) hydrolysis of the three peptidases already characterized in R. microplus eggs (Boophilus Yolk pro-cathepsin (BYC), Tick Heme Binding Aspartic Proteinase (THAP) and VTDCE). VTDCE activity was assessed in vitro using the natural substrate and a synthetic substrate (N-Cbz-Phe-Arg-MCA). The activity was inhibited by anti-VTDCE antibodies. In the present study, it was shown that VTDCE acts differently from BYC and THAP in VT hydrolysis and that the vaccination of bovines with VTDCE induces a partial protective immune response against R. microplus infestation. Immunized bovines challenged with R. microplus larvae presented an overall protection of 21%, and a reduction in the weight of fertile eggs of 17.6% was observed. The data obtained indicate that VTDCE seems to be important for tick physiology, and that it induces partial protective immune response when inoculated in bovines. This suggests that VTDCE can be useful to improve the protective capacity observed for other antigens.
Assuntos
Doenças dos Bovinos/parasitologia , Cisteína Endopeptidases/imunologia , Rhipicephalus/imunologia , Infestações por Carrapato/veterinária , Vacinas/imunologia , Animais , Anticorpos/sangue , Ácido Aspártico Endopeptidases/imunologia , Western Blotting/veterinária , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/prevenção & controle , Cisteína Endopeptidases/metabolismo , Precursores Enzimáticos/imunologia , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Infestações por Carrapato/imunologia , Infestações por Carrapato/prevenção & controle , Vacinas/farmacologia , Vitelinas/metabolismoRESUMO
Boophilus Yolk pro-Cathepsin (BYC) is an aspartic proteinase found in Boophilus microplus eggs that is involved in the embryogenesis and has been tested as antigen to compose an anti-tick vaccine. The vaccine potential of a recombinant BYC expressed in Escherichia coli (rBYC) was investigated. rBYC was purified and used to immunize Hereford cattle. The sera of bovines immunized with rBYC recognized the native BYC with a titer ranging from 125 to 4000. Furthermore, immunized bovines challenged with 20,000 larvae presented an overall protection of 25.24%. The partial protection obtained against B. microplus infestation with the recombinant protein immunization was similar to the already described for native BYC immunization.
Assuntos
Ácido Aspártico Endopeptidases/imunologia , Doenças dos Bovinos/prevenção & controle , Doenças dos Bovinos/parasitologia , Precursores Enzimáticos/imunologia , Ixodidae/imunologia , Infestações por Carrapato/prevenção & controle , Infestações por Carrapato/veterinária , Vacinação/veterinária , Animais , Formação de Anticorpos , Ácido Aspártico Endopeptidases/genética , Western Blotting/veterinária , Bovinos , Doenças dos Bovinos/imunologia , Precursores Enzimáticos/genética , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Imunoglobulina G/sangue , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Infestações por Carrapato/imunologia , Infestações por Carrapato/parasitologiaRESUMO
The scope of the present study was to evaluate the presence and activation of proacrosin/acrosin as a tool to determine the acrosomal status of fresh and frozen/thawed dog spermatozoa. Monoclonal antibody C5F11, directed against human acrosin, cross-reacted with dog spermatozoa and labeled the acrosome of both fresh and frozen/thawed dog spermatozoa. Frozen/thawed spermatozoa had a lesser proportion of labeled spermatozoa than fresh spermatozoa (P<0.05). When live spermatozoa were labeled with soybean trypsin inhibitor conjugated with Alexa 488 (SBTI-Alexa 488), the proportion of acrosome-labeled fresh spermatozoa was less than frozen/thawed spermatozoa (P<0.05). By using Western blots and enzymatic activity, frozen/thawed spermatozoa had a greater proportion of active acrosin than fresh spermatozoa. In addition, beta 1,4-galactosyl-transferase (GalT), a plasma membrane bound protein, remained attached to frozen/thawed spermatozoa. Proacrosin is activated during freezing/thawing of dog spermatozoa, and that proacrosin/acrosin may be a good indicator of acrosomal integrity of frozen/thawed spermatozoa.
Assuntos
Acrosina/imunologia , Acrossomo , Anticorpos Monoclonais/imunologia , Cães/fisiologia , Precursores Enzimáticos/imunologia , Espermatozoides/fisiologia , Acrosina/metabolismo , Acrossomo/imunologia , Acrossomo/fisiologia , Reação Acrossômica/fisiologia , Animais , Western Blotting/veterinária , Precursores Enzimáticos/metabolismo , Masculino , Preservação do Sêmen/veterinária , Espermatozoides/enzimologia , Espermatozoides/imunologiaRESUMO
OBJECTIVE: To determine the contribution of the polysulfate-binding domain (PSBD) of acrosin during sperm penetration. DESIGN: To inhibit the in vitro fertilization of mouse zona-intact oocytes by using a polyclonal antibody raised against an 18-amino acid peptide of proacrosin (anti-PSBD). SETTING: Unit of Reproduction and Development, Faculty of Biological Sciences, Pontifical Catholic University of Chile. PATIENT(S): None. INTERVENTION(S): A polyclonal antibody against the 43IFMYHNNRRYHTCGGILL(60) peptide was raised in New Zealand female rabbits. The specificity of the antibody was evaluated by an ELISA. Zona-intact mouse oocytes were coincubated with capacitated spermatozoa for 3 hours in the presence of 0.63 mg/mL of the antibody or preimmune serum. As a control, we used zona-free mouse oocytes under the same experimental conditions. MAIN OUTCOME MEASURE(S): We evaluated the fertilization rate of zona-intact and zona-free mouse oocytes by phase-contrast microscopy. An oocyte was considered fertilized when at least one decondensed sperm head was found within the egg cytoplasm. We evaluated 50-60 mouse oocytes in each group in three independent experiments. RESULT(S): The anti-PSBD antibody inhibited the fertilization of zona-intact, but not zona-free, mouse oocytes, by capacitated spermatozoa. In addition, the binding of the anti-PSBD to proacrosin/acrosin in a solid-phase assay was inhibited in the presence of polysulfates (fucoidan). CONCLUSION(S): The anti-PSBD directed against the PSBD of proacrosin/acrosin inhibited the penetration of capacitated mouse spermatozoa through the zona pellucida. This antibody may be a useful tool to define the roles of the different domains of proacrosin/acrosin during gamete interaction.
Assuntos
Acrosina/imunologia , Anticorpos/farmacologia , Precursores Enzimáticos/imunologia , Fertilização in vitro , Fragmentos de Peptídeos/imunologia , Sulfatos/metabolismo , Acrosina/química , Acrosina/fisiologia , Sequência de Aminoácidos , Animais , Anticorpos/metabolismo , Sítios de Ligação/imunologia , Precursores Enzimáticos/química , Precursores Enzimáticos/fisiologia , Feminino , Fertilização/fisiologia , Imunofluorescência , Masculino , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Polissacarídeos/metabolismo , Capacitação Espermática , Zona Pelúcida/fisiologiaRESUMO
Cathepsin L1, a cysteine protease secreted by the gastrodermis of juvenile and adult Fasciola hepatica, was expressed in Escherichia coli as a fusion protein containing the proregion, supplied with six histidyl residues at the N-terminal end (rproCL1). In this study we tested its potential as antigen for the serologic diagnosis of F. hepatica infections by enzyme-linked immunosorbent assay (ELISA). The analyzed human sera included 16 positive samples, 99 negative controls and 111 from individuals affected by other parasitic and non parasitic diseases. The sensitivity and specificity of the rproCL1-ELISA were 100%. We also assessed the ability to detect antibodies in sera from 10 experimentally infected sheep, obtaining preliminary results that shown a response since the third week post infection in all the studied animals. Therefore, the recombinant rproCL1-based ELISA could be a standardized test for the accurate diagnosis of fasciolosis.
Assuntos
Anticorpos Anti-Helmínticos/sangue , Catepsinas/imunologia , Cisteína Endopeptidases/imunologia , Precursores Enzimáticos/imunologia , Fasciola hepatica/imunologia , Fasciolíase/diagnóstico , Sequência de Aminoácidos , Animais , Anticorpos Anti-Helmínticos/biossíntese , Catepsina L , Catepsinas/biossíntese , Catepsinas/química , Cisteína Endopeptidases/biossíntese , Cisteína Endopeptidases/química , Precursores Enzimáticos/biossíntese , Precursores Enzimáticos/química , Ensaio de Imunoadsorção Enzimática , Fasciola hepatica/isolamento & purificação , Fasciolíase/imunologia , Fasciolíase/veterinária , Humanos , Imunoglobulina G/sangue , RNA de Helmintos , Proteínas Recombinantes de Fusão/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Ovinos , Doenças dos Ovinos/imunologiaRESUMO
Proacrosin is a multifunctional protein present in the sperm acrosome. This study characterizes the expression of human proacrosin in bacteria and assesses zona pellucida binding activity. The cDNA encoding human proacrosin was subcloned in pGEX-3X and pET-22b vectors. In the pGEX system, expression of the full-length fusion protein was not detected. In the pET system, an expression product with an apparent molecular size similar to that expected for the proenzyme (Rec-40, 42-44 kDa) was recognized by a monoclonal antibody to human acrosin, AcrC5F10. A 32-34-kDa protein (Rec-30), not recognized by AcrC5F10 on Western blots, was the major expression product. Proteins of 21 (Rec-20) and 18 (Rec-10) kDa were recovered as insoluble expression products as were Rec-40 and Rec-30, and truncated products from the C terminus were detected in the soluble fraction. Rec-40 and Rec-30 coexisted at any culture time tested. Immune serum raised against Rec-30 (AntiRec-30) stained the acrosomal region of permeabilized human spermatozoa and recognized the recombinant proteins and proacrosin from human sperm extracts. Amino acid sequence analysis indicated that Rec-30, Rec-20, and Rec-10 are N-terminal fragments of proacrosin. The recombinant proteins Rec-40, -30, -20, and -10 were found to interact with homologous (125)I-zona pellucida glycoproteins.
Assuntos
Acrosina/genética , Acrosina/metabolismo , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Escherichia coli/genética , Zona Pelúcida/metabolismo , Acrosina/imunologia , Precursores Enzimáticos/imunologia , Feminino , Vetores Genéticos , Humanos , Soros Imunes , Radioisótopos do Iodo , Masculino , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de Proteína , Fatores de TempoRESUMO
BACKGROUND: Cationic streptococcal proteinase (erythrotoxin B) and its precursor, zymogen, are putative nephritogenic antigens. The present study was designed to test whether serum titers to these antigens were good markers of streptococcal infection associated with glomerulonephritis. METHODS: We studied 153 patients (male/female = 104/49, age range, 2 to 23 years old) with acute poststreptococcal glomerulonephritis (APSGN) from three countries (Venezuela, Chile and Argentina). The site of the initial infection was the skin in 84 patients, the throat in 55 patients and was unknown in 14 patients. In addition, we studied 23 patients (1 to 24 years old) with streptococcal infection not associated with glomerulonephritis (14 patients with impetigo and 9 patients with pharyngitis). As control group, 93 healthy individuals (54 males, 2 to 19 years old) were studied. Anti-zymogen and anti-proteinase titers were determined in a single laboratory by ELISA, and the intra- and interassay coefficients of variation were 5.3% and 8.5%, respectively. ASO titers and anti-DNAse B titers were also done. RESULTS: Anti-zymogen titers of 1:800 to 1:3200 had likelihood ratios (sensitivity/1-specificity) for detection of streptococcal infection in APSGN patients ranging from 2.00 to 44.2 in Argentina, Chile and Venezuela. Anti-zymogen titers decreased one to two months after APSGN and they were 1 to 3 log2 dilutions higher that anti-proteinase titers. Receiver operating characteristic (ROC) curves showed that anti-zymogen titers were consistently superior to anti-streptolysin O and anti-DNAse B titers as markers for streptococcal infection in APSGN. CONCLUSIONS: These results suggest that increased anti-zymogen antibody titers are the best available marker for streptococcal infection associated with acute glomerulonephritis.
Assuntos
Anticorpos Antibacterianos/sangue , Proteínas de Bactérias , Cisteína Endopeptidases/imunologia , Precursores Enzimáticos/imunologia , Exotoxinas/imunologia , Glomerulonefrite/microbiologia , Proteínas de Membrana , Streptococcus pyogenes/imunologia , Doença Aguda , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Impetigo/microbiologia , Masculino , Sensibilidade e EspecificidadeRESUMO
The capacity of the Boophilus Yolk pro-Cathepsin (BYC) to induce a protective immune response in cattle against Boophilus microplus infestation was tested by vaccination experiments and by inoculation of monoclonal antibody (MAb) against BYC into fully engorged tick females. In immunization experiments the measurement of various biological parameters demonstrated a partial protection against B. microplus. A continuous decrease in the levels of specific antibodies was observed over 11 months when six bovines were maintained in field conditions. The inoculation of the MAb into tick females produced a dose-dependent decrease in oviposition and survival of the ectoparasite compared to the control.