RESUMO
For more than a decade, the cytokine interleukin-12 (IL-12) has been utilized, either alone or in combination with other drugs, as a treatment for cancer. The numerous anti-tumor properties of IL-12 still generate interest in the clinical use of this cytokine, even though it has demonstrated toxicity when administrated systemically. As an approach to overcome this toxicity, numerous laboratories have attempted to induce IL-12 expression at the site of the tumor. However for tumors that are difficult to remove surgically or for the treatment of disseminated metastases, systemic expression of this cytokine still remains as the most efficient method of administration. Nevertheless, finding alternative approaches for the use of IL-12 in the treatment of cancer and unraveling the basis of IL-12-side effects remain a challenge. In the present work we demonstrate that systemic expression of IL-12 through hydrodynamic injection of IL-12 cDNA is able to induce different types of liver lesions associated with a toxic pathology. However we report here that hepatic toxicity is diminished and survival of mice enhanced in the absence of tumor necrosis factor alpha (TNFα). This observation is in contrast to several murine models and clinical trials that postulate interferon gamma (IFNγ) as the main cytokine responsible for IL-12 toxicity. Moreover, our work demonstrates that when IL-12 cDNA is co-injected with IL-18 cDNA or when mice are pre-treated with a low dose of IL-12 cDNA prior to receiving a high dose of IL-12 cDNA, systemic levels of TNFα are almost completely abrogated, resulting in improved survival and less hepatic damage. Importantly, abrogation of TNFα signaling does not affect the strong anti-tumor activity of IL-12. Thus, neutralizing TNFα with antagonists already approved for human use offers a promising approach to abrogate IL-12 side effects during the use of this cytokine for the treatment of cancer.
Assuntos
DNA Complementar/administração & dosagem , Imunoterapia/métodos , Interleucina-12/imunologia , Interleucina-18/imunologia , Melanoma Experimental/terapia , Neoplasias Esplênicas/terapia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , DNA Complementar/imunologia , Expressão Gênica , Hidrodinâmica , Injeções Intravenosas , Interleucina-12/biossíntese , Interleucina-12/genética , Interleucina-18/biossíntese , Interleucina-18/genética , Fígado/efeitos dos fármacos , Fígado/patologia , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Neoplasias Esplênicas/imunologia , Neoplasias Esplênicas/patologia , Cauda , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Digoxin is a pharmaceutical used in the control of cardiac dysfunction. Its therapeutic window is narrow, with effect dosage very close to the toxic dosage. To counteract the toxic effect, polyclonal Fab fragments are commercially available. Our study is based on a monoclonal anti-digoxin antibody, which would provide a product with a specific potency and more precise dosage for the detoxification of patients under digoxin treatment. Phage display technology was used to select variants with high affinity. From an anti-digoxin hybridoma, RNA was extracted for subsequent cDNA synthesis. Specific primers were used for the LC and Fd amplifications, then cloned sequentially in a phagemid vector (pComb3X) for the combinatorial Fab library construction. Clones were selected for their ability to bind to digoxin-BSA. The presence of light and heavy chains was checked, randomly selected clones then sequenced and induced to produce soluble Fabs, and subsequently analyzed for anti-digoxin expression. Out of ten clones randomly chosen, six resulted positive expression of the product. The sequencing of these revealed two identical clones and one presenting a pseudogene in the LC. Four clones presenting variations in the framework1 showed binding to digoxin-BSA by ELISA and western blotting. The specific binding was further confirmed by Biacore(®), which allowed ranking of the clones. The development of these clones allowed the selection of variants with higher affinity than the original version.
Assuntos
Anticorpos Monoclonais/genética , Técnicas de Visualização da Superfície Celular/métodos , Digoxina/imunologia , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/imunologia , Anticorpos Monoclonais/imunologia , DNA Complementar/genética , DNA Complementar/imunologia , Escherichia coli/genética , Escherichia coli/imunologia , Variação Genética , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Humanos , Hibridomas/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/imunologia , Biblioteca de PeptídeosRESUMO
The protozoan Trypanosoma cruzi is the etiological agent of Chagas' disease, a major chronic infection in Latin America. Currently, there are neither effective drugs nor vaccines for the treatment or prevention of the disease. Several T. cruzi surface antigens are being tested as vaccines but none of them proved to be completely protective, probably because they represent only a limited repertoire of all the possible T. cruzi target molecules. Taking into account that the trypomastigote stage of the parasite must express genes that allow the parasite to disseminate into the tissues and invade cells, we reasoned that genes preferentially expressed in trypomastigotes represent potential targets for immunization. Here we screened an epimastigote-subtracted trypomastigote cDNA expression library by genetic immunization, in order to find new vaccine candidates for Chagas' disease. After two rounds of immunization and challenge with trypomastigotes, this approach led to the identification of a pool of 28 gene fragments that improved in vivo protection. Sequence analysis of these putative candidates revealed that 19 out of 28 (67.85%) of the genes were hypothetical proteins or unannotated T. cruzi open reading frames, which certainly would not have been identified by other methods of vaccine discovery.
Assuntos
Doença de Chagas/imunologia , DNA Complementar/genética , DNA de Protozoário/imunologia , Vacinas Protozoárias/uso terapêutico , Trypanosoma cruzi/imunologia , Animais , Anticorpos Antiprotozoários/análise , Anticorpos Antiprotozoários/biossíntese , Formação de Anticorpos , Doença de Chagas/epidemiologia , DNA Complementar/imunologia , Biblioteca Gênica , Humanos , América Latina/epidemiologia , Camundongos , Camundongos Endogâmicos C3HRESUMO
Zinc-dependent metalloproteinases play a key role in the hemorrhage induced by viperid bite envenoming. In this work we report the cloning and sequencing of the cDNA from a novel P-III type metalloproteinase from Crotalus durissus durissus venom glands. The recombinant plasmid was used for DNA immunization in mice using accelerate DNA-coated microparticles with the Gene Gun system. The results showed that there is no significant difference in the efficiency of the immunization in mice when gold or tungsten microparticles were used. A pool of the sera from mice immunized with the metalloproteinase encoding DNA neutralized the hemorrhagic activity of C. d. durissus venom. The co-immunization with DNA encoding the metalloproteinase and a plasmid encoding the murine IL-2 increased the number of mice which show a specific antibody response towards C. d. durissus venom antigens. The neutralizing ability of the produced antibodies demonstrates that DNA immunization with tungsten microparticles may be used in strategies for antivenom production.
Assuntos
Antivenenos/farmacologia , Venenos de Crotalídeos/enzimologia , DNA Complementar/imunologia , Hemorragia/prevenção & controle , Metaloproteases/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Venenos de Crotalídeos/antagonistas & inibidores , Venenos de Crotalídeos/toxicidade , DNA Complementar/genética , Hemorragia/induzido quimicamente , Hemorragia/patologia , Metaloproteases/classificação , Metaloproteases/genética , Camundongos , Dados de Sequência Molecular , Testes de Neutralização , Análise de Sequência de DNA , Pele/efeitos dos fármacos , Pele/patologiaRESUMO
Considering the scarcity of defined antigens, actually useful and reliable for use in the field studies, we propose an alternative method for selection of cDNA clones with potential use in the diagnosis of schistosomiasis. Human antibodies specific to a protein fraction of 31/32 kDa (Sm31/32), dissociated from immune complexes, are used for screening of clones from an adult worm cDNA library. Partial sequencing of five clones, selected through this strategy, showed to be related to Schistosoma mansoni: two were identified as homologous to heat shock protein 70, one to glutathione S-transferase, one to homeodomain protein, and one to a previously described EST (expressed sequence tag) of S. mansoni. This last clone was the most consistently reactive during the screening process with the anti-Sm31/32 antibodies dissociated from the immune complexes. The complete sequence of this clone was obtained and the translation data yielded only one ORF (open reading frame) that code for a protein with 57 amino acids. Based on this amino acid sequence two peptides were chemically synthesized and evaluated separately against a pool of serum samples from schistosomiasis patients and non-schistosomiasis individuals. Both peptides showed strong reactivity only against the positive pool, suggesting that these peptides may be useful as antigens for the diagnosis of schistosomiasis mansoni.
Assuntos
DNA Complementar/genética , Biblioteca de Peptídeos , Schistosoma mansoni/genética , Schistosoma mansoni/imunologia , Esquistossomose mansoni/diagnóstico , Animais , Anticorpos Anti-Helmínticos/genética , Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/genética , Antígenos de Helmintos/imunologia , Clonagem Molecular/métodos , DNA Complementar/imunologia , Etiquetas de Sequências Expressas , Biblioteca Gênica , Proteínas de Choque Térmico HSP70/imunologia , Humanos , Fases de Leitura AbertaRESUMO
Considering the scarcity of defined antigens, actually useful and reliable for use in the field studies, we propose an alternative method for selection of cDNA clones with potential use in the diagnosis of schistosomiasis. Human antibodies specific to a protein fraction of 31/32 kDa (Sm31/32), dissociated from immune complexes, are used for screening of clones from an adult worm cDNA library. Partial sequencing of five clones, selected through this strategy, showed to be related to Schistosoma mansoni: two were identified as homologous to heat shock protein 70, one to glutathione S-transferase, one to homeodomain protein, and one to a previously described EST (expressed sequence tag) of S. mansoni. This last clone was the most consistently reactive during the screening process with the anti-Sm31/32 antibodies dissociated from the immune complexes. The complete sequence of this clone was obtained and the translation data yielded only one ORF (open reading frame) that code for a protein with 57 amino acids. Based on this amino acid sequence two peptides were chemically synthesized and evaluated separately against a pool of serum samples from schistosomiasis patients and non-schistosomiasis individuals. Both peptides showed strong reactivity only against the positive pool, suggesting that these peptides may be useful as antigens for the diagnosis of schistosomiasis mansoni.
Considerando a escassez de antígenos quimicamente definidos, realmente úteis e confiáveis para aplicação na soroepidemiologia da esquistossomose em larga escala, foi proposto, neste trabalho, um método alternativo para a seleção de clones de cDNA que expressam proteínas com putativo potencial diagnóstico na esquistossomose. Empregando anticorpos específicos contra uma fração proteica de 31/32 kDa (Sm31/32), purificados através da dissociação de imunocomplexos, foram selecionados cinco clones de cDNA a partir de genoteca de verme adulto de Schistosoma mansoni. O seqüenciamento parcial destes clones demonstrou que todos eram relacionados ao S. mansoni: dois apresentaram homologia com a proteína de choque térmico de 70 kDa e os demais com glutationa S-transferase, "homeodomain protein" e uma etiqueta de seqüência expressa (EST). Este último foi o clone que melhor reagiu, durante o processo de seleção, com os anticorpos anti-Sm31/32 dissociados de imunocomplexos. Baseado na seqüência de aminoácidos deste clone, dois peptídeos foram quimicamente sintetizados e analisados separadamente frente a misturas de soros de indivíduos normais e de pacientes com esquistossomose mansoni. Ambos os peptídeos demonstraram uma intensa reatividade somente contra a mistura de soros positivos, sugerindo que estes peptídeos podem ser úteis como antígenos para o diagnóstico da esquistossomose mansoni.
Assuntos
Humanos , Animais , DNA Complementar/genética , Biblioteca de Peptídeos , Schistosoma mansoni/genética , Schistosoma mansoni/imunologia , Esquistossomose mansoni/diagnóstico , Anticorpos Anti-Helmínticos/genética , Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/genética , Antígenos de Helmintos/imunologia , Clonagem Molecular/métodos , DNA Complementar/imunologia , Etiquetas de Sequências Expressas , Biblioteca Gênica , /imunologia , Fases de Leitura AbertaRESUMO
The kappa light chain locus of swine has been mapped to chromosome 3q12-q14 but at this time, there is not enough information comprising the variable region genes or their transcripts. Here we report the sequences of five transcripts of swine kappa light chain variable region obtained from the spleen of two adult Yorkshire pigs. The lengths of the deduced sequences of these transcripts were variable (between 107 to 112 amino acids). Comparisons of the nucleotide sequences of their variable regions with other species like human and murine VL genes shows a high degree of identity, indicating the use of at least two different families of variable light genes. The contribution of these genes to the generation of variability in swine light chain variable genes as well as their therapeutic use in humans is discussed. An interesting possibility would be the development of an antiretroviral vaccine, useful to protect against a potential risk of infections due to xenotransplantation.
Assuntos
Região Variável de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/genética , Animais , Diversidade de Anticorpos/genética , Diversidade de Anticorpos/imunologia , Sequência de Bases , DNA Complementar/genética , DNA Complementar/imunologia , Humanos , Região Variável de Imunoglobulina/imunologia , Cadeias kappa de Imunoglobulina/imunologia , Dados de Sequência Molecular , Análise de Sequência de DNA , SuínosRESUMO
The central objective of this research was to test molecularly defined, live attenuated Venezuelan equine encephalitis virus (VEEV) vaccine candidates that were produced through precise genetic manipulation of rationally selected viral nucleotide sequences. Molecular clones of vaccine candidates were constructed by inserting either three independently attenuating mutations or a PE2 cleavage-signal mutation with a second-site resuscitating mutation into full-length cDNA clones. Vaccine candidate viruses were recovered through DNA transcription and RNA transfection of cultured cells, and assessed in rodent and non-human primate models. Based on results from this assessment, one of the PE2 cleavage-signal mutants, V3526, was determined to be the best vaccine candidate for further evaluation for human use.
Assuntos
Vírus da Encefalite Equina Venezuelana/genética , Vírus da Encefalite Equina Venezuelana/imunologia , Encefalomielite Equina Venezuelana/prevenção & controle , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/análise , Anticorpos Antivirais/biossíntese , Clonagem Molecular , Cricetinae , DNA Complementar/imunologia , Vírus da Encefalite Equina Venezuelana/patogenicidade , Feminino , Macaca fascicularis , Mesocricetus , Camundongos , Camundongos Endogâmicos C57BL , Mutação/imunologia , Engenharia de Proteínas , Vacinas Atenuadas/imunologia , Vacinas Virais/genéticaRESUMO
The kappa light chain locus of swine has been mapped to chromosome 3q12-q14 but at this time, there is not enough information comprising the variable region genes or their transcripts. Here we report the sequences of five transcripts of swine kappa light chain variable region obtained from the spleen of two adult Yorkshire pigs. The lengths of the deduced sequences of these transcripts were variable (between 107 to 112 amino acids). Comparisons of the nucleotide sequences of their variable regions with other species like human and murine VL genes shows a high degree of identity, indicating the use of at least two different families of variable light genes. The contribution of these genes to the generation of variability in swine light chain variable genes as well as their therapeutic use in humans is discussed. An interesting possibility would be the development of an antiretroviral vaccine, useful to protect against a potential risk of infections due to xenotransplantation (au)
Assuntos
Animais , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Análise de Sequência de DNA , Suínos , Diversidade de Anticorpos , DNA Complementar/genética , DNA Complementar/imunologiaRESUMO
BACKGROUND: In the last 10 years type-I allergy against proteins from Hevea brasiliensis latex has become an acknowledged medical issue. Fruit-allergic patients represent one risk group for developing latex allergy. Class I chitinases have been identified from chestnut, avocado and banana as relevant allergens. The chitin binding (hevein) domain from these class I chitinases has been postulated to bear the important IgE binding epitopes. OBJECTIVE: To clone the cDNA of an allergenic latex class I chitinase, to express the recombinant protein and to determine its IgE cross-reactivity with hevein (Hev b 6.02). METHODS: A full-length cDNA coding for a class I chitinase has been isolated from Hevea latex RNA by reverse transcription followed by PCR. The chitinase encoding sequence has been subcloned into the pMAL expression vector and expressed in E. coli as a fusion protein to maltose binding protein. The highly enriched recombinant protein fraction has been tested for its IgE binding capacity in immunoblots and ELISA. Furthermore, the pathogenesis-related function of the recombinant protein was tested in a fungal growth inhibition assay. RESULTS: The Hevea brasiliensis latex chitinase, designated Hev b 11, displays 70% identity to the endochitinase from avocado and its hevein-domain 58% to hevein (Hev b 6.02). The recombinant Hev b 11-maltose binding protein is recognized by latex- and fruit-allergic patients with IgE binding in both, ELISA and immunoblots. Pre-incubation of sera with rHev b 11-maltose binding protein showed an overall 16% inhibition of subsequent binding to rHev b 6.02-maltose binding protein on solid phase. The growth of F. oxysporum was inhibited in a dose dependent manner by addition of rHev b 11-maltose binding protein to the culture. CONCLUSIONS: Hev b 11, a class I chitinase, is another allergen from Hevea latex with a chitin binding domain and displays a different IgE binding capacity compared with hevein.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Alérgenos/química , Alérgenos/imunologia , Alérgenos/isolamento & purificação , Quitinases/química , Quitinases/imunologia , Quitinases/isolamento & purificação , Clonagem Molecular , Proteínas de Escherichia coli , Hevea/imunologia , Proteínas de Transporte de Monossacarídeos , Proteínas de Plantas/química , Sequência de Aminoácidos , Antígenos de Plantas , Sequência de Bases , Sítios de Ligação/imunologia , Proteínas de Transporte/imunologia , Proteínas de Transporte/isolamento & purificação , DNA Complementar/imunologia , Ensaio de Imunoadsorção Enzimática , Hipersensibilidade Alimentar/imunologia , Frutas/imunologia , Humanos , Immunoblotting , Imunoglobulina E/imunologia , Hipersensibilidade ao Látex/imunologia , Proteínas Ligantes de Maltose , Dados de Sequência Molecular , Proteínas de Plantas/imunologia , Proteínas de Plantas/isolamento & purificação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
cDNA expression library immunization (cDELI), based on the use of a large number of pathogen's cDNA clones, has been previously used in our laboratory in an experimental model of murine Taenia crassiceps cysticercosis. In this study we show that ex vivo immunization of mice with macrophages pulsed in vitro with plasmid DNA containing cDNA expression library (2x10(4) clones) leads to significant reduction of parasite load. Additionally, pathogen-specific proliferation of splenocytes from immunized mice is demonstrated indicating the induction of cellular protective immune response and the efficacy of the proposed strategy to identify vaccine candidate antigens. Our method may represent an attractive tool in vaccine discovery applicable to any host-pathogen system and may be useful especially for the pathogens with large genomes and complicated life cycles.
Assuntos
DNA Complementar/imunologia , Macrófagos/imunologia , Vacinas de DNA/imunologia , Animais , Feminino , Biblioteca Gênica , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Teníase/prevenção & controle , TransfecçãoRESUMO
The mite Blomia tropicalis is a potent source of allergens in tropical and subtropical regions. So far, most of these allergens have only been studied by immunoblotting. To characterize them at the molecular level, a lambda gt11 complementary DNA library was constructed from messenger RNA isolated from whole B. tropicalis mites. This library was screened by using pooled sera from patients allergic to B. tropicalis in a plaque IgE radioimmunoassay. A B. tropicalis IgE-positive clone (Bt-M) was selected for immunologic studies. After subcloning into pBluescript (Stratagene, La Jolla, Calif.), it produced a sequence of 310 bp, with a probable amino acid sequence of 72 residues for the expressed peptide. The recombinant protein was transferred to nitrocellulose filters and probed with 100 sera from patients allergic to B. tropicalis. Forty-seven percent of sera reacted with the recombinant allergen. Immunoblottings performed with allergic serum and B. tropicalis-affinity-purified IgE demonstrated that the recombinant protein shares allergenic epitopes with the 11-13, 14, and 16 kd native allergens of B. tropicalis, which are known to be important allergens of this mite.
Assuntos
Alérgenos/genética , Alérgenos/isolamento & purificação , Sítios de Ligação de Anticorpos , DNA Complementar/isolamento & purificação , Ácaros/imunologia , Adolescente , Adulto , Alérgenos/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Plantas , Sequência de Bases , Clonagem Molecular , DNA Complementar/biossíntese , DNA Complementar/imunologia , Feminino , Humanos , Immunoblotting , Imunoglobulina E/química , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas Recombinantes/imunologia , Análise de Sequência de DNARESUMO
The Flaviviridae is a family of about 70 mostly arthropod-borne viruses many of which are major public health problems with members present in most continents. Among the most important are yellow fever (YF), dengue (DEN) with its 4 serotypes and Japanese escephalitis (JE) virus. A live attenuated virus is used as a cost-effective, safe and efficacious vaccine against YF but no other live flavivirus vaccines have been licensed. The development of recombinant DNA technology and its application to study flavivirus genome structure and expression has opened new possibilities for flavivirus vaccine development. The new approaches include the use of cDNAs encompassing the whole viral genome to generate infectious RNA after in vitro transcription. This methodology allows the genetic mapping of specific viral functions and the design of viral mutants with considerable potential as new live attenuated virues. The use of infectious cDNA as a carrier for heterologous antigens is a gaining importance since chimeric viruses are shown to be viable, immunogenic and less virulent in some cases as compared to the parental viruses. The use of DNA to overcome intrinsic mutation rates of RNA virus populations in conjunction with vaccine production in cell culture should improve the reliability and lower the cost of live attenuated vaccines.