Avaliação da resposta imune em camundongos utilizando diferentes protocolos de imunização com antígenos recombinantes de Leishmania chagasi

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2012-06-25T20:55:47Z No. of bitstreams: 1 Cristiane Garboggini Pinheiro Avaliação da resposta imune em camnundongos....pdf: 3469982 bytes, checksum: 4041d40a1d0e55867e25c7be92f3dd02 (MD5)Made available in DSpace on 2012-06-25T20:55:47Z (GMT). No. of bitstreams: 1 Cristiane Garboggini Pinheiro Avaliação da resposta imune em camnundongos....pdf: 3469982 bytes, checksum: 4041d40a1d0e55867e25c7be92f3dd02 (MD5) Previous issue date: 2011Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, BrasilA leishmaniose visceral é uma doença parasitária causada por Leishmania chagasi. Os cães são considerados como principais reservatórios do parasito. Uma vacina efetiva para o cão pode contribuir para o controle da LV tanto no homem como no cão. Em um estudo prévio, antígenos recombinantes, obtidos a partir de uma biblioteca de cDNA da forma amastigota de Leishmania chagasi, foram selecionados a fim de serem avaliados como candidatos a componente de uma vacina contra leishmaniose visceral canina. No presente trabalho, a resposta imune humoral e celular de camundongos foi avaliada após injeção de alguns desses antígenos recombinantes, em cinco diferentes protocolos de imunização. Grupos de camundongos BALB/c foram injetados com as proteínas rLci4A-NH6 ou rLci2B-NH6, isoladamente ou associadas aos adjuvantes saponina, ODN 1826 ou diferentes doses de um plasmídeo codificando IL-12 murina. Além disso, animais foram sensibilizados com plasmídeo codificando Lci2B e receberam reforço com a proteína rLci2B-NH6 associada à saponina. Por último, os animais foram injetados com plasmídeos codificando novos antígenos, Lci2-NT-5R-CT ou Lci2-NT-CT, e recebram reforço com as respectivas proteínas recombinantes rLci2-NT-5R-CT-NH6 ou rLci2-NT-CT-NH6 associadas à saponina. Nesse último protocolo, os plasmídeos utilizados continham o gene para uma proteína transmembrana associada a lisossomos, LAMP, visando à produção in vivo de quimeras de dos antígenos de Leishmania com LAMP, a fim de direcionar a apresentação dos peptídeos para linfócitos T CD4+ via moléculas de MHC-II. Em um dos protocolos, os animais foram desafiados com 1 x 107 Leishmanias para avaliação da carga parasitária no baço após imunização. Os resultados encontrados mostraram que animais que receberam rLci4A-NH6, isoladamente ou com diferentes doses de plasmídeo codificando IL-12, apresentaram níveis signficantes de IgG e IgG1, ao passo que o uso do antígeno com os adjuvantes saponina ou ODN 1826 induziu a produção de IgG, IgG2a e IgG1, além de produção de IL-5 pelo grupo que recebeu saponina. Animais que receberam rLci2B-NH6, isoladamente ou associado aos plasmídeos contendo inserto de IL-12, produziram IgG e IgG1 antígeno específicos e falharam também na produção de citocinas e proliferação celular. A utilização de saponina com esse antígeno induziu produção de IgG, IgG2a e IgG1, assim como de IFN-γ. Já o uso de ODN 1826, induziu a produção de IgG e IgG1, favoreceu a produção de IgG2a, mas falhou na indução de resposta imune celular significante. Por outro lado, animais que receberam plasmídeo codificando Lci2B e reforço com a proteína associada à saponina apresentaram uma resposta imune com predominância de anticorpos IgG2a e produção de IFN-γ por alguns animais, a qual não conferiu proteção contra infecção pelo parasito. Por fim, a utilização de quimeras de LAMP/Lci2-NT-5R-CT ou LAMP/Lci2-5R-CT induziu uma fraca resposta imune humoral, com produção de IgG, IgG1 e IgG2a por alguns animais. No entanto, a utilização da quimera LAMP/Lci2-NT-CT induziu proliferação celular e tendência a produção de IFN-γ. Em conclusão, nenhum dos cinco protocolos utilizados foi capaz de induzir uma resposta imune específica predominantemente celular do tipo Th1 ou capaz de conferir proteção contra infecção dos animais.Visceral leishmaniasis is a disease caused by Leishmania chagasi. Dogs are the main reservoir of the parasite. A canine vaccine may contribute to the desease control in humans and dogs. In a previous study, recombinant antigens were selected from a cDNA library of Leishmania chagasi amastigotes in order to be evaluated as components of a vaccine against canine visceral leishmaniasis. In this study, five immunization protocols were assessed in mice with some of these antigens. BALB/c mice were injected with either rLci4A-NH6 or rLci2B-NH6 proteins, associated or not with the adjuvants saponin, ODN 1826 and different doses of a plasmid encoding murine IL-12 (pIL-12). Moreover, animals were primed with a plasmid encoding Lci2B (pLci2B) and boosted with rLci2B combined with saponin. Finally, all mice were primed with new antigens, Lci2-NT-5R-CT or Lci2-NT-CT, and they were boosted with the following recombinant proteins rLci2-NT-5R-CT-NH6 or rLci2-NT-CTNH6 associated with saponin. In this late experiment, the used plasmids had an insert of a lisossome associated membrane protein, LAMP, to produce in vivo chimeras composed of LAMP with the antigens and to direct the presentation of the peptides to CD4+ T cell through MHC-II molecules. The humoral immune response was assessed by antibodies production (IgG, IgG1 and IgG2a) in animal serum samples and the cellular immune response by cellular proliferation and cytokine production (IFN-γ and IL-5). The results showed that animals that were injected with rLci4A-NH6 alone or combined with pIL-12 generated IgG and IgG1 specific antibodies, while others that received saponin and ODN 1826 as adjuvants produced IgG, IgG2a and IgG1. Furthermore, IL-5 was detected in rLci4A-NH6/saponin group, but only in one of two experiments. Animals that were injected with rLci2B-NH6, alone or associated with pIL-12, presented IgG and IgG1 antibodies but no cellular immune response. The saponin used as adjuvant induced IgG, IgG2a and IgG1 antibodies, as well as IFN-γ. The use of ODN 1826 induced IgG and IgG1, and favored IgG2a production, but failed in inducing IFN-γ. On the other hand, animals that were primed with pLci2B and boosted with the protein associated with saponin produced IgG IgG1 and more intensively IgG2a, and only some animals produced IFN-γ. Nevertheless, such immune response did not protect the animals against the parasite infection. At last, LAMP/Lci2-NT-5R-CT or LAMP/Lci2-5R-CT chimeras induced a weak humoral immune response, in which IgG, IgG1 and IgG2a antibodies were detected in only a few animals. However, mice that were injected with LAMP/Lci2-NT-CT presented cellular proliferation and some animals produced IFN-γ. In conclusion, in spite of the fact that the antigens are immunogenic in a murine model, none of the used protocols elicited an intense Th1 cellular immune response or a protective one

Optimization of canine interleukin-12 production using a baculovirus insect cell expression system

Background: Interleukin-12 is an important cytokine in mediating cellular immune responses. Results: Recombinant single-chain canine IL-12 was produced in a baculovirus-insect cell system with the aim of conducting further studies on modulation of immune responses in dogs. To optimize the production of recombinant canine IL-12, a classical baculovirus and a modified vector (chitinase A and v-cathepsin knockout) were used containing a native or an optimized insert of canine IL-12. The optimized IL-12 construct contained the GP64 signal peptide and was synthesized with optimized codons for expression in Trichoplusia ni cells. Dot-blot and Western blot analysis showed the highest production levels of recombinant IL-12 protein by the use of the modified baculovirus vector containing the optimized insert, at a multiplicity of infection of five and at 48 h after infection. The recombinant cytokine was successfully purified and showed a good degree of purity, integrity, folding, and yield, with very little endotoxin contamination. Recombinant canine IL-12 induced IFN-γ in canine lymphocytes, indicating that it was biologically active. Conclusion: Therefore, this study describes an efficient method to produce adequate amounts of biologically active canine IL-12, useful for immunomodulation studies in dogs.</p

Requirement of dual stimulation by homologous recombinant IL-2 and recombinant IL-12 for the in vitro production of interferon gamma by canine peripheral blood mononuclear cells.

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2014-09-30T17:25:47Z No. of bitstreams: 1 Pereira A Requirement of dual stimulation.........pdf: 724685 bytes, checksum: d93ffeee109acbadf1748e48f588bb43 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2014-09-30T17:26:04Z (GMT) No. of bitstreams: 1 Pereira A Requirement of dual stimulation.........pdf: 724685 bytes, checksum: d93ffeee109acbadf1748e48f588bb43 (MD5)Made available in DSpace on 2014-09-30T17:59:21Z (GMT). No. of bitstreams: 1 Pereira A Requirement of dual stimulation.........pdf: 724685 bytes, checksum: d93ffeee109acbadf1748e48f588bb43 (MD5) Previous issue date: 2014Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Laboratório de Patologia e Biointervenção. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Laboratório de Patologia e Biointervenção. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Laboratório de Patologia e Biointervenção. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Laboratório de Patologia e Biointervenção. Salvador, BA, BrasilInstituto Nacional de Ciência e Tecnologia de Doenças Tropicais. INCT-DT. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Laboratório de Patologia e Biointervenção. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Laboratório de Patologia e Biointervenção. Salvador, BA, Brasil / Instituto Nacional de Ciência e Tecnologia de Doenças Tropicais. INCT-DT. Salvador, BA, BrasilBACKGROUND: Very few studies have been carried out so far aiming at modulating cellular immune responses in dogs. In this study, we evaluated the ability of recombinant canine IL-2 (rcaIL-2) and IL-12, in the form of a single-chain fusion protein (rsccaIL-12), to stimulate peripheral blood mononuclear cells (PBMC) of healthy mongrel dogs. RESULTS: Recombinant canine IL-2 purified from Escherichia coli or present in the supernatant of COS-7 cells transfected with pcDNA3.1-caIL-2 (COS-7 caIL-2 supernatant) was able to induce proliferation of CTLL-2 cells, thus showing their functional activity. In addition, purified rcaIL-2 and COS-7 caIL-2 supernatant stimulated resting canine PBMC proliferation to a level higher than baseline level. Neither COS-7 sccaIL-12 supernatant nor COS-7 caIL-2 supernatant alone was able to induce significant production of interferon gamma by resting PBMC. However, COS-7 sccaIL-12 supernatant in combination with COS-7 caIL-2 supernatant induced production of IFN-γ by those cells. CONCLUSIONS: The data shown herein suggest that the combination of canine recombinant IL-12 and IL-2 can be useful to promote cellular immune responses in dogs

IFN-gamma expression is up-regulated by peripheral blood mononuclear cells (PBMC) from non-exposed dogs upon Leishmania chagasi promastigote stimulation in vitro.

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2014-06-11T17:23:43Z No. of bitstreams: 1 Rodrigues CAT IFN-y expression....pdf: 284804 bytes, checksum: 8deea07db1b090684bd9aeacd243dab0 (MD5)Made available in DSpace on 2014-06-11T17:23:43Z (GMT). No. of bitstreams: 1 Rodrigues CAT IFN-y expression....pdf: 284804 bytes, checksum: 8deea07db1b090684bd9aeacd243dab0 (MD5) Previous issue date: 2009Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Laboratório Integrado de Patologia e Biointervenção. Salvador, BA, Brasil / Universidade Federal da Bahia. Faculdade de Medicina. Salvador, BA, Brasil / UFMS-MS. Depto. de Morfofisiologia. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Laboratório Integrado de Patologia e Biointervenção. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Laboratório Integrado de Patologia e Biointervenção. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Laboratório de Imunoparasitologia. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Laboratório Integrado de Patologia e Biointervenção. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Laboratório Integrado de Patologia e Biointervenção. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Laboratório Integrado de Patologia e Biointervenção. Salvador, BA, Brasil / UFBA. Faculdade de Medicina. Salvador, BA, BrasilWhile the response to Leishmania spp. is well characterized in mice and humans, much less is known concerning the canine immune response, particularly soon after exposure to the parasite. Early events are considered to be a determinant of infection outcome. To investigate the dog’s early immune response to L. chagasi, an in vitro priming system (PIV) using dog naı¨ve PBMC was established. Until now, dog PIV immune response to L. chagasi has not been assessed. We co-cultivated PBMC primarily stimulated with L. chagasi in vitro with autologous infected macrophages and found that IFN-g mRNA is up-regulated in these cells compared to control unstimulated cells. IL-4 and IL-10 mRNA expression by L. chagasi-stimulated PBMC was similar to control unstimulated PBMCwhen incubated with infected macrophages. Surprisingly, correlation studies showed that a lower IFN-g/IL-4 expression ratio correlated with a lower percentage of infection. We propose that the direct correlation between IFN-g/IL-4 ratio and parasite load is dependent on the higher correlation of both IFN-g and IL-4 expression with lower parasite infection. This PIV system was shown to be useful in evaluating the dog immune response to L. chagasi, and results indicate that a balance between IFN-g and IL-4 is associated with control of parasite infection in vitro

Parasitic load and histological aspects in different regions of the spleen of dogs with visceral leishmaniasis

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2018-02-26T13:48:42Z No. of bitstreams: 1 Bagues NC Parasitic load and histological aspects....pdf: 688389 bytes, checksum: 1bb681c1e1dd7799e6f764a3a9bfeaaf (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2018-02-26T13:58:56Z (GMT) No. of bitstreams: 1 Bagues NC Parasitic load and histological aspects....pdf: 688389 bytes, checksum: 1bb681c1e1dd7799e6f764a3a9bfeaaf (MD5)Made available in DSpace on 2018-02-26T13:58:56Z (GMT). No. of bitstreams: 1 Bagues NC Parasitic load and histological aspects....pdf: 688389 bytes, checksum: 1bb681c1e1dd7799e6f764a3a9bfeaaf (MD5) Previous issue date: 2018Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Grant CNPq 573839/2008-5).Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. ,Escola de Medicina Veterinária. Salvador, BA, Brasil / Instituto Nacional de Ciência e Tecnologia de Doenças Tropicais. Salvador, BA, BrasilFundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / Instituto Nacional de Ciência e Tecnologia de Doenças Tropicais. Salvador, BA, BrasilFundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / Instituto Nacional de Ciência e Tecnologia de Doenças Tropicais. Salvador, BA, BrasilLeishmania infantum causes from subclinical infection to severe disease in humans and dogs. The spleen is one of the organs most affected by the infection. Although evidence exists that the parasitic load distribution and histological alterations may not be homogeneous in the affected organs of naturally infected individuals, it has not been formally demonstrated using the current techniques used for studying the disease. In six dogs naturally infected with Leishmania, parasitic load and histological changes were compared in samples collected from the lower, middle and upper third of the spleen. Parasitic load in the spleen of the group of dogs was variable, revealing a difference of 61 times between animals with the lowest and the highest parasitism. The set of parasitic load values of each dog showed a cluster trend, when compared to the other animals. Nevertheless, the parasitic load values of each dog showed a variation ranging from 3.2 to 34.7 times between lowest and highest value. Histological changes showed recognizable variation in frequency (granulomas) or intensity (perisplenitis) in the spleen of 2 out of the 6 dogs. The agreement of histological findings between samples collected from the different thirds of the spleen was good (kappa coeficient, 0.61-0.80) very good (0.81-0.99) or perfect (1.00), for most of the parameters analyzed. Variability of parasitic load and, to a lesser extent, histological changes in spleen of dogs with visceral leishmaniasis is observed. Such variability may be taken in account in the design of studies on pathogenesis, vaccine and therapeutic drug development

Blocking IL-10 signaling with soluble IL-10 receptor restores in vitro specific lymphoproliferative response in dogs with leishmaniasis caused by Leishmania infantum.

rIL-10 plays a major role in restricting exaggerated inflammatory and immune responses, thus preventing tissue damage. However, the restriction of inflammatory and immune responses by IL-10 can also favor the development and/or persistence of chronic infections or neoplasms. Dogs that succumb to canine leishmaniasis (CanL) caused by L. infantum develop exhaustion of T lymphocytes and are unable to mount appropriate cellular immune responses to control the infection. These animals fail to mount specific lymphoproliferative responses and produce interferon gamma and TNF-alpha that would activate macrophages and promote destruction of intracellular parasites. Blocking IL-10 signaling may contribute to the treatment of CanL. In order to obtain a tool for this blockage, the present work endeavored to identify the canine casIL-10R1 amino acid sequence, generate a recombinant baculovirus chromosome encoding this molecule, which was expressed in insect cells and subsequently purified to obtain rcasIL-10R1. In addition, rcasIL-10R1 was able to bind to homologous IL-10 and block IL-10 signaling pathway, as well as to promote lymphoproliferation in dogs with leishmaniasis caused by L. infantum