RESUMO
BACKGROUND: Leptospirosis is a zoonotic disease caused by infection with spirochetes from Leptospira genus. It has been classified into at least 17 pathogenic species, with more than 250 serologic variants. This wide distribution may be a result of leptospiral ability to colonize the renal tubules of mammalian hosts, including humans, wildlife, and many domesticated animals. Previous studies showed that the expression of proteins belonging to the microbial heat shock protein (HSP) family is upregulated during infection and also during various stress stimuli. Several proteins of this family are known to have important roles in the infectious processes in other bacteria, but the role of HSPs in Leptospira spp. is poorly understood. In this study, we have evaluated the capacity of the protein GroEL, a member of HSP family, of interacting with host proteins and of stimulating the production of cytokines by macrophages. RESULTS: The binding experiments demonstrated that the recombinant GroEL protein showed interaction with several host components in a dose-dependent manner. It was also observed that GroEL is a surface protein, and it is secreted extracellularly. Moreover, two cytokines (tumor necrosis factor-α and interleukin-6) were produced when macrophages cells were stimulated with this protein. CONCLUSIONS: Our findings showed that GroEL protein may contribute to the adhesion of leptospires to host tissues and stimulate the production of proinflammatory cytokines during infection. These features might indicate an important role of GroEL in the pathogen-host interaction in the leptospirosis.
Assuntos
Chaperonina 60/imunologia , Citocinas/imunologia , Interações Hospedeiro-Patógeno/imunologia , Leptospira/metabolismo , Macrófagos/imunologia , Macrófagos/microbiologiaRESUMO
Paracoccidioides species cause paracoccidioidomycosis (PCM), a systemic mycosis highly prevalent in Brazil. Therapy of PCM has some issues that make studies for new therapeutic and vaccine targets relevant, such as the P. brasiliensis 60-kDa-heat-shock protein (PbHsp60), an immunogenic antigen that induces protection in experimental mice infection. Here, we investigated the relative expression of mRNA for PbHsp60 in P. brasiliensis in the different morphotypes of P. brasiliensis and in morphological transition phases. In addition, antibodies to rPbHsp60 were produced and used to analyze the location of PbHsp60 in yeast and hyphae by electron microscopy. The analyses showed a substantial increase in the relative amounts of HSP60 mRNA in yeast when compared to mycelium and an intermediate expression in transitional forms. Regarding the cell location, immunoelectron microscopy analysis revealed that PbHsp60 is within the cell wall. These observations suggest that this protein may be involved in the maintenance of the cell wall integrity and the interaction with the host for colonization, infection and pathogenesis.
Assuntos
Chaperonina 60/imunologia , Paracoccidioides/imunologia , RNA Mensageiro/imunologia , Animais , Antígenos de Fungos/imunologia , Expressão Gênica/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Paracoccidioides/patogenicidade , Reação em Cadeia da PolimeraseRESUMO
The present study is aimed at evaluating serological method using scFv anti-Strongyloides sp. and reporting the frequencies of the results with conventional parasitological technique (faeces) in elderly individuals. Among 112 elderly individuals (≥60 years of age), 14.28% were positive for at least one enteroparasite, with one individual positive for S. stercoralis. Sera were evaluated for the presence of anti-Strongyloides sp. antibodies using total or detergent fraction extracts of Strongyloides venezuelensis, which presented positivity rates of 19.64% and 10.71%, respectively. An anti-HSP60 single-chain variable fragment from Strongyloides sp. was used to detect parasite antigens, with 5.36% (6 individuals) of ELISA-positive individuals returning a positive result. While the serological test indicates previous or recent infection and may be limited by antigen purification, the anti-HSP60 method reflects the presence of Strongyloides sp. immune complexes and exhibits greater sensitivity and specificity. Our results demonstrate the variable occurrence of enteroparasites in elderly individuals residing in long-term nursing homes and validate a novel epidemiological tool to describe infection cases by Strongyloides sp.
Assuntos
Anticorpos Anti-Helmínticos/sangue , Complexo Antígeno-Anticorpo/sangue , Antígenos de Helmintos/sangue , Chaperonina 60/sangue , Anticorpos de Cadeia Única/sangue , Estrongiloidíase/diagnóstico , Idoso , Idoso de 80 Anos ou mais , Animais , Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/imunologia , Brasil , Chaperonina 60/imunologia , Fezes/parasitologia , Feminino , Instituição de Longa Permanência para Idosos , Humanos , Masculino , Pessoa de Meia-Idade , Casas de Saúde , Sensibilidade e Especificidade , Anticorpos de Cadeia Única/imunologia , Strongyloides/crescimento & desenvolvimento , Strongyloides/imunologia , Strongyloides/patogenicidade , Estrongiloidíase/sangue , Estrongiloidíase/imunologia , Estrongiloidíase/parasitologiaRESUMO
Shigellosis is a diarrheal disease that causes high mortality every year, especially in children, elderly and immunocompromised patients. Recently, resistance strains to antibiotic therapy are in the rise and the World Health Organization prioritizes the development of a safe vaccine against the most common causal agent of shigellosis, Shigella flexneri. This pathogen uses autotransporter proteins such as SigA, Pic and Sap to increase virulence and some of them have been described as highly immunogenic proteins. In this study, we used immune-informatics analysis to identify the most antigenic epitope as a vaccine candidate on three passenger domains of auto-transporter proteins encoded on the pathogenic island SHI-1, to induce immunity against S. flexneri. Epitope identification was done using various servers such as Bepipred, Bcepred, nHLAPRED, NetMHCII, Rankpep and IEDB and the final selection was done based on its antigenicity using the VaxiJen server. Moreover, to enhance immunity, the GroEL adjuvant was added to the final construct as a Toll-like receptor 2 (TLR2) agonist. On the other hand, to predict the tertiary structure, the I-TASSER server was used, and the best model was structurally validated using the ProSA-web software and the Ramachandran plot. Subsequently, the model was refined and used for docking and molecular dynamics analyses with TLR2, which demonstrated an appropriate and stable interaction. In summary, a potential subunit vaccine candidate, that contains B and T cell epitopes with proper physicochemical properties was designed. This multiepitope vaccine is expected to elicit robust humoral and cellular immune responses and vest protective immunity against S. flexneri.
Assuntos
Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Disenteria Bacilar/terapia , Serina Proteases/imunologia , Shigella flexneri/imunologia , Sistemas de Secreção Tipo V/imunologia , Adjuvantes Imunológicos/farmacologia , Antígenos de Bactérias/imunologia , Vacinas Bacterianas/uso terapêutico , Chaperonina 60/imunologia , Chaperonina 60/farmacologia , Biologia Computacional , Simulação por Computador , Disenteria Bacilar/microbiologia , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Humanos , Imunidade Celular , Imunidade Humoral , Imunogenicidade da Vacina , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Domínios Proteicos/imunologia , Receptor 2 Toll-Like/agonistas , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/uso terapêuticoRESUMO
Paracoccidioidomycosis (PCM) is a systemic mycosis autochthonous to Latin America and endemic to Brazil, which has the majority of the PCM cases. PCM is acquired through the inhalation of propagules of fungi from genus Paracoccidioides spp. and mainly affects the lungs. We have previously shown that P. brasiliensis-infected mice treated with single-dose of recombinant 60-kDa-heat shock protein from P. brasiliensis (rPbHsp60) had a worsening infection in comparison to animals only infected. In this study, we investigate whether the treatment of infected mice with PB_HSP60 gene cloned into a plasmid (pVAX1-PB_HSP60) would result in efficient immune response and better control of the disease. The harmful impact of single-dose therapy with protein was not seen with plasmid preparations. Most importantly, three doses of pVAX1-PB_HSP60 and protein induced a beneficial effect in experimental PCM with a reduction in fungal load and lung injury when compared with infected mice treated with pVAX1 or PBS. The increase of the cytokines IFN-γ, TNF, and IL-17 and the decrease of IL-10 observed after treatment with three doses of pVAX1-PB_HSP60 appears to be responsible for the control of infection. These results open perspectives of the therapeutic use of Hsp60 in PCM.
Assuntos
Chaperonina 60/imunologia , Vacinas Fúngicas/imunologia , Paracoccidioides/imunologia , Paracoccidioidomicose/imunologia , Paracoccidioidomicose/prevenção & controle , Vacinas de DNA/imunologia , Animais , Antígenos de Fungos/imunologia , Chaperonina 60/genética , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Vacinas Fúngicas/genética , Imunização , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Paracoccidioides/genética , Paracoccidioidomicose/genética , Paracoccidioidomicose/microbiologia , Prognóstico , Vacinas de DNA/genéticaRESUMO
BACKGROUND: The 90-kDa heat-shock protein (Hsp90) from Nicotiana benthamiana (NbHsp90.3) is a promising adjuvant, especially for those vaccines that require a T cell-mediated immune response. Toxoplasma gondii SAG1 is considered one of the most important antigens for the development of effective subunit vaccines. Some epitopes located in the SAG1 C-terminus region have showed a strong humoral and cellular immune response. In the present study, we aimed to assess the efficacy of NbHsp90.3 as carrier/adjuvant of SAG1-derived peptide (SAG1HC) in a T. gondii infection murine model. METHODS: In the present study, C57BL/6 mice were intraperitoneal immunized with the NbHsp90.3-SAG1HC fusion protein (NbHsp90.3-SAG1HC group), mature SAG1 (SAG1m group), NbHsp90.3 (NbHsp90.3 group) or PBS buffer 1× (PBS group). The levels of IgG antibodies and the cytokine profile were determined by ELISA. Two weeks after the last immunization, all mice were orally challenged with 20 cysts of T. gondii Me49 strain and the number of brain cysts was determined. In addition, both humoral and cellular immune responses were also evaluated during the acute and chronic phase of T. gondii infection by ELISA. RESULTS: The characterization of the immune response generated after vaccination with NbHsp90.3 as an adjuvant showed that NbHsp90.3-SAG1HC-immunized mice produced antibodies that were able to recognize not only rSAG1m but also the native SAG1 present in the total lysate antigen extract (SAG1TLA) from T. gondii tachyzoites, while control groups did not. Furthermore, anti-rSAG1m IgG2a/2b antibodies were significantly induced. In addition, only the spleen cell cultures from NbHsp90.3-SAG1HC-immunized mice showed a significantly increased production of IFN-γ. During the chronic phase of T. gondii infection, the antibodies generated by the infection were unable to detect the recombinant protein, but they did react with TLA extract. In addition, splenocytes from all groups showed a high production of IFN-γ when stimulated with rGRA4, but only those from NbHsp90.3-SAG1HC group stimulated with rSAG1m showed high production of IFN-γ. Finally, NbHsp90.3-SAG1HC-immunized mice exhibited a significant reduction in the cyst load (56%) against T. gondii infection. CONCLUSIONS: We demonstrated that NbHsp90.3 enhances the humoral and cell-mediated immune response through a Th1 type cytokine production. Mice vaccinated with NbHsp90.3-SAG1HC exhibited a partial protection against T. gondii infection and it was correlated with the induction of memory immune response. We developed and validated a vaccine formulation which, to our knowledge, for the first time includes the NbHsp90.3 protein covalently fused to a peptide from T. gondii SAG1 protein that contains T- and B-cell epitopes.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Antígenos de Protozoários/imunologia , Chaperonina 60/imunologia , Nicotiana/química , Proteínas de Protozoários/imunologia , Vacinas Protozoárias/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Citocinas/imunologia , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Feminino , Imunidade Celular , Imunidade Humoral , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos C57BL , Vacinas Protozoárias/administração & dosagem , Toxoplasma , Toxoplasmose Animal/prevenção & controleRESUMO
The conventional treatment for fungal diseases usually shows long periods of therapy and the high frequency of relapses and sequels. New strategies of the treatment are necessary. We have shown that the Mycobacterium leprae HSP65 gene can be successfully used as therapy against murine Paracoccidioidomycosis (PCM). Here, we described the methodology of DNAhsp65 immunotherapy in mice infected with the dimorphic fungus Paracoccidioides brasiliensis, one of PCM agent, evaluating cytokines levels, fungal burden, and lung injury. Our results provide a new prospective on the immunotherapy of mycosis.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Chaperonina 60/imunologia , Vacinas Fúngicas/imunologia , Paracoccidioidomicose/imunologia , Vacinas de DNA/imunologia , Animais , Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Chaperonina 60/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Vacinas Fúngicas/genética , Imunoterapia/métodos , Ativação Linfocitária/imunologia , Camundongos , Óxido Nítrico/metabolismo , Paracoccidioidomicose/microbiologia , Paracoccidioidomicose/prevenção & controle , Paracoccidioidomicose/terapia , Plasmídeos/genética , Baço/imunologia , Baço/metabolismo , Baço/patologia , Vacinas de DNA/genéticaRESUMO
Previously we showed that 65-kDa Mycobacterium leprae heat shock protein (Hsp65) is a target for the development of a tuberculosis vaccine. Here we evaluated peripheral blood mononuclear cells (PBMC) from healthy individuals or tuberculosis patients stimulated with two forms of Hsp65 antigen, recombinant DNA that encodes Hsp65 (DNA-HSP65) or recombinant Hsp65 protein (rHsp65) in attempting to mimic a prophylactic or therapeutic study in vitro, respectively. Proliferation and cytokine-producing CD4+ or CD8+ cell were assessed by flow cytometry. The CD4+ cell proliferation from healthy individuals was stimulated by DNA-HSP65 and rHsp65, while CD8+ cell proliferation from healthy individuals or tuberculosis patients was stimulated by rHSP65. DNA-HSP65 did not improve the frequency of IFN-gamma+ cells from healthy individuals or tuberculosis patients. Furthermore, we found an increase in the frequency of IL-10-producing cells in both groups. These findings show that Hsp65 antigen activates human lymphocytes and plays an immune regulatory role that should be addressed as an additional antigen for the development of antigen-combined therapies.
Assuntos
Proteínas de Bactérias/imunologia , Chaperonina 60/imunologia , Imunidade Celular , Ativação Linfocitária , Tuberculose/imunologia , Adulto , Proteínas de Bactérias/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Chaperonina 60/genética , Citotoxicidade Imunológica , Feminino , Voluntários Saudáveis , Humanos , Interferon gama/biossíntese , Interferon gama/imunologia , Interleucina-10/biossíntese , Interleucina-10/imunologia , Leucócitos Mononucleares/imunologia , Macrófagos Alveolares/imunologia , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/imunologia , Proteínas Recombinantes/imunologia , Vacinas contra a Tuberculose/imunologia , Regulação para Cima , Vacinas de DNA/farmacologia , Adulto JovemRESUMO
Phage display is a powerful technology that selects specific proteins or peptides to a target. We have used Phage Display to select scFv (single-chain variable fragment) clones from a combinatorial library against total proteins of Strongyloides venezuelensis. After scFv characterization, further analysis demonstrated that this recombinant fragment of antibody was able to bind to an S. venezuelensis antigenic fraction of ~65 kDa, present in the body periphery and digestive system of infective larvae (L3), as demonstrated by immunofluorescence. Mass spectrometry results followed by bioinformatics analysis showed that this antigenic fraction was a heat shock protein 60 (HSP60) of Strongyloides sp. The selected scFv was applied in serodiagnosis by immune complexes detection in serum samples from individuals with strongyloidiasis using a sandwich enzyme-linked immunosorbent assay (ELISA), showing sensitivity of 97.5% (86.84-99.94), specificity of 98.81 (93.54-99.97), positive likelihood ratio of 81.60 and an area under the curve of 0.9993 (0.9973-1.000). Our study provided a novel monoclonal scFv antibody fragment which specifically bound to HSP60 of Strongyloides sp. and was applied in the development of an innovative serodiagnosis method for the human strongyloidiasis.
Assuntos
Antígenos de Helmintos/imunologia , Chaperonina 60/imunologia , Anticorpos de Cadeia Única/metabolismo , Strongyloides/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Animais , Antígenos de Helmintos/sangue , Feminino , Humanos , Larva/metabolismo , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Ratos , Ratos Wistar , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética , Strongyloides/crescimento & desenvolvimento , Adulto JovemRESUMO
Despite substantial efforts in recent years toward the development of new vaccines and drugs against tuberculosis (TB), success has remained elusive. Immunotherapy of TB with mycobacterial Hsp65 as a DNA vaccine (DNA-hsp65) results in a reduction of systemic bacterial loads and lung tissue damage, but the high homology of Hsp65 with the mammalian protein raises concern that pathological autoimmune responses may also be triggered. We searched for autoimmune responses elicited by DNA-hsp65 immunotherapy in mice chronically infected with TB by evaluating the humoral immune response and comprehensive histopathology using stereology. Cross-reactive antibodies between mycobacterial and mammalian Hsp60/65 were detected; however, no signs of pathological autoimmunity were found up to 60 days after the end of the therapy.
Assuntos
Anticorpos Antibacterianos/imunologia , Autoimunidade/imunologia , Proteínas de Bactérias/imunologia , Chaperonina 60/imunologia , Proteínas Mitocondriais/imunologia , Mycobacterium leprae/imunologia , Vacinas de DNA/imunologia , Animais , Autoimunidade/efeitos dos fármacos , Proteínas de Bactérias/administração & dosagem , Chaperonina 60/administração & dosagem , Chaperonina 60/antagonistas & inibidores , Reações Cruzadas/efeitos dos fármacos , Reações Cruzadas/imunologia , Imunidade Humoral/efeitos dos fármacos , Imunidade Humoral/imunologia , Imunoterapia/métodos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Mitocondriais/antagonistas & inibidores , Vacinas contra a Tuberculose/administração & dosagem , Vacinas contra a Tuberculose/imunologia , Vacinas de DNA/administração & dosagemRESUMO
Tuberculosis (TB) is a serious public health problem. Development of experimental models and vaccines are essential to elucidate physiopathological mechanisms and to control the disease. Vascular endothelial growth factor (VEGF) is a potent activator of vascular permeability and angiogenesis. VEGF seems to participate in breakdown of the blood brain-barrier (BBB) in tuberculous meningitis (TBM), contributing to worsening of disease. Therefore, the objective here was to extent the characterization of our previously described murine model of central nervous system TB (CNS-TB) by describing the VEGF participation in the CNS disease, and suggesting a vaccination plan in mice. Plasmid encoding DNA protein antigen DNA-hsp65 has been described as a protector against TB infection and was used here to test its effectiveness in the prevention of VEGF production and TB disease. Vaccinated mice and its controls were injected with Mycobacterium bovis bacillus Calmette-Guerin (BCG) in cerebellum. Four weeks after BCG injection, mice were perfused and brains were paraffin-embedded for VEGF expression analysis. We observed VEGF immunohistochemical expression in TBM and granulomas in non-vaccinated mice. The DNA-hsp65 treatment blocked the expression of VEGF in mice TBM. Therefore, our murine model indicated the VEGF participation in the physiopathology of CNS-TB and the potential prevention of the DNA-hsp65 in the disease progression.
Assuntos
Proteínas de Bactérias/imunologia , Chaperonina 60/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose do Sistema Nervoso Central/metabolismo , Vacinas de DNA/imunologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Animais , Proteínas de Bactérias/genética , Doenças Cerebelares/metabolismo , Doenças Cerebelares/prevenção & controle , Cerebelo/metabolismo , Chaperonina 60/genética , Modelos Animais de Doenças , Esquemas de Imunização , Masculino , Camundongos , Mycobacterium bovis , Tuberculoma Intracraniano/metabolismo , Tuberculoma Intracraniano/prevenção & controle , Tuberculose do Sistema Nervoso Central/prevenção & controle , Tuberculose Meníngea/metabolismo , Tuberculose Meníngea/prevenção & controleRESUMO
Chromoblastomycosis (CBM) is a chronic subcutaneous mycosis, caused by several dimorphic, pigmented dematiaceous fungi. Patients with the disease are still considered a therapeutic challenge, mainly due to its recalcitrant nature. There is no "gold standard" treatment for this neglected mycosis, but rather there are several treatment options. Chemotherapy alternatives include 5-flucytosine, itraconazole, terbinafine, fluconazole, thiabendazole, ketoconazole and amphotericin B, although the healing of severe cases is still uncommon. However, several studies have reported the DNA vaccine to be promising in the treatment for fungal infections; this vaccine allows the host to restore depressed cellular immunity, minimizing the toxic effects from conventional antifungal therapies. This work was therefore carried out aiming to establish a suitable model for experimental CBM, suggesting also new therapies, including DNA-hsp65 vaccine. By analyzing the morphometrical and histopathological aspects and by quantifying the fungal burden, the results showed the establishment of a chronic, although transitory, experimental CBM model with lesions similar to those presented in humans. A treatment regimen using intralesional itraconazole or amphotericin B was effective in treating experimental CBM, as was a therapy using naked DNA-hsp65 vaccine. It has also been shown that chemotherapy associated with DNA-hsp65 vaccine is promising in the treatment for CBM.
Assuntos
Proteínas de Bactérias/imunologia , Chaperonina 60/imunologia , Cromoblastomicose/terapia , Fatores Imunológicos/administração & dosagem , Vacinas de DNA/administração & dosagem , Animais , Antifúngicos/uso terapêutico , Proteínas de Bactérias/genética , Chaperonina 60/genética , Cromoblastomicose/microbiologia , Cromoblastomicose/patologia , Contagem de Colônia Microbiana , Modelos Animais de Doenças , Histocitoquímica , Fatores Imunológicos/genética , Masculino , Ratos Wistar , Resultado do Tratamento , Vacinas de DNA/genéticaRESUMO
We evaluated associations between the concentrations of heat shock proteins (hsp60 and hsp70) and their respective antibodies, alterations in maternal reproductive performance, and fetal malformations in pregnant rats with hyperglycemia. Mild diabetes (MD) or severe diabetes (SD) was induced in Sprague-Dawley rats prior to mating; non-treated non-diabetic rats (ND) served as controls. On day 21 of pregnancy, maternal blood was analyzed for hsp60 and hsp70 and their antibodies; and fetuses were weighed and analyzed for congenital malformations. Hsp and anti-hsp levels were correlated with blood glucose levels during gestation. There was a positive correlation between hsp60 and hsp70 levels and the total number of malformations (R = 0.5908, P = 0.0024; R = 0.4877, P = 0.0134, respectively) and the number of malformations per fetus (R = 0.6103, P = 0.0015; R = 0.4875, P = 0.0134, respectively). The anti-hsp60 IgG concentration was correlated with the number of malformations per fetus (R = 0.3887, P = 0.0451) and the anti-hsp70 IgG level correlated with the total number of malformations (R = 0.3999, P = 0.0387). Moreover, both hsp and anti-hsp antibodies showed negative correlations with fetal weight. The results suggest that there is a relationship between hsp60 and hsp70 levels and their respective antibodies and alterations in maternal reproductive performance and impaired fetal development and growth in pregnancies associated with diabetes.
Assuntos
Chaperonina 60/metabolismo , Diabetes Mellitus Experimental/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Prenhez , Animais , Anticorpos/sangue , Anticorpos/imunologia , Glicemia/análise , Chaperonina 60/imunologia , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/patologia , Feminino , Desenvolvimento Fetal , Proteínas de Choque Térmico HSP70/imunologia , Gravidez , Ratos , Ratos Sprague-DawleyRESUMO
In the last several years, the use of dendritic cells has been studied as a therapeutic strategy against tumors. Dendritic cells can be pulsed with peptides or full-length protein, or they can be transfected with DNA or RNA. However, comparative studies suggest that transfecting dendritic cells with messenger RNA (mRNA) is superior to other antigen-loading techniques in generating immunocompetent dendritic cells. In the present study, we evaluated a new therapeutic strategy to fight tuberculosis using dendritic cells and macrophages transfected with Hsp65 mRNA. First, we demonstrated that antigen-presenting cells transfected with Hsp65 mRNA exhibit a higher level of expression of co-stimulatory molecules, suggesting that Hsp65 mRNA has immunostimulatory properties. We also demonstrated that spleen cells obtained from animals immunized with mock and Hsp65 mRNA-transfected dendritic cells were able to generate a mixed Th1/Th2 response with production not only of IFN-γ but also of IL-5 and IL-10. In contrast, cells recovered from mice immunized with Hsp65 mRNA-transfected macrophages were able to produce only IL-5. When mice were infected with Mycobacterium tuberculosis and treated with antigen-presenting cells transfected with Hsp65 mRNA (therapeutic immunization), we did not detect any decrease in the lung bacterial load or any preservation of the lung parenchyma, indicating the inability of transfected cells to confer curative effects against tuberculosis. In spite of the lack of therapeutic efficacy, this study reports for the first time the use of antigen-presenting cells transfected with mRNA in experimental tuberculosis.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Proteínas de Bactérias/administração & dosagem , Chaperonina 60/administração & dosagem , Mycobacterium tuberculosis/imunologia , RNA Mensageiro/imunologia , Vacinas contra a Tuberculose/administração & dosagem , Tuberculose/imunologia , Animais , Proteínas de Bactérias/efeitos adversos , Proteínas de Bactérias/imunologia , Chaperonina 60/efeitos adversos , Chaperonina 60/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/efeitos adversos , Baço/imunologia , Transfecção , Tuberculose/prevenção & controle , Vacinas contra a Tuberculose/efeitos adversos , Vacinas contra a Tuberculose/imunologiaRESUMO
CD4(+) Foxp3(+) regulatory T cells inhibit the production of interferon-γ, which is the major mediator of protection against Mycobacterium tuberculosis infection. In this study, we evaluated whether the protection conferred by three different vaccines against tuberculosis was associated with the number of spleen and lung regulatory T cells. We observed that after homologous immunization with the 65 000 molecular weight heat-shock protein (hsp 65) DNA vaccine, there was a significantly higher number of spleen CD4(+) Foxp3(+) cells compared with non-immunized mice. Heterologous immunization using bacillus Calmette-Guérin (BCG) to prime and DNA-hsp 65 to boost (BCG/DNA-hsp 65) or BCG to prime and culture filtrate proteins (CFP)-CpG to boost (BCG/CFP-CpG) induced a significantly higher ratio of spleen CD4(+) /CD4(+) Foxp3(+) cells compared with non-immunized mice. In addition, the protection conferred by either the BCG/DNA-hsp 65 or the BCG/CFP-CpG vaccines was significant compared with the DNA-hsp 65 vaccine. Despite the higher ratio of spleen CD4(+) /CD4(+) Foxp3(+) cells found in BCG/DNA-hsp 65-immunized or BCG/CFP-CpG-immunized mice, the lungs of both groups of mice were better preserved than those of DNA-hsp 65-immunized mice. These results confirm the protective efficacy of BCG/DNA-hsp 65 and BCG/CFP-CpG heterologous prime-boost vaccines and the DNA-hsp 65 homologous vaccine. Additionally, the prime-boost regimens assayed here represent a promising strategy for the development of new vaccines to protect against tuberculosis because they probably induce a proper ratio of CD4(+) and regulatory (CD4(+) Foxp3(+) ) cells during the immunization regimen. In this study, this ratio was associated with a reduced number of regulatory cells and no injury to the lungs.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Vacinas contra a Tuberculose/imunologia , Vacinas de DNA/imunologia , Animais , Proteínas de Bactérias/imunologia , Chaperonina 60/imunologia , Feminino , Fatores de Transcrição Forkhead/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium tuberculosis/imunologia , Baço/imunologia , Tuberculose/imunologia , Tuberculose/patologiaRESUMO
BACKGROUND: Previous studies have established that mycobacterial infections ameliorate allergic inflammation. However, a non-infectious approach that controls allergic responses might represent a safer and more promising strategy. The 60-65 kDa heat shock protein (Hsp) family is endowed with anti-inflammatory properties, but it is still unclear whether and how single mycobacterial Hsp control allergic disorders. OBJECTIVE: Therefore, in this study we determined whether the administration of Mycobacterial leprae Hsp65 expressed by recombinant a DNA plasmid could attenuate a previously established allergic response. METHODS: We used an experimental model of airway allergic inflammation to test the effects of immunotherapy with DNA encoding Hsp65. Allergic mice, previously sensitized and challenged with ovalbumin, were treated with tree intramuscular doses of recombinant DNA encoding Hsp65. After treatment, mice received a second allergen challenge and the allergic response was measured. RESULTS: We found that immunotherapy attenuated eosinophilia, pulmonary inflammation, Th2 cytokine and mucus production. Moreover, we showed that the inhibition of allergic response is dependent on IL-10 production. Both Hsp65 and allergen-specific IL-10-producing cells contributed to this effect. Cells transferred from DNA-immunized mice to allergic mice migrated to allergic sites and down-modulated the Th2 response. CONCLUSIONS AND CLINICAL RELEVANCE: Our findings clearly show that immunotherapy with DNA encoding Hsp65 can attenuate an established Th2 allergic inflammation through an IL-10-dependent mechanism; moreover, the migration of allergen- and Hsp65-specific cells to the allergic sites exerts a fundamental role. This work represents a novel contribution to the understanding of immune regulation by Hsp65 in allergic diseases.
Assuntos
Proteínas de Bactérias , Chaperonina 60 , DNA Recombinante/administração & dosagem , Imunoterapia/métodos , Interleucina-10/metabolismo , Hipersensibilidade Respiratória/terapia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Chaperonina 60/genética , Chaperonina 60/imunologia , DNA Recombinante/imunologia , Modelos Animais de Doenças , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Interleucina-10/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mycobacterium leprae/imunologia , Hipersensibilidade Respiratória/imunologia , Resultado do TratamentoRESUMO
Hsp60 is an abundant and highly conserved family of intracellular molecules. Increased levels of this family of proteins have been observed in the extracellular compartment in chronic inflammation. Administration of M. leprae Hsp65 [WT] in [NZBxNZW]F(1) mice accelerates the Systemic Lupus Erythematosus [SLE] progression whereas the point mutated K(409)A Hsp65 protein delays the disease. Here, the biological effects of M. leprae Hsp65 Leader pep and K(409)A pep synthetic peptides, which cover residues 352-371, are presented. Peptides had immunomodulatory effects similar to that observed with their respective proteins on survival and the combined administration of K(409)A+Leader pep or K(409)A pep+WT showed that the mutant forms were able to inhibit the deleterious effect of WT on mortality, indicating the neutralizing potential of the mutant molecules in SLE progression. Molecular modeling showed that replacing Lysine by Alanine affects the electrostatic potential of the 352-371 region. The number of interactions observed for WT is much higher than for Hsp65 K(409)A and mouse Hsp60. The immunomodulatory effects of the point-mutated protein and peptide occurred regardless of the catalytic activity. These findings may be related to the lack of effect on survival when F(1) mice were inoculated with Hsp60 or K(409)A pep. Our findings indicate the use of point-mutated Hsp65 molecules, such as the K(409)A protein and its corresponding peptide, that may minimize or delay the onset of SLE, representing a new approach to the treatment of autoimmune diseases.
Assuntos
Proteínas de Bactérias/imunologia , Chaperonina 60/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Mutação de Sentido Incorreto , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/genética , Chaperonina 60/administração & dosagem , Chaperonina 60/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Imunização , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Lúpus Eritematoso Sistêmico/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos NZB , Camundongos Endogâmicos , Dados de Sequência Molecular , Peptídeos/administração & dosagem , Peptídeos/genética , Peptídeos/imunologia , Sinais Direcionadores de Proteínas/genéticaRESUMO
Investigating the proteolytic activity of the recombinant Mycobacterium leprae Heat Shock Protein of 65 kDa (rHsp65), chaperonin 2 (cpn2), we observed that it displays high instability. The fragmentation process starts at the C-terminus followed by progressive degradation of the N-terminus, which leads to a stable fragment comprising the middle region of the molecule. Urea was able to prevent autolysis, probably due to its denaturing action, while EDTA increased degradation levels indicating the need for metal ions. Peptides originated from autolysis were purified and analyzed by mass spectrometry, generating a continuous map. Since the bacteria and mammalian Hsp60 are known to be targets of the immune response and have been implicated in autoimmune diseases and chronic inflammation, the in vivo effect of rHsp65 peptides was evaluated in the spontaneous Systemic Lupus Erythematosus (SLE) model developed by the (NZB/NZW)F(1) mouse hybrids, and their individual anti-rHsp65 IgG2a/IgG1 antibody titer ratio was determined. The results showed orientation toward a T(H)1 responsiveness, and the treatment with the rHsp65 peptides diminished the environmental variance of the survival time of treated animals. These results outline the fact that environmental factors may also act through the modified stability expression of Heat Shock Proteins intervening during autoimmune processes.
Assuntos
Autoanticorpos/sangue , Doenças Autoimunes/diagnóstico , Proteínas de Bactérias/imunologia , Biomarcadores/sangue , Chaperonina 60/imunologia , Mycobacterium leprae/imunologia , Animais , Modelos Animais de Doenças , Feminino , Camundongos , Doenças dos Roedores/imunologia , Análise de SobrevidaRESUMO
Induction of immune tolerance as therapeutic approach for autoimmune diseases constitutes a current research focal point. In this sense, we aimed to evaluate an altered peptide ligand (APL) for induction of peripheral tolerance in patients with rheumatoid arthritis (RA). A novel T-cell epitope from human heat-shock protein 60 (Hsp60), an autoantigen involved in the pathogenesis of RA, was identified by bioinformatics tools and an APL was design starting from this epitope. We investigated the ability of this APL for inducing regulatory T cells (Treg cells) in mice and evaluated the therapeutic effect of this peptide in an adjuvant-induced arthritis (AA) rat model. Clinical score, TNFα levels and histopathology were monitored, as well as the capacity of this APL for inducing Treg cells. Finally, the potentialities of the APL for inducing Treg cells were evaluated in ex vivo assays using mononuclear cells isolated from peripheral blood (PBMC). The APL induced an increase of the proportions of Treg cells in the draining lymph nodes of the injected site in mice. The APL efficiently inhibited the course of AA, with significant reduction of the clinical and histopathology score. This effect was associated with an increase of the proportions of Treg cells and a decrease of TNFα levels in spleen. Finally, stimulation of PBMCs from RA patients by the APL increases the proportions of the CD4(+)CD25(high)FoxP3(+) Treg cells. These results indicate a therapeutic potentiality of APL and support further investigation of this candidate drug for treatment of RA.
Assuntos
Artrite Experimental/tratamento farmacológico , Artrite Experimental/imunologia , Chaperonina 60/imunologia , Epitopos de Linfócito T/imunologia , Linfócitos T Reguladores/imunologia , Adolescente , Adulto , Idoso , Animais , Chaperonina 60/uso terapêutico , Feminino , Humanos , Tolerância Imunológica , Ligantes , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Masculino , Camundongos , Pessoa de Meia-Idade , Peptídeos/imunologia , Ratos , Ratos Endogâmicos Lew , Linfócitos T Reguladores/metabolismoRESUMO
This work investigated the effect of previous Mycobacterium avium exposure on the protective ability of the DNA vaccine pVAXhsp65 against inflammation in the pulmonary parenchyma. BALB/c mice were presensitized with heat-killed M. avium and then immunized with three doses of pVAXhsp65 prior to challenge with Mycobacterium tuberculosis. M. avium sensitization induced high levels of spontaneous IL-5 production that were concomitant with a positive delayed-type hypersensitivity reaction; antigen-specific IFN-γ production was also observed upon splenocyte stimulation. Prior exposure to M. avium resulted in altered cytokine and antibody production induced by immunization with pVAXhsp65; instead of a Th1 response, vaccinated mice previously exposed to M. avium developed a strong Th2 response. This switch to a Th2 response coincided with the loss of the anti-inflammatory effect of pVAXhsp65 vaccination previously observed in the pulmonary parenchyma of mice infected with M. tuberculosis. These results suggest that exposure to environmental mycobacteria can modulate immune responses induced by mycobacterial vaccines other than bacillus Calmette-Guérin.