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1.
Arthrosc Tech ; 9(1): e71-e78, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32021777

RESUMO

The prevalence of ulnar collateral ligament injuries and reconstructions among overhead throwing athletes has significantly risen in recent years. Surgical reconstruction has become the main treatment modality for athletes who have failed conservative treatment and wish to return to their sport. There has been an increased interest in graft augmentation in ligament reconstruction surgeries as surgeons search for ways to decrease the chance of graft failure. Augmented graft techniques have been described for other procedures. We present a technique that incorporates a cross-linked suture tape into either a palmaris longus or gracilis tendon autograft or allograft for ulnar collateral ligament reconstruction. This may allow for a biomechanically stronger construct because it appears this is the case in other settings. The goal is that this would lead to decreased rates of failure or possibly allow athletes to return at an accelerated rate.

2.
Front Immunol ; 10: 1456, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31293599

RESUMO

Background: Chagas cardiomyopathy is caused by Trypanosoma cruzi (Tc). Two antigenic candidates, TcG2 and TcG4, are recognized by antibodies in naturally infected dogs and humans; and these vaccine candidates provided protection from Tc infection in mice and dogs. Trypanosoma rangeli (Tr) is non-pathogenic to mammals and shown to elicit cross-reactive anti-Tc antibodies. In this study, we investigated if fixed Tr (fTr) can further enhance the efficacy of the TcG2/TcG4 DNA vaccine. Methods and Results: C57BL/6 mice were immunized with TcG2/TcG4 DNA vaccine and fTr (delivered as an adjuvant or in prime-boost approach), and challenged with Tc. Serology studies showed that fTr (±quil-A) elicited Tc- and Tr-reactive IgGs that otherwise were not stimulated by TcG2/TcG4 vaccine only, and quil-A had suppressive effects on fTr-induced IgGs. After challenge infection, TcG2/TcG4-vaccinated mice exhibited potent expansion of antigen- and Tc-specific IgGs that were not boosted by fTr±quil-A. Flow cytometry analysis showed that TcG2/TcG4-induced dendritic cells (DC) and macrophages (Mφ) responded to challenge infection by expression of markers of antigen uptake, processing, and presentation, and production of pro-inflammatory cytokines. TcG2/TcG4-induced CD4+T cells acquired Th1 phenotype and expressed markers that orchestrate adaptive immunity. A fraction of vaccine-induced CD4+T cells exhibited iTreg phenotype responsible for aversion of self-injurious immune responses. Further, TcG2/TcG4-vaccinated mice exhibited potent expansion of poly-functional CD8+T cells with TNF-α/IFN-γ production and cytolytic phenotype post-infection. Subsequently, tissue parasites and pathology were hardly detectable in TcG2/TcG4-vaccinated/infected mice. Inclusion of fTr±quil-A had no clear additive effects in improving the Tc-specific adaptive immunity and parasite control than was noted in mice vaccinated with TcG2/TcG4 alone. Non-vaccinated mice lacked sufficient activation of Th1 CD4+/CD8+T cells, and exhibited >10-fold higher levels of tissue parasite burden than was noted in vaccinated/infected mice. Conclusion: TcG2/TcG4 vaccine elicits highly effective immunity, and inclusion of fTr is not required to improve the efficacy of DNA vaccine against acute Tc infection in mice.


Assuntos
Antígenos de Protozoários/farmacologia , Doença de Chagas/prevenção & controle , Imunidade Celular/efeitos dos fármacos , Imunização Secundária , Vacinas Protozoárias/farmacologia , Células Th1/imunologia , Trypanosoma cruzi/imunologia , Vacinas de DNA/farmacologia , Animais , Antígenos de Protozoários/imunologia , Doença de Chagas/imunologia , Doença de Chagas/patologia , Feminino , Camundongos , Vacinas Protozoárias/imunologia , Células Th1/patologia , Vacinas de DNA/imunologia
3.
J Innate Immun ; 9(2): 203-216, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27902980

RESUMO

BACKGROUND: Chronic inflammation and oxidative stress are hallmarks of chagasic cardiomyopathy (CCM). In this study, we determined if microparticles (MPs) generated during Trypanosoma cruzi (Tc) infection carry the host's signature of the inflammatory/oxidative state and provide information regarding the progression of clinical disease. METHODS: MPs were harvested from supernatants of human peripheral blood mononuclear cells in vitro incubated with Tc (control: LPS treated), plasma of seropositive humans with a clinically asymptomatic (CA) or symptomatic (CS) disease state (vs. normal/healthy [NH] controls), and plasma of mice immunized with a protective vaccine before challenge infection (control: unvaccinated/infected). Macrophages (mφs) were incubated with MPs, and we probed the gene expression profile using the inflammatory signaling cascade and cytokine/chemokine arrays, phenotypic markers of mφ activation by flow cytometry, cytokine profile by means of an ELISA and Bioplex assay, and oxidative/nitrosative stress and mitotoxicity by means of colorimetric and fluorometric assays. RESULTS: Tc- and LPS-induced MPs stimulated proliferation, inflammatory gene expression profile, and nitric oxide (∙NO) release in human THP-1 mφs. LPS-MPs were more immunostimulatory than Tc-MPs. Endothelial cells, T lymphocytes, and mφs were the major source of MPs shed in the plasma of chagasic humans and experimentally infected mice. The CS and CA (vs. NH) MPs elicited >2-fold increase in NO and mitochondrial oxidative stress in THP-1 mφs; however, CS (vs. CA) MPs elicited a more pronounced and disease-state-specific inflammatory gene expression profile (IKBKB, NR3C1, and TIRAP vs. CCR4, EGR2, and CCL3), cytokine release (IL-2 + IFN-γ > GCSF), and surface markers of mφ activation (CD14 and CD16). The circulatory MPs of nonvaccinated/infected mice induced 7.5-fold and 40% increases in ∙NO and IFN-γ production, respectively, while these responses were abolished when RAW264.7 mφs were incubated with circulatory MPs of vaccinated/infected mice. CONCLUSION: Circulating MPs reflect in vivo levels of an oxidative, nitrosative, and inflammatory state, and have potential utility in evaluating disease severity and the efficacy of vaccines and drug therapies against CCM.


Assuntos
Micropartículas Derivadas de Células/metabolismo , Doença de Chagas/imunologia , Inflamação/imunologia , Leucócitos Mononucleares/imunologia , Macrófagos/imunologia , Trypanosoma cruzi/imunologia , Vacinas/imunologia , Animais , Doenças Assintomáticas , Linhagem Celular , Micropartículas Derivadas de Células/imunologia , Citocinas/metabolismo , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Humanos , Inflamação/genética , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Estresse Oxidativo , Vacinação
4.
Int J Proteomics ; 2016: 1384523, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27635260

RESUMO

Nitric oxide (NO) protects the heart against ischemic injury; however, NO- and superoxide-dependent S-nitrosylation (S-NO) of cysteines can affect function of target proteins and play a role in disease outcome. We employed 2D-GE with thiol-labeling FL-maleimide dye and MALDI-TOF MS/MS to capture the quantitative changes in abundance and S-NO proteome of HF patients (versus healthy controls, n = 30/group). We identified 93 differentially abundant (59-increased/34-decreased) and 111 S-NO-modified (63-increased/48-decreased) protein spots, respectively, in HF subjects (versus controls, fold-change | ≥1.5|, p ≤ 0.05). Ingenuity pathway analysis of proteome datasets suggested that the pathways involved in phagocytes' migration, free radical production, and cell death were activated and fatty acid metabolism was decreased in HF subjects. Multivariate adaptive regression splines modeling of datasets identified a panel of proteins that will provide >90% prediction success in classifying HF subjects. Proteomic profiling identified ATP-synthase, thrombospondin-1 (THBS1), and vinculin (VCL) as top differentially abundant and S-NO-modified proteins, and these proteins were verified by Western blotting and ELISA in different set of HF subjects. We conclude that differential abundance and S-NO modification of proteins serve as a mechanism in regulating cell viability and free radical production, and THBS1 and VCL evaluation will potentially be useful in the prediction of heart failure.

5.
PLoS Negl Trop Dis ; 10(2): e0004490, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26919708

RESUMO

Trypanosoma cruzi (Tc) infection causes chagasic cardiomyopathy; however, why 30-40% of the patients develop clinical disease is not known. To discover the pathomechanisms in disease progression, we obtained the proteome signature of peripheral blood mononuclear cells (PBMCs) of normal healthy controls (N/H, n = 30) and subjects that were seropositive for Tc-specific antibodies, but were clinically asymptomatic (C/A, n = 25) or clinically symptomatic (C/S, n = 28) with cardiac involvement and left ventricular dysfunction. Protein samples were labeled with BODIPY FL-maleimide (dynamic range: > 4 orders of magnitude, detection limit: 5 f-mol) and resolved by two-dimensional gel electrophoresis (2D-GE). After normalizing the gel images, protein spots that exhibited differential abundance in any of the two groups were analyzed by mass spectrometry, and searched against UniProt human database for protein identification. We found 213 and 199 protein spots (fold change: |≥ 1.5|, p< 0.05) were differentially abundant in C/A and C/S individuals, respectively, with respect to N/H controls. Ingenuity Pathway Analysis (IPA) of PBMCs proteome dataset identified an increase in disorganization of cytoskeletal assembly and recruitment/activation and migration of immune cells in all chagasic subjects, though the invasion capacity of cells was decreased in C/S individuals. IPA predicted with high probability a decline in cell survival and free radical scavenging capacity in C/S (but not C/A) subjects. The MYC/SP1 transcription factors that regulate hypoxia and oxidative/inflammatory stress were predicted to be key targets in the context of control of Chagas disease severity. Further, MARS-modeling identified a panel of proteins that had >93% prediction success in classifying infected individuals with no disease and those with cardiac involvement and LV dysfunction. In conclusion, we have identified molecular pathways and a panel of proteins that could aid in detecting seropositive individuals at risk of developing cardiomyopathy.


Assuntos
Cardiomiopatia Chagásica/metabolismo , Leucócitos Mononucleares/química , Proteínas/química , Proteoma/química , Cardiomiopatia Chagásica/parasitologia , Doença Crônica , Eletroforese em Gel Bidimensional , Humanos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/parasitologia , Proteínas/metabolismo , Proteoma/metabolismo , Proteômica , Trypanosoma cruzi/fisiologia
6.
PLoS One ; 10(6): e0130562, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26075398

RESUMO

Trypanosoma cruzi-induced oxidative and inflammatory responses are implicated in chagasic cardiomyopathy. In this study, we examined the therapeutic utility of a subunit vaccine against T. cruzi and determined if glutathione peroxidase (GPx1, antioxidant) protects the heart from chagasic pathogenesis. C57BL/6 mice (wild-type (WT) and GPx1 transgenic (GPxtg) were infected with T. cruzi and at 45 days post-infection (dpi), immunized with TcG2/TcG4 vaccine delivered by a DNA-prime/Protein-boost (D/P) approach. The plasma and tissue-sections were analyzed on 150 dpi for parasite burden, inflammatory and oxidative stress markers, inflammatory infiltrate and fibrosis. WT mice infected with T. cruzi had significantly more blood and tissue parasite burden compared with infected/GPxtg mice (n = 5-8, p<0.01). Therapeutic vaccination provided >15-fold reduction in blood and tissue parasites in both WT and GPxtg mice. The increase in plasma levels of myeloperoxidase (MPO, 24.7%) and nitrite (iNOS activity, 45%) was associated with myocardial increase in oxidant levels (3-4-fold) and non-responsive antioxidant status in chagasic/WT mice; and these responses were not controlled after vaccination (n = 5-7). The GPxtg mice were better equipped than the WT mice in controlling T. cruzi-induced inflammatory and oxidative stress markers. Extensive myocardial and skeletal tissue inflammation noted in chagasic/WT mice, was significantly more compared with chagasic/GPxtg mice (n = 4-6, p<0.05). Vaccination was equally effective in reducing the chronic inflammatory infiltrate in the heart and skeletal tissue of infected WT and GPxtg mice (n = 6, p<0.05). Hypertrophy (increased BNP and ANP mRNA) and fibrosis (increased collagen) of the heart were extensively present in chronically-infected WT and GPxtg mice and notably decreased after therapeutic vaccination. We conclude the therapeutic delivery of D/P vaccine was effective in arresting the chronic parasite persistence and chagasic pathology; and GPx1 over-expression provided additive benefits in reducing the parasite burden, inflammatory/oxidative stress and cardiac remodeling in Chagas disease.


Assuntos
Cardiomiopatia Chagásica/terapia , Doença de Chagas/imunologia , Glutationa Peroxidase/metabolismo , Vacinas Protozoárias/imunologia , Vacinas de Subunidades/imunologia , Animais , Antioxidantes/metabolismo , Linfócitos T CD8-Positivos/imunologia , Cardiomiopatia Chagásica/imunologia , Cardiomiopatia Chagásica/parasitologia , Doença de Chagas/parasitologia , Doença de Chagas/prevenção & controle , Modelos Animais de Doenças , Glutationa Peroxidase/biossíntese , Inflamação/imunologia , Inflamação/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miocárdio/patologia , Estresse Oxidativo/imunologia , Espécies Reativas de Oxigênio/metabolismo , Trypanosoma cruzi/imunologia , Vacinação
7.
PLoS Pathog ; 11(5): e1004828, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25951312

RESUMO

In this study, we evaluated the long-term efficacy of a two-component subunit vaccine against Trypanosoma cruzi infection. C57BL/6 mice were immunized with TcG2/TcG4 vaccine delivered by a DNA-prime/Protein-boost (D/P) approach and challenged with T. cruzi at 120 or 180 days post-vaccination (dpv). We examined whether vaccine-primed T cell immunity was capable of rapid expansion and intercepting the infecting T. cruzi. Our data showed that D/P vaccine elicited CD4+ (30-38%) and CD8+ (22-42%) T cells maintained an effector phenotype up to 180 dpv, and were capable of responding to antigenic stimulus or challenge infection by a rapid expansion (CD8>CD4) with type 1 cytokine (IFNγ+ and TFNα+) production and cytolytic T lymphocyte (CTL) activity. Subsequently, challenge infection at 120 or 180 dpv, resulted in 2-3-fold lower parasite burden in vaccinated mice than was noted in unvaccinated/infected mice. Co-delivery of IL-12- and GMCSF-encoding expression plasmids provided no significant benefits in enhancing the anti-parasite efficacy of the vaccine-induced T cell immunity. Booster immunization (bi) with recombinant TcG2/TcG4 proteins 3-months after primary vaccine enhanced the protective efficacy, evidenced by an enhanced expansion (1.2-2.8-fold increase) of parasite-specific, type 1 CD4+ and CD8+ T cells and a potent CTL response capable of providing significantly improved (3-4.5-fold) control of infecting T. cruzi. Further, CD8+T cells in vaccinated/bi mice were predominantly of central memory phenotype, and capable of responding to challenge infection 4-6-months post bi by a rapid expansion to a poly-functional effector phenotype, and providing a 1.5-2.3-fold reduction in tissue parasite replication. We conclude that the TcG2/TcG4 D/P vaccine provided long-term anti-T. cruzi T cell immunity, and bi would be an effective strategy to maintain or enhance the vaccine-induced protective immunity against T. cruzi infection and Chagas disease.


Assuntos
Doença de Chagas/imunologia , DNA de Protozoário/genética , Imunização Secundária/métodos , Linfócitos T/imunologia , Trypanosoma cruzi/imunologia , Animais , Doença de Chagas/patologia , Citocinas/imunologia , Citocinas/metabolismo , DNA de Protozoário/imunologia , Ativação Linfocitária/imunologia , Camundongos Endogâmicos C57BL , Plasmídeos/imunologia , Vacinação/métodos , Vacinas de DNA/imunologia
8.
PLoS Negl Trop Dis ; 9(4): e0003625, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25853654

RESUMO

Chagas disease, caused by Trypanosoma cruzi, is endemic in southern parts of the American continent. Herein, we have tested the protective efficacy of a DNA-prime/T. rangeli-boost (TcVac4) vaccine in a dog (Canis familiaris) model. Dogs were immunized with two-doses of DNA vaccine (pcDNA3.1 encoding TcG1, TcG2, and TcG4 antigens plus IL-12- and GM-CSF-encoding plasmids) followed by two doses of glutaraldehyde-inactivated T. rangeli epimastigotes (TrIE); and challenged with highly pathogenic T. cruzi (SylvioX10/4) isolate. Dogs given TrIE or empty pcDNA3.1 were used as controls. We monitored post-vaccination and post-challenge infection antibody response by an ELISA, parasitemia by blood analysis and xenodiagnosis, and heart function by electrocardiography. Post-mortem anatomic and pathologic evaluation of the heart was conducted. TcVac4 induced a strong IgG response (IgG2>IgG1) that was significantly expanded post-infection, and moved to a nearly balanced IgG2/IgG1 response in chronic phase. In comparison, dogs given TrIE or empty plasmid DNA only developed high IgG titers with IgG2 predominance in response to T. cruzi infection. Blood parasitemia, tissue parasite foci, parasite transmission to triatomines, electrocardiographic abnormalities were significantly lower in TcVac4-vaccinated dogs than was observed in dogs given TrIE or empty plasmid DNA only. Macroscopic and microscopic alterations, the hallmarks of chronic Chagas disease, were significantly decreased in the myocardium of TcVac4-vaccinated dogs. We conclude that TcVac4 induced immunity was beneficial in providing resistance to T. cruzi infection, evidenced by control of chronic pathology of the heart and preservation of cardiac function in dogs. Additionally, TcVac4 vaccination decreased the transmission of parasites from vaccinated/infected animals to triatomines.


Assuntos
Doença de Chagas/prevenção & controle , Vacinas Protozoárias/imunologia , Trypanosoma cruzi/imunologia , Vacinas de DNA/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Doença de Chagas/imunologia , Cães , Ensaio de Imunoadsorção Enzimática , Imunoglobulina G/sangue , Interleucina-12/imunologia , Miocárdio/patologia , Parasitemia/imunologia , Plasmídeos/genética , Vacinas Protozoárias/administração & dosagem , Trypanosoma cruzi/genética , Vacinação , Vacinas de DNA/administração & dosagem
9.
PLoS One ; 9(11): e111539, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25372293

RESUMO

In this study, we have utilized wild-type (WT), ASC-/-, and NLRP3-/- macrophages and inhibition approaches to investigate the mechanisms of inflammasome activation and their role in Trypanosoma cruzi infection. We also probed human macrophages and analyzed published microarray datasets from human fibroblasts, and endothelial and smooth muscle cells for T. cruzi-induced changes in the expression genes included in the RT Profiler Human Inflammasome arrays. T. cruzi infection elicited a subdued and delayed activation of inflammasome-related gene expression and IL-1ß production in mφs in comparison to LPS-treated controls. When WT and ASC-/- macrophages were treated with inhibitors of caspase-1, IL-1ß, or NADPH oxidase, we found that IL-1ß production by caspase-1/ASC inflammasome required reactive oxygen species (ROS) as a secondary signal. Moreover, IL-1ß regulated NF-κB signaling of inflammatory cytokine gene expression and, subsequently, intracellular parasite replication in macrophages. NLRP3-/- macrophages, despite an inability to elicit IL-1ß activation and inflammatory cytokine gene expression, exhibited a 4-fold decline in intracellular parasites in comparison to that noted in matched WT controls. NLRP3-/- macrophages were not refractory to T. cruzi, and instead exhibited a very high basal level of ROS (>100-fold higher than WT controls) that was maintained after infection in an IL-1ß-independent manner and contributed to efficient parasite killing. We conclude that caspase-1/ASC inflammasomes play a significant role in the activation of IL-1ß/ROS and NF-κB signaling of cytokine gene expression for T. cruzi control in human and mouse macrophages. However, NLRP3-mediated IL-1ß/NFκB activation is dispensable and compensated for by ROS-mediated control of T. cruzi replication and survival in macrophages.


Assuntos
Caspase 1/metabolismo , Doença de Chagas/metabolismo , Doença de Chagas/parasitologia , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , NF-kappa B/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Trypanosoma cruzi , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Adaptadoras de Sinalização CARD , Proteínas de Transporte/genética , Doença de Chagas/genética , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Humanos , Inflamassomos/genética , Macrófagos/parasitologia , Camundongos , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR
10.
Infect Immun ; 82(4): 1382-9, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24421046

RESUMO

Chagas disease is endemic in Latin America and an emerging infectious disease in the United States. No effective treatments are available. The TcG1, TcG2, and TcG4 antigens are highly conserved in clinically relevant Trypanosoma cruzi isolates and are recognized by B and T cells in infected hosts. Delivery of these antigens as a DNA prime/protein boost vaccine (TcVac2) elicited lytic antibodies and type 1 CD8(+) T cells that expanded upon challenge infection and provided >90% control of parasite burden and myocarditis in chagasic mice. Here we determined if peripheral blood can be utilized to capture the TcVac2-induced protection from Chagas disease. We evaluated the serum levels of T. cruzi kinetoplast DNA (TckDNA), T. cruzi 18S ribosomal DNA (Tc18SrDNA), and murine mitochondrial DNA (mtDNA) as indicators of parasite persistence and tissue damage and monitored the effect of sera on macrophage phenotype. Circulating TckDNA/Tc18SrDNA and mtDNA were decreased by >3- to 5-fold and 2-fold, respectively, in vaccinated infected mice compared to nonvaccinated infected mice. Macrophages incubated with sera from vaccinated infected mice exhibited M2 surface markers (CD16, CD32, CD200, and CD206), moderate proliferation, a low oxidative/nitrosative burst, and a regulatory/anti-inflammatory cytokine response (interleukin-4 [IL-4] plus IL-10 > tumor necrosis factor alpha [TNF-α]). In comparison, macrophages incubated with sera from nonvaccinated infected mice exhibited M1 surface markers, vigorous proliferation, a substantial oxidative/nitrosative burst, and a proinflammatory cytokine response (TNF-α ≫ IL-4 plus IL-10). Cardiac infiltration of macrophages and TNF-α and oxidant levels were significantly reduced in TcVac2-immunized chagasic mice. We conclude that circulating TcDNA and mtDNA levels and macrophage phenotype mediated by serum constituents reflect in vivo levels of parasite persistence, tissue damage, and inflammatory/anti-inflammatory state and have potential utility in evaluating disease severity and efficacy of vaccines and drug therapies.


Assuntos
Doença de Chagas/prevenção & controle , Ativação de Macrófagos/imunologia , Vacinas Protozoárias/imunologia , Trypanosoma cruzi/imunologia , Vacinas de DNA/imunologia , Animais , Antígenos CD/imunologia , Doença de Chagas/imunologia , Citocinas/metabolismo , DNA de Cinetoplasto/sangue , DNA Mitocondrial/sangue , Feminino , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , RNA Ribossômico 18S/sangue
11.
PLoS One ; 8(3): e59434, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23555672

RESUMO

We characterized the immune responses elicited by a DNA-prime/MVA-boost vaccine (TcVac3) constituted of antigenic candidates (TcG2 and TcG4), shown to be recognized by B and T cell responses in Trypanosoma cruzi (Tc) infected multiple hosts. C57BL/6 mice immunized with TcVac3 elicited a strong antigen-specific, high-avidity, trypanolytic antibody response (IgG2b>IgG1); and a robust antigen- and Tc-specific CD8(+)T cell response with type-1 cytokine (IFN-γ(+)TNF-α>IL-4(+)IL-10) and cytolytic effector (CD8(+)CD107a(+)IFN-γ(+)Perforin(+)) phenotype. The vaccine-induced effector T cells significantly expanded upon challenge infection and provided >92% control of T. cruzi. Co-delivery of IL-12 and GMCSF cytokine adjuvants didn't enhance the TcVac3-induced resistance to T. cruzi. In chronic phase, vaccinated/infected mice exhibited a significant decline (up to 70%) in IFN-γ(+)CD8(+)T cells, a predominance of immunoregulatory IL-10(+)/CD4(+)T and IL10(+)/CD8(+)T cells, and presented undetectable tissue parasitism, inflammatory infiltrate, and fibrosis in vaccinated/infected mice. In comparison, control mice responded to challenge infection by a low antibody response, mixed cytokine profile, and consistent activation of pro-inflammatory CD8(+)T cells associated with parasite persistence and pathologic damage in the heart. We conclude that TcVac3 elicited type-1 effector T cell immunity that effectively controlled T. cruzi infection, and subsequently, predominance of anti-inflammatory responses prevented chronic inflammation and myocarditis in chagasic mice.


Assuntos
Cardiomiopatia Chagásica/prevenção & controle , Imunização Secundária , Vacinas Protozoárias/imunologia , Trypanosoma cruzi/fisiologia , Vacinas de DNA/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antiprotozoários/imunologia , Afinidade de Anticorpos , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Cardiomiopatia Chagásica/imunologia , Cardiomiopatia Chagásica/metabolismo , Doença Crônica/prevenção & controle , Citocinas/metabolismo , Feminino , Camundongos , Trypanosoma cruzi/imunologia , Vacinação
12.
PLoS Negl Trop Dis ; 7(1): e2018, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23350012

RESUMO

BACKGROUND: Chagas disease, caused by Trypanosoma cruzi, is endemic in Latin America and an emerging infectious disease in the US and Europe. We have shown TcG1, TcG2, and TcG4 antigens elicit protective immunity to T. cruzi in mice and dogs. Herein, we investigated antigenicity of the recombinant proteins in humans to determine their potential utility for the development of next generation diagnostics for screening of T. cruzi infection and Chagas disease. METHODS AND RESULTS: Sera samples from inhabitants of the endemic areas of Argentina-Bolivia and Mexico-Guatemala were analyzed in 1(st)-phase for anti-T. cruzi antibody response by traditional serology tests; and in 2(nd)-phase for antibody response to the recombinant antigens (individually or mixed) by an ELISA. We noted similar antibody response to candidate antigens in sera samples from inhabitants of Argentina and Mexico (n=175). The IgG antibodies to TcG1, TcG2, and TcG4 (individually) and TcG(mix) were present in 62-71%, 65-78% and 72-82%, and 89-93% of the subjects, respectively, identified to be seropositive by traditional serology. Recombinant TcG1- (93.6%), TcG2- (96%), TcG4- (94.6%) and TcG(mix)- (98%) based ELISA exhibited significantly higher specificity compared to that noted for T. cruzi trypomastigote-based ELISA (77.8%) in diagnosing T. cruzi-infection and avoiding cross-reactivity to Leishmania spp. No significant correlation was noted in the sera levels of antibody response and clinical severity of Chagas disease in seropositive subjects. CONCLUSIONS: Three candidate antigens were recognized by antibody response in chagasic patients from two distinct study sites and expressed in diverse strains of the circulating parasites. A multiplex ELISA detecting antibody response to three antigens was highly sensitive and specific in diagnosing T. cruzi infection in humans, suggesting that a diagnostic kit based on TcG1, TcG2 and TcG4 recombinant proteins will be useful in diverse situations.


Assuntos
Antígenos de Protozoários/imunologia , Doença de Chagas/diagnóstico , Doença de Chagas/prevenção & controle , Vacinas Protozoárias/administração & dosagem , Vacinas Protozoárias/imunologia , Trypanosoma cruzi/imunologia , Adolescente , Adulto , Idoso , Anticorpos Antiprotozoários/sangue , América Central , Doença de Chagas/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina G/sangue , Pessoa de Meia-Idade , Sensibilidade e Especificidade , América do Sul , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Adulto Jovem
13.
Vaccine ; 30(50): 7179-86, 2012 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-23079191

RESUMO

In this study, we have characterized the immune mechanisms elicited by antigenic candidates, TcG2 and TcG4, delivered by a DNA-prime/MVA-boost approach, and evaluated the host responses to Trypanosoma cruzi infection in C57BL/6 mice. Immunization of mice with antigenic candidates elicited antigen-specific, high-avidity, trypanolytic antibody response (IgG2b>IgG1) and CD8(+)T cells that exhibited type-1 cytolytic effector (CD8(+)CD107a(+)IFN-γ(+)Perforin(+)) phenotype. The extent of TcG2-dependent type 1 B and T cell immunity was higher than that noted in TcG4-immunized mice, and expanded accordingly in response to challenge infection with T. cruzi. The progression of chronic phase in immunized mice was associated with persistence of IgGs, 55-90% reduction in the frequency of proinflammatory (IFN-γ(+) or TNF-α(+)) CD8(+)T cells, and an increase or emergence of immunoregulatory (IL-10(+)) CD4/CD8 T cells. The tissue parasitism, infiltration of inflammatory infiltrate, parasite persistence, and fibrosis were decreased by 82-92% in heart and skeletal muscle of immunized/chronically infected mice. Control mice exhibited a significantly low antibody response, consistent activation of effector CD8(+)T cells dominated by pro-inflammatory phenotype and mixed cytokine profile (IFN-γ+TNF-α>IL-4+IL-10), parasite persistence and pathologic damage in chagasic hearts. We conclude that delivery of TcG2 or TcG4 by DNA-rMVA approach elicits effective antibody and CD8(+)T cell mediated immunity against T. cruzi and Chagas disease. The emergence of type 2 cytokine and T cell response in chronic phase was indicative of prevention of clinical disease.


Assuntos
Antígenos de Protozoários/imunologia , Linfócitos T CD8-Positivos/imunologia , Doença de Chagas/prevenção & controle , Vacinas Protozoárias/imunologia , Trypanosoma cruzi/imunologia , Vacinas de DNA/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/genética , Doença de Chagas/patologia , Modelos Animais de Doenças , Portadores de Fármacos , Feminino , Vetores Genéticos , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos C57BL , Vacinas Protozoárias/administração & dosagem , Trypanosoma cruzi/genética , Vacinas de DNA/administração & dosagem , Vacinas de Subunidades/administração & dosagem , Vacinas de Subunidades/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Vírus Vaccinia/genética
14.
Mol Cell Proteomics ; 11(8): 435-52, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22543060

RESUMO

Chagas disease is initiated upon infection by Trypanosoma cruzi. Among the health consequences is a decline in heart function, and the pathophysiological mechanisms underlying this manifestation are not well understood. To explore the possible mechanisms, we employed IgY LC10 affinity chromatography in conjunction with ProteomeLab PF2D and two-dimensional gel electrophoresis to resolve the proteome signature of high and low abundance serum proteins in chagasic patients. MALDI-TOF MS/MS analysis yielded 80 and 14 differentially expressed proteins associated with cardiomyopathy of chagasic and other etiologies, respectively. The extent of oxidative stress-induced carbonyl modifications of the differentially expressed proteins (n = 26) was increased and coupled with a depression of antioxidant proteins. Functional annotation of the top networks developed by ingenuity pathway analysis of proteome database identified dysregulation of inflammation/acute phase response signaling and lipid metabolism relevant to production of prostaglandins and arachidonic acid in chagasic patients. Overlay of the major networks identified prothrombin and plasminogen at a nodal position with connectivity to proteome signature indicative of heart disease (i.e., thrombosis, angiogenesis, vasodilatation of blood vessels or the aorta, and increased permeability of blood vessel and endothelial tubes), and inflammatory responses (e.g., platelet aggregation, complement activation, and phagocyte activation and migration). The detection of cardiac proteins (myosin light chain 2 and myosin heavy chain 11) and increased levels of vinculin and plasminogen provided a comprehensive set of biomarkers of cardiac muscle injury and development of clinical Chagas disease in human patients. These results provide an impetus for biomarker validation in large cohorts of clinically characterized chagasic patients.


Assuntos
Biomarcadores/sangue , Doença de Chagas/sangue , Proteoma/análise , Proteômica/métodos , Adolescente , Adulto , Idoso , Biomarcadores/metabolismo , Cardiomiopatia Chagásica/sangue , Cardiomiopatia Chagásica/metabolismo , Doença de Chagas/metabolismo , Eletroforese em Gel Bidimensional , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Carbonilação Proteica , Proteoma/classificação , Proteoma/metabolismo , Transdução de Sinais , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Adulto Jovem
15.
J Am Heart Assoc ; 1(6): e003855, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23316324

RESUMO

BACKGROUND: Mitochondrial dysfunction is a key determinant in chagasic cardiomyopathy development in mice; however, its relevance in human Chagas disease is not known. We determined if defects in mitochondrial biogenesis and dysregulation of peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1 (PGC-1)-regulated transcriptional pathways constitute a mechanism or mechanisms underlying mitochondrial oxidative-phosphorylation (OXPHOS) deficiency in human Chagas disease. METHODS AND RESULTS: We utilized human cardiomyocytes and left-ventricular tissue from chagasic and other cardiomyopathy patients and healthy donors (n>6/group). We noted no change in citrate synthase activity, yet mRNA and/or protein levels of subunits of the respiratory complexes were significantly decreased in Trypanosoma cruzi-infected cardiomyocytes (0 to 24 hours) and chagasic hearts. We observed increased mRNA and decreased nuclear localization of PGC-1-coactivated transcription factors, yet the expression of genes for PPARγ-regulated fatty acid oxidation and nuclear respiratory factor (NRF1/2)-regulated mtDNA replication and transcription machinery was enhanced in infected cardiomyocytes and chagasic hearts. The D-loop formation was normal or higher, but mtDNA replication and mtDNA content were decreased by 83% and 40% to 65%, respectively. Subsequently, we noted that reactive oxygen species (ROS), oxidative stress, and mtDNA oxidation were significantly increased, yet NRF1/2-regulated antioxidant gene expression remained compromised in infected cardiomyocytes and chagasic hearts. CONCLUSIONS: The replication of mtDNA was severely compromised, resulting in a significant loss of mtDNA and expression of OXPHOS genes in T cruzi-infected cardiomyocytes and chagasic hearts. Our data suggest increased ROS generation and selective functional incapacity of NRF2-mediated antioxidant gene expression played a role in the defects in mtDNA replication and unfitness of mtDNA for replication and gene expression in Chagas disease.


Assuntos
Doença de Chagas/fisiopatologia , Replicação do DNA/fisiologia , DNA Mitocondrial/fisiologia , Renovação Mitocondrial/fisiologia , Trypanosoma cruzi , Western Blotting , Células Cultivadas , Doença de Chagas/genética , Doença de Chagas/metabolismo , DNA Mitocondrial/metabolismo , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Microscopia de Fluorescência , Doenças Mitocondriais/genética , Doenças Mitocondriais/fisiopatologia , Miócitos Cardíacos/fisiologia , Miócitos Cardíacos/ultraestrutura , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/fisiologia , Fator 1 Nuclear Respiratório/genética , Fator 1 Nuclear Respiratório/metabolismo , Fator 1 Nuclear Respiratório/fisiologia , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia
16.
Cardiovasc Pathol ; 21(2): 83-8, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22001052

RESUMO

BACKGROUND: Rheumatic fever and chronic rheumatic heart disease (RHD) remains one of the most important causes of cardiovascular morbidity leading to a major public health problem, especially in developing countries. This was a pilot study to assess the presence of inflammation and expression of adhesion molecules by immunohistochemistry (IHC) in endomyocardial biopsy specimens of patients with chronic RHD. METHODS: Endomyocardial biopsy was obtained from 14 patients of chronic RHD with no features of activity clinically. Biopsies were processed for histology and IHC. IHC was carried using monoclonal antibodies against CD3, CD4, CD8, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1. RESULTS: Histomorphologically, varying degree of interstitial and perivascular fibrosis was seen in all the 13 patients (100%). Mild fibrosis (1+) was seen in five patients (38.5%); moderate interstitial fibrosis (2+) was present in four patients (30.8%).There was no Aschoff nodule or evidence of active myocarditis in any of the biopsy specimens. IMMUNOHISTOCHEMISTRY: Moderate positivity of (2+) and intense positivity of (3+) for intercellular adhesion molecule-1 was seen in 11 and 2 patients, respectively. With vascular cell adhesion molecule-1, four showed mild positivity (1+), and three showed intense positivity (3+). The phenotypic analysis of the inflammatory cells in our study revealed CD8(+) cells in 77%, CD4(+) in 23.1%, and CD3(+) in 38.5% of total patients, which suggests chronicity. CONCLUSION: The nonspecific histomorphological changes and increased adhesion molecules expression could be a part of the ventricular remodeling due to the hemodynamic stress by the stenotic or regurgitant lesions of RHD itself.


Assuntos
Molécula 1 de Adesão Intercelular/metabolismo , Cardiopatia Reumática/patologia , Molécula 1 de Adesão de Célula Vascular/metabolismo , Remodelação Ventricular/fisiologia , Adolescente , Adulto , Biomarcadores/metabolismo , Biópsia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/patologia , Doença Crônica , Progressão da Doença , Feminino , Fibrose/metabolismo , Fibrose/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Miocardite/metabolismo , Miocardite/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Projetos Piloto , Estudos Prospectivos , Cardiopatia Reumática/metabolismo , Nódulo Reumático/patologia , Adulto Jovem
17.
Adv Parasitol ; 76: 153-70, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21884891

RESUMO

Inflammation is a host defence activated by exogenous (e.g. pathogen-derived, pollutants) or endogenous (e.g. reactive oxygen species-ROS) danger signals. Mostly, endogenous molecules (or their derivatives) have well-defined intracellular function but become danger signal when released or exposed following stress or injury. In this review, we discuss the potential role of ROS in chronic evolution of inflammatory cardiovascular diseases, using our experiences working on chagasic cardiomyopathy as a focus-point.


Assuntos
Cardiomiopatia Chagásica/imunologia , Citocinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Trypanosoma cruzi/imunologia , Animais , Cardiomiopatia Chagásica/patologia , Humanos , Inflamação/imunologia , Inflamação/patologia , Trypanosoma cruzi/patogenicidade
18.
Adv Parasitol ; 75: 121-46, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21820554

RESUMO

The pathology of Chagas disease presents a complicated and diverse picture in humans. The major complications and destructive evolutionary outcomes of chronic infection by Trypanosoma cruzi in humans include ventricular fibrillation, thromboembolism and congestive heart failure. Studies in animal models and human patients have revealed the pathogenic mechanisms during disease progression, pathology of disease and features of protective immunity. Accordingly, several antigens, antigen-delivery vehicles and adjuvants have been tested to elicit immune protection to T. cruzi in experimental animals. This review summarizes the research efforts in vaccine development against Chagas disease during the past decade.


Assuntos
Doença de Chagas/prevenção & controle , Vacinas Protozoárias/uso terapêutico , Trypanosoma cruzi/imunologia , Adjuvantes Imunológicos/farmacologia , Animais , Antígenos de Protozoários/imunologia , Antígenos CD8/imunologia , Doença de Chagas/imunologia , Doença Crônica/terapia , Humanos , Imunidade Celular , Camundongos , Trypanosoma cruzi/patogenicidade , Vacinas de Subunidades/imunologia
19.
PLoS Negl Trop Dis ; 5(5): e1050, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21625470

RESUMO

BACKGROUND: Trypanosoma cruzi, the etiologic agent of Chagas Disease, is a major vector borne health problem in Latin America and an emerging infectious disease in the United States. METHODS: We tested the efficacy of a multi-component DNA-prime/DNA-boost vaccine (TcVac1) against experimental T. cruzi infection in a canine model. Dogs were immunized with antigen-encoding plasmids and cytokine adjuvants, and two weeks after the last immunization, challenged with T. cruzi trypomastigotes. We measured antibody responses by ELISA and haemagglutination assay, parasitemia and infectivity to triatomines by xenodiagnosis, and performed electrocardiography and histology to assess myocardial damage and tissue pathology. RESULTS: Vaccination with TcVac1 elicited parasite-and antigen-specific IgM and IgG (IgG2>IgG1) responses. Upon challenge infection, TcVac1-vaccinated dogs, as compared to non-vaccinated controls dogs, responded to T. cruzi with a rapid expansion of antibody response, moderately enhanced CD8(+) T cell proliferation and IFN-γ production, and suppression of phagocytes' activity evidenced by decreased myeloperoxidase and nitrite levels. Subsequently, vaccinated dogs controlled the acute parasitemia by day 37 pi (44 dpi in non-vaccinated dogs), and exhibited a moderate decline in infectivity to triatomines. TcVac1-immunized dogs did not control the myocardial parasite burden and electrocardiographic and histopatholgic cardiac alterations that are the hallmarks of acute Chagas disease. During the chronic stage, TcVac1-vaccinated dogs exhibited a moderate decline in cardiac alterations determined by EKG and anatomo-/histo-pathological analysis while chronically-infected/non-vaccinated dogs continued to exhibit severe EKG alterations. CONCLUSIONS: Overall, these results demonstrated that TcVac1 provided a partial resistance to T. cruzi infection and Chagas disease, and provide an impetus to improve the vaccination strategy against Chagas disease.


Assuntos
Doença de Chagas/prevenção & controle , Imunização Secundária/métodos , Vacinas Protozoárias/imunologia , Vacinação/métodos , Vacinas de DNA/imunologia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/genética , Animais , Anticorpos Antiprotozoários/sangue , Linfócitos T CD8-Positivos/imunologia , Doença de Chagas/imunologia , Citocinas/administração & dosagem , Citocinas/genética , Modelos Animais de Doenças , Cães , Ensaio de Imunoadsorção Enzimática , Feminino , Testes de Hemaglutinação , Masculino , Miocárdio/patologia , Parasitemia/imunologia , Parasitemia/prevenção & controle , Plasmídeos , Vacinas Protozoárias/administração & dosagem , Células Th1/imunologia , Vacinas de DNA/administração & dosagem
20.
PLoS Negl Trop Dis ; 4(8): e797, 2010 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-20706586

RESUMO

BACKGROUND: Chagas disease is a major health problem in Latin America, and an emerging infectious disease in the US. Previously, we have screened the Trypanosoma cruzi sequence database by a computational/bioinformatics approach, and identified antigens that exhibited the characteristics of vaccine candidates. METHODOLOGY: We investigated the protective efficacy of a multi-component DNA-prime/protein-boost vaccine (TcVac2) constituted of the selected candidates and cytokine (IL-12 and GM-CSF) expression plasmids in a murine model. C57BL/6 mice were immunized with antigen-encoding plasmids plus cytokine adjuvants, followed by recombinant proteins; and two-weeks later, challenged with T. cruzi trypomastigotes. ELISA and flow cytometry were employed to measure humoral (antibody isotypes) and cellular (lymphocyte proliferation, CD4(+) and CD8(+) T cell phenotype and cytokines) responses. Myocardial pathology was evaluated by H&E and Masson's trichrome staining. PRINCIPAL FINDINGS: TcVac2 induced a strong antigen-specific antibody response (IgG2b>IgG1) and a moderate level of lymphocyte proliferation in mice. Upon challenge infection, TcVac2-vaccinated mice expanded the IgG2b/IgG1 antibodies and elicited a substantial CD8(+) T cell response associated with type 1 cytokines (IFN-gamma and TNF-alpha) that resulted in control of acute parasite burden. During chronic phase, antibody response persisted, splenic activation of CD8(+) T cells and IFN-gamma/TNF-alpha cytokines subsided, and IL-4/IL-10 cytokines became dominant in vaccinated mice. The tissue parasitism, inflammation, and fibrosis in heart and skeletal muscle of TcVac2-vaccinated chronic mice were undetectable by histological techniques. In comparison, mice injected with vector or cytokines only responded to T. cruzi by elicitation of a mixed (type 1/type 2) antibody, T cell and cytokine response, and exhibited persistent parasite burden and immunopathology in the myocardium. CONCLUSION: TcVac2-induced activation of type 1 antibody and lymphocyte responses provided resistance to acute T. cruzi infection, and consequently, prevented the evolution of chronic immunopathology associated with parasite persistence in chagasic hearts.


Assuntos
Antígenos de Protozoários/imunologia , Doença de Chagas/prevenção & controle , Vacinas Protozoárias/imunologia , Trypanosoma cruzi/imunologia , Vacinas de DNA/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células , Doença de Chagas/imunologia , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Imunização Secundária/métodos , Interleucina-12/genética , Interleucina-12/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/patologia , Plasmídeos , Vacinas Protozoárias/administração & dosagem , Vacinação/métodos , Vacinas de DNA/administração & dosagem , Vacinas de Subunidades/administração & dosagem , Vacinas de Subunidades/imunologia
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