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1.
Front Microbiol ; 9: 3320, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30697201

RESUMO

Trypanosoma cruzi (Tc) infection causes Chagas disease (ChD) presented by dilated cardiomyopathy and heart failure. During infection, oxidative and nitrosative stresses are elicited by the immune cells for control the pathogen; however, excess nitric oxide and superoxide production can result in cysteine S-nitrosylation (SNO) of host proteins that affects cellular homeostasis and may contribute to disease development. To identify the proteins with changes in SNO modification levels as a hallmark of ChD, we obtained peripheral blood mononuclear cells (PBMC) from seronegative, normal healthy (NH, n = 30) subjects, and from seropositive clinically asymptomatic (ChD CA, n = 25) or clinically symptomatic (ChD CS, n = 28) ChD patients. All samples were treated (Asc+) or not-treated (Asc-) with ascorbate (reduces nitrosylated thiols), labeled with the thiol-labeling BODIPY FL-maleimide dye, resolved by two-dimensional electrophoresis (total 166 gels), and the protein spots that yielded significant differences in abundance or SNO level at p-value of ≤ 0.05 t-test/Welch/BH were identified by MALDI-TOF/TOF MS or OrbiTrap LC-MS/MS. Targeted analysis of a new cohort of PBMC samples (n = 10-14/group) was conducted to verify the differential abundance/SNO levels of two of the proteins in ChD (vs. NH) subjects. The multivariate adaptive regression splines (MARS) modeling, comparing differences in relative SNO level (Asc-/Asc+ ratio) of the protein spots between any two groups yielded SNO biomarkers that exhibited ≥90% prediction success in classifying ChD CA (582-KRT1 and 884-TPM3) and ChD CS (426-PNP, 582-KRT1, 486-ALB, 662-ACTB) patients from NH controls. Ingenuity Pathway Analysis (IPA) of the SNO proteome dataset normalized to changes in protein abundance suggested the proteins belonging to the signaling networks of cell death and the recruitment and migration of immune cells were most affected in ChD CA and ChD CS (vs. NH) subjects. We propose that SNO modification of the select panel of proteins identified in this study have the potential to identify ChD severity in seropositive individuals exposed to Tc infection.

2.
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
3.
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
4.
Guatem. pediátr. ; 2(1): 17-25, 2016.
Artigo em Espanhol | LILACS | ID: biblio-981219

RESUMO

La enterocolitis necrosante (ECN) es la urgencia gastrointestinal más frecuente en lactantes pretérmino. En el Hospital Roosevelt la mortalidad secundaria a la misma fue del 40% en el año 2009. Según literatura internacional, de los pacientes que sobreviven, aproximadamente 25% presentarán secuelas de larga duración como retraso en el neurodesarrollo y desnutrición; sin embargo, en Guatemala no se tienen datos al respecto.


Assuntos
Recém-Nascido , Estado Nutricional , Enterocolite Necrosante , Crescimento , Neurologia
5.
Rev Biol Trop ; 59(1): 37-52, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21513192

RESUMO

In tropical and subtropical regions of the world, parasitic diseases are a main cause of losses in livestock productivity. The increased acquired resistence to anthelmintics by gastrointestinal nematodes, requires biological control be considered as a potential feasible and effective alternative. The most effective natural soil enemies of nematodes are nematophagous fungi. In order to collect and identify predator nematophagous fungi (PNF), samples were obtained from 51 farms distributed throughout the seven provinces of Costa Rica. The origin samples included: soil from different crops (potatoes, tomatoes, bananas, ornamental plants, squash and coffee); animal feces (cattle, sheep, goat and horse); soil and fallen leaves from forest; and plants with signs of nematode infection. Each sample was processed using three techniques for the extraction of fungi from soil: sprinkling technique, soil dilution and humidity chamber. Twenty four strains of nematophagous fungi were found in 19 farms; 83.3% of the fungi were isolated by sprinkling technique. The following fungi were identified: Arthrobotrys oligospora (n = 13); Candelabrella musiformis (n = 9); and for the first time there was isolation of A. conoides (n = 1) and A. dactyloides (n = 1) in the country. Moreover, 16 strains from Trichoderma (n=13), Beauveria (n = 1), Clonostachys (n = 1) and Lecanicillium (n = 1) were obtained. In addition, pH of each possible fungal isolation source was measured, and it varied from 5.2 to 9.9, however PNF isolates fell within the range of 5.6 to 7.5. The PNF strains were cultivated in four different media for the production of chhlamydospores: potato dextrose agar (PDA); corn meal agar (CMA); malt extract agar (MEA) and potato carrot agar (PCA). Out of these cultures, 95.8% of the strains formed chlamydospores primarily in the PCA. Of these strains, the profilic spore producers were subjected to ruminant artificial gastrointestinal conditions. A total of 14 fungi were tested, out of which 42.9% survived the digestive analysis. Neither A. conoides nor A. dactyloides were viable following the in vitro gastrointestinal test. The PNF isolated in this study demonstrated an action against ovine and caprine gastrointestinal nematodes and are candidates for use in biological control of these organisms. Among these microorganisms, Candelabrella musiformis appears to be the most promising fungi for use as a biological control agent in Costa Rica.


Assuntos
Fungos/fisiologia , Doenças das Cabras/prevenção & controle , Nematoides/microbiologia , Infecções por Nematoides/veterinária , Controle Biológico de Vetores/métodos , Doenças dos Ovinos/prevenção & controle , Animais , Costa Rica , Fezes/microbiologia , Fungos/isolamento & purificação , Doenças das Cabras/parasitologia , Cabras , Infecções por Nematoides/prevenção & controle , Ovinos , Doenças dos Ovinos/parasitologia , Microbiologia do Solo
6.
Rev. biol. trop ; 59(1): 37-52, mar. 2011. ilus, graf, tab
Artigo em Inglês | LILACS | ID: lil-638044

RESUMO

In tropical and subtropical regions of the world, parasitic diseases are a main cause of losses in livestock productivity. The increased acquired resistence to anthelmintics by gastrointestinal nematodes, requires biological control be considered as a potential feasible and effective alternative. The most effective natural soil enemies of nematodes are nematophagous fungi. In order to collect and identify predator nematophagous fungi (PNF), samples were obtained from 51 farms distributed throughout the seven provinces of Costa Rica. The origin samples included: soil from different crops (potatoes, tomatoes, bananas, ornamental plants, squash and coffee); animal feces (cattle, sheep, goat and horse); soil and fallen leaves from forest; and plants with signs of nematode infection. Each sample was processed using three techniques for the extraction of fungi from soil: sprinkling technique, soil dilution and humidity chamber. Twenty four strains of nematophagous fungi were found in 19 farms; 83.3% of the fungi were isolated by sprinkling technique. The following fungi were idenified: Arthrobotrys oligospora (n=13); Candelabrella musiformis (n=9); and for the first time there was isolation of A. conoides (n=1) and A. dactyloides (n=1) in the country. Moreover, 16 strains from Trichoderma (n=13), Beauveria (n=1), Clonostachys (n=1) and Lecanicillium (n=1) were obtained. In addition, pH of each possible fungal isolation source was measured, and it varied from 5.2 to 9.9, however PNF isolates fell within the range of 5.6 to 7.5. The PNF strains were cultivated in four different media for the production of chhlamydospores: potato dextrose agar (PDA); corn meal agar (CMA); malt extract agar (MEA) and potato carrot agar (PCA). Out of these cultures, 95.8% of the strains formed chlamydospores primarily in the PCA. Of these strains, the profilic spore producers were subjected to ruminant artificial gastrointestinal conditions. A total of 14 fungi were tested, out of which 42.9% survived the digestive analysis. Neither A. conoides nor A. dactyloides were viable following the in vitro gastrointestinal test. The PNF isolated in this study demostrated an action against ovine and caprine gastrointestinal nematodes and are candidates for use in biological control of these organisms. Among these microorganisms, Candelabrella musiformis appears to be the most promising fungi for use as a biological control agent in Costa Rica. Rev. Biol. Trop. 59 (1): 37-52. Epub 2011 March 01.


El control biológico es en la actualidad una alternativa para el control de los nematodos gastrointestinales que desarrollaron resistencia a los principales grupos de antihelmínticos. Para el aislamiento e identificación de hongos nematófagos depredadores, se tomaron muestras de 51 fincas distribuidas entre todas las provincias de Costa Rica. La naturaleza de las muestras incluyó: suelos de diferentes sembradíos (papa, tomate, banano, plantas ornamentales, chayote y café), heces de animales (bovinos, ovinos, caprinos y equinos), suelo y hojarasca de bosques y plantas con signos de enfermedad causada por nematodos. Las muestras se procesaron mediante 3 técnicas diferentes para la extracción de hongos a partir del suelo: espolvoreado en placa, dilución de suelos y cámara húmeda. Veinticuatro cepas de hongos nematófagos fueron aisladas de 19 fincas; el 83.3% de éstos fueron aislados mediante las técnica de espolvoreado en placa. Los hongos fueron identificados como: Arthrobotrys oligospora (n=13), Candelabrella musiformis (n=9) y por primera vez se reporta el aislamiento de A. conoides (n=1) y A. dactyloides (n=1) en el país. Asimismo, se aislaron 16 cepas de hongos de los géneros Trichoderma (n=13), Beauveria (n=1), Clonostachys (n=1) y Lecanicillium (n=1). Adicionalmente se les midió el pH, el cual varió entre 5.2-9.9, ubicándose los HND dentro de un rango entre 5.6-7.5. Las cepas de HND fueron cultivadas en 4 medios diferentes para la producción de clamidosporas: papa dextrosa agar, harina de maíz, extracto de malta y agar papa-zanahoria. El 95.8% de las cepas aisladas produjeron clamidosporas, principalmente en el medio agar papa-zanahoria. De estas cepas, se escogieron las de mayor producción para ser sometidas a la prueba de digestibilidad in vitro. Un total de 14 cepas fueron sometidos a esta prueba, de las cuales el 42.9% resultaron viables; de éstas, las cepas de A. conoides y A. dactyloides no sobrevivieron a la prueba de digestibilidad in vitro. De los microorganismos aislados, Candelabrella musiformis se considera el más promisorio de los hongos como agente biológico en Costa Rica.


Assuntos
Animais , Fungos/fisiologia , Doenças das Cabras/prevenção & controle , Nematoides/microbiologia , Infecções por Nematoides/veterinária , Controle Biológico de Vetores/métodos , Doenças dos Ovinos/prevenção & controle , Costa Rica , Fezes/microbiologia , Fungos/isolamento & purificação , Cabras , Doenças das Cabras/parasitologia , Infecções por Nematoides/prevenção & controle , Ovinos , Microbiologia do Solo , Doenças dos Ovinos/parasitologia
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