Nuevo método de evaluación del efecto antiparasitario en modelos in vitro e in vivo, a través de la visualización de Trypanosoma cruzi-GFP / New method of evaluation of the antiparasitic effect models in in vitro and in vivo, through the viewing of Trypanosoma cruzi-GFP

Los tratamientos de primera línea para la enfermedad de Chagas generan importantes efectos adversos que acentúan el deterioro de la salud en los pacientes. La necesidad de generar fármacos alternativos ha permitido desarrollar estudios donde se emplean parásitos capaces de expresar una proteína fluorescente, a fin de correlacionar fluorescencia con población de protozoarios. En este sentido, ideamos una metodología para el seguimiento de la proliferación de Trypanosoma cruzi-GFP (Green Fluorescent Protein) en modelos in vitro e in vivo, empleando el equipo iBox- UVP. Los ensayos in vitro se iniciaron con una curva de calibración usando concentraciones entre 5x105 y 5x107 parásitos/mL. Seguidamente, con una curva de proliferación evidenciamos a través de la fluorescencia la susceptibilidad de los parásitos frente a la droga comercial Benznidazol (IC50= 5,3±1,3 μM). En el ensayo in vivo se corroboró cualitativamente el efecto quimioterapéutico del Benznidazol (100 mg/kg/día) en ratones C57BL/6, partiendo de un inóculo de 2,5x105 parásitos, haciendo captura de imágenes de fluorescencia cada dos días a partir del día 1, e inicio del tratamiento por vía oral el sexto día. El coeficiente de correlación cercano a 1 obtenido en la curva de calibración habla de un método de cuantificación parasitario sencillo y robusto; también los ensayos en modelos in vitro e in vivo permitieron monitorear el efecto dosis-dependiente de Benznidazol sobre T. cruzi-GFP. En síntesis, elaboramos una metodología novedosa, rápida, no invasiva y que sigue en tiempo real la respuesta quimioterapéutica de drogas anti-T. cruzi.

The first-line treatments for Chagas disease generate significant adverse effects that accentuate the health deterioration in patients. The need to generate alternative drugs has led to the development of studies in which parasites will express a fluorescent protein, and correlate this expression with protozoan population. We devised a methodology for monitoring the proliferation of Trypanosoma cruzi- GFP (Green Fluorescent Protein) in models in vitro and in vivo, using the equipment iBox-UVP. In vitro assays were initiated with a calibration curve using concentrations between 5x105 and 5x107 parasites/mL. Subsequently, with a proliferation curve, through fluorescence we determined the susceptibility of the parasites against the commercial drug Benznidazol (IC50= 5,3±1,3 μM). In vivo assays corroborated qualitatively the chemotherapeutic effect of Benznidazol (100 mg/kg/day) in C57BL/6 mice, starting from an inoculum of 2.5x105 parasites, making capture of fluorescence imaging every two days from day 1, and starting oral treatment on the sixth day. The correlation coefficient close to 1 obtained in the calibration curve showed that this quantification method of parasites is simple and robust; assays in vitro and in vivo allowed monitoring dose-dependent effects of Benznidazol agains T. cruzi-GFP. We have produced an innovative, rapid, non-invasive method that monitors in real time the chemotherapeutic response of anti-T. cruzi drugs.

Outcomes of cataract surgery in diabetic patients: results of the Pan American Collaborative Retina Study Group / Resultados da cirurgia de catarata em pacientes diabéticos: resultados do Pan-American Collaborative Retina Study Group

Purpose: This study was designed to evaluate the visual and anatomical outcomes after cataract surgery in diabetic patients with different intraoperative therapeutic strategies. Methods: The research design comprised of a multicentric, retrospective, interventional study conducted at 6 centers in Argentina, Brazil, Costa Rica, Puerto Rico, Spain, and Venezuela. We included 138 diabetic patients with at least 6-month follow-up following phacoemulsification and intraocular lens implantation. Best-corrected visual acuity (BCVA) and central subfield thickness were collected at baseline and at 1-, 2-, 3-, and 6-month follow-up. Of these, 42 cases were not treated with any intraoperative coadjuvant medication (Group 1), 59 patients received intraoperative bevacizumab (Group 2) and 37 patients received intraoperative triamcinolone (4 mg/0.1 ml) (Group 3). Results: The mean logMAR [± standard deviation (SD)] BCVA improved from 0.82 (± 0.43) at baseline, to 0.14 (± 0.23) at 6-month follow-up (p<0.001) in Group 1; from 0.80 (± 0.48) to 0.54 (± 0.45) (p<0.001) in Group 2; and from 1.0 (± 0.40) to 0.46 (± 0.34) (p<0.001) in Group 3. The mean central subfield thickness increased from 263.57 µm (± 35.7) at baseline to 274.57 µm (± 48.7) at 6-month follow-up (p=0.088) in Group 1; from 316.02 µm (± 100.4) to 339.56 µm (± 145.3) (p=0.184) in Group 2; and from 259.18 µm (± 97.9) to 282.21 µm (± 87.24) (p=0.044) in Group 3. Conclusion: Diabetic patients may significantly benefit from cataract surgery. This study provides evidence to support the use of intravitreal triamcinolone or bevacizumab at the time of cataract surgery in cases with pre-existent diabetic macular edema or moderate-severe non-proliferative diabetic retinopathy. .

Objetivo: Avaliar os resultados visuais e anatômicos após a cirurgia de catarata em pacientes diabéticos com estratégias terapêuticas intraoperatórias diferentes. Métodos: Estudo multicêntrico, retrospectivo, de intervenção realizado em 6 centros da Argentina, Brasil, Costa Rica, Porto Rico, Espanha e Venezuela. Foram incluídos 138 pacientes diabéticos com pelo menos 6 meses de seguimento após facoemulsificação com implante de lente intraocular. Acuidade visual melhor corrigida (BCVA) e a espessura subcampo central (CST ) foram coletadas no início e em 1, 2, 3 e 6 meses de seguimento. Destes, 42 casos não foram tratadas com qualquer co-adjuvante de medicamentos intra-operatório (Grupo 1), 59 pacientes receberam bevacizumab intraoperatório (Grupo 2), e 37 pacientes receberam triancinolona intraoperatória (4 mg/0,1 ml) (Grupo 3). Resultados: A média logMAR (± desvio-padrão [DP]) BCVA melhorou de 0,82 (± 0,43) no início do estudo, para 0,14 (± 0,23) aos 6 meses de seguimento (p<0,001) no Grupo 1; de 0,80 (± 0,48) para 0,54 (± 0,45) (p<0,001) no Grupo 2; e de 1,0 (± 0,40) para 0,46 (± 0,34) (p<0,001) no Grupo 3. A CST média aumentou de 263,57 µm (± 35,7) na linha de base para 274,57±48,7 µm em 6 meses acompanhamento (p=0,088) no Grupo 1; de 316,02 µm (± 100,4), para 339,56 µm (± 145,3) (p=0,184) no Grupo 2; e de 259,18 µm (± 97,9), para 282,21 µm (±87,24) (p=0,044) no grupo 3. Conclusões: Pacientes diabéticos podem se beneficiar significativamente da cirurgia de catarata. Este estudo parece fornecer evidências para apoiar o uso de triancinolona intravítrea ou bevacizumab no momento da cirurgia de catarata em casos com edema macular diabético preexistente (DME) ou retinopatia diabética não-proliferativa moderada a grave. .