Early-Phase Perceptions of COVID-19's Impact on Ophthalmology Practice Patterns: A Survey from the Pan-American Association of Ophthalmology.

Purpose: The COVID-19 pandemic affected medical practice worldwide due to interventions to prevent spreading. Its effect on ophthalmology practices in Latin America has not yet been explored. We aimed to assess the perceptions about the pandemic from countries' ophthalmological national and subspecialty retina societies affiliated to the Pan-American Association of Ophthalmology (PAAO). Patients and Methods: A survey-based study of leaders of national ophthalmological and retinal societies was conducted. The survey was sent by email to 30 societies, from which 20 responded (12 countries, 66.6% response rate). It included closed- and open-ended questions about (1) operational capacity and precautions, (2) telemedicine and virtual care, (3) procedures, and (4) post-pandemic considerations. Results: There was a marked decline in ophthalmology patient visits (80-95%) and elective surgeries (90%) during 2020 compared to before the pandemic. Precautions like temperature checks, mask usage, and social distancing were widely implemented while personal protective equipment (PPE) availability varied. Telemedicine use was limited due to lack of experience with it. Reopening plans focused on maintaining precautions and gradually resuming activities. Economic and security concerns were raised, and adherence to guidelines was emphasized. Respondents acknowledged the need to adapt to a "new normal". Long duration drugs, fewer imaging studies, and shorter wait times were preferred; however, availability of long duration drugs was limited. Conclusion: The pandemic impacted ophthalmology in Latin America, with reduced patient visits, procedures, and surgeries. Delayed treatment and complications were likely the result of the pandemic.

[Prenatal rescue dose of betamethasone in the preterm infant with intrauterine growth restriction]. / Dosis prenatal de rescate de betametasona en el prematuro con restricción del crecimiento intrauterino.

Antenatal corticosteroids reduce mortality and respiratory distress syndrome (RDS) in preterm newborns. These benefits decrease after a week of administration, recommending a rescue therapy if there is a new threat of premature delivery. Repeated administration of antenatal corticosteroids may have deleterious effects and their benefits are controversial in intrauterine growth restriction (IUGR). OBJECTIVE: to verify the effects in the IUGR population of antenatal betamethasone rescue therapy on neonatal morbidity and mortality, RDS, and neurodevelopment at 2 years. PATIENTS AND METHOD: Retrospective study including ≤ 34 weeks and ≤ 1,500g preterm newborns divided according to antenatal betamethasone exposure: Single-cycle (2 doses) vs Rescue therapy (3 doses). Subgroups were created for those ≥ 30 weeks. Both cohorts were followed up to 24 months of corrected age. The Ages & Stages Questionnaires (ASQ)® was administered to assess neurodevelopment. RESULTS: 62 preterm infants with a diagnosis of IUGR were included. The rescue therapy group compared with the single-dose group showed no differences in morbidity and mortality and less intubation rate at birth (p = 0.02), with no differences in respiratory support at 7 days of life. Preterm newborns ≥ 30 weeks exposed to rescue therapy showed higher morbidity and mortality (p = 0.03) and bronchopulmonary dysplasia (BPD) (p = 0.02), showing no differences in RDS. The rescue therapy group showed worse mean scores on the ASQ-3 scale, with no significant differences in cerebral palsy or sensory deficits. CONCLUSIONS: Rescue therapy reduces intubation at birth but does not reduce morbidity and mortality. However, at > 30 weeks, this benefit is not observed and the IUGR population exposed to rescue therapy presented more BPD and lower scores on the ASQ-3 scale at 2 years. Future studies should be aimed at the individualization of antenatal corticosteroid therapy.

PHOTODYNAMIC THERAPY WITH VERTEPORFIN PLUS INTRAVITREAL BEVACIZUMAB FOR CIRCUMSCRIBED CHOROIDAL HEMANGIOMA: 4 YEARS OF FOLLOW-UP.

PURPOSE: To report the anatomical and functional response of combined photodynamic therapy and intravitreal injection of bevacizumab in a patient with symptomatic circumscribed choroidal hemangioma. METHODS: The patient received a single-session full-fluence photodynamic therapy immediately followed by an intravitreal injection of bevacizumab (1.25 mg/0.05 mL). RESULTS: One week after combined therapy, an improvement of best-corrected visual acuity from count fingers to 20/60 and a significant decrease in subretinal fluid were noted. One month later, we observed decreased leakage on fluorescein angiography in all phases of the study. Three months after treatment, the best-corrected visual acuity improved to 20/25 and spectral domain optical coherence tomography scans showed return to normal foveal architecture with no subretinal fluid and completely flat tumor. These findings were maintained during 4 years of follow-up. CONCLUSION: Combination therapy was associated with a rapid and persistent resolution of subretinal fluid, improvement of best-corrected visual acuity, and visual stability at 4 years of follow-up.

Anti-leishmanial effect of spiro dihydroquinoline-oxindoles on volume regulation decrease and sterol biosynthesis of Leishmania braziliensis.

Diverse spiro dihydroquinoline-oxindoles (JS series) were prepared using the BF3•OEt2-catalyzed imino Diels-Alder reaction between ketimine-isatin derivatives and trans-isoeugenol. Ten spiro-oxiindole derivatives were selected and evaluated at different stages of the life cycle of Leishmania braziliensis parasites, responsible for cutaneous leishmaniasis in South America. Among them, the 8'-ethyl-4'-(4-hydroxy-3-methoxyphenyl)-3'-methyl-3',4'-dihydro-1'H-spiro[indoline-3,2'-quinolin]-2-one called JS87 was able to inhibit the growth of promastigotes without affecting the mammalian cells viability, and to decrease the number of intracellular amastigotes of L. braziliensis. This spiro compound was found to act through the alteration of parasite internal regulation by disrupting the regulatory volume decrease (RVD), and to affect the sterol biosynthetic pathway at level of squalene epoxidase (SE) enzyme. These results revealed that the spiro annulation between quinoline and oxindole scaffolds enhances the anti-leishmanial activity, and could assist in the development of potent quinoline-oxindole hybrids against Leishmania braziliensis, the main etiological agent of cutaneous leishmaniasis in South America.

Yellow (577 nm) micropulse laser versus half-dose verteporfin photodynamic therapy in eyes with chronic central serous chorioretinopathy: results of the Pan-American Collaborative Retina Study (PACORES) Group.

PURPOSE: To compare the functional and anatomical outcomes of eyes with chronic central serous chorioretinopathy treated with yellow micropulse (MP) laser versus half-dose verteporfin photodynamic therapy (PDT). METHODS: This is a multicentre, retrospective comparative study of 92 eyes treated with yellow MP laser (duty cycle of 5%, zero spacing between spots, spot size varied from 100 to 200 µm, power varied from 320 to 660 mW, and the pulse burst duration was 200 ms) and 67 eyes treated with PDT (half-dose verteporfin (3 mg/m2) infused over 10 min), followed by laser activation for 83 s. Spot sizes varied from 400 to 2000 µm. RESULTS: In the MP group, at 12 months of follow-up, the mean best corrected visual acuity (BCVA) improved from the logarithm of the minimum angle of resolution (logMAR) of 0.41±0.27 at baseline to 0.21±0.26 (P<0.0001), 48.9% (45/92) of eyes had an improvement of ≥3 lines of BCVA from baseline, 48.9% (45/92) of eyes remained within 2 lines of baseline BCVA, and only 2.2% (2/92) of eyes lost ≥3 lines of BCVA from baseline. In the PDT group, at 12 months of follow-up, the mean BCVA changed from logMAR of 0.50±0.34 at baseline to 0.47±0.34 (P=0.89), 19% (13/67) of eyes had an improvement of ≥3 lines of BCVA from baseline, 73% (49/67) of eyes remained within 2 lines of baseline BCVA, and 7% (5/67) of eyes lost ≥3 lines of BCVA from baseline. There were no adverse events attributable to the yellow MP laser treatment. One eye in the PDT group developed choroidal neovascularisation, which was treated with three intravitreal bevacizumab injections. CONCLUSIONS: Both PDT and MP are effective in restoring the macular anatomy. In places where PDT is not available, yellow MP laser may be an adequate treatment alternative.

Mechanism of Action of Miltefosine on Leishmania donovani Involves the Impairment of Acidocalcisome Function and the Activation of the Sphingosine-Dependent Plasma Membrane Ca2+ Channel.

Leishmania donovani is the causing agent of visceral leishmaniasis, a common infection that affects millions of people from the most underdeveloped countries. Miltefosine is the only oral drug to treat infections caused by L. donovani Nevertheless, its mechanism of action is not well understood. While miltefosine inhibits the synthesis of phosphatidylcholine and also affects the parasite mitochondrion, inhibiting the cytochrome c oxidase, it is to be expected that this potent drug also produces its effect through other targets. In this context, it has been reported that the disruption of the intracellular Ca2+ homeostasis represents an important object for the action of drugs in trypanosomatids. Recently, we have described a plasma membrane Ca2+ channel in Leishmania mexicana, which is similar to the L-type voltage-gated Ca2+ channel (VGCC) present in humans. Remarkably, the parasite Ca2+ channel is activated by sphingosine, while the L-type VGCC is not affected by this sphingolipid. In the present work we demonstrated that, similarly to sphingosine, miltefosine is able to activate the plasma membrane Ca2+ channel from L. donovani Interestingly, nifedipine, the classical antagonist of the human channel, was not able to fully block the parasite plasma membrane Ca2+ channel, indicating that the mechanism of interaction is not identical to that of sphingosine. In this work we also show that miltefosine is able to strongly affect the acidocalcisomes from L. donovani, inducing the rapid alkalinization of these important organelles. In conclusion, we demonstrate two new mechanisms of action of miltefosine in L. donovani, both related to disruption of parasite Ca2+ homeostasis.

Un-explained visual loss following silicone oil removal: results of the Pan American Collaborative Retina Study (PACORES) Group.

PURPOSE: To report the incidence and clinical features of patients that experienced un-explained visual loss following silicone oil (SO) removal. METHODS: Multicenter retrospective study of patients that underwent SO removal during 2000-2012. Visual loss of ≥2 lines was considered significant. RESULTS: A total of 324 eyes of 324 patients underwent SO removal during the study period. Forty two (13%) eyes suffered a significant visual loss following SO removal. Twenty three (7.1%) of these eyes lost vision secondary to known causes. In the remaining 19 (5.9%) eyes, the loss of vision was not explained by any other pathology. Eleven of these 19 patients (57.9%) were male. The mean age of this group was 49.2 ± 16.4 years. Eyes that had an un-explained visual loss had a mean IOP while the eye was filled with SO of 19.6 ± 6.9 mm Hg. The length of time that the eye was filled with SO was 14.8 ± 4.4 months. In comparison, eyes that did not experience visual loss had a mean IOP of 14 ± 7.3 mm Hg (p < 0.0002) and a mean tamponade duration of 9.3 ± 10.9 months (p < 0.0001). CONCLUSIONS: An un-explained visual loss after SO removal was observed in 5.9% of eyes. Factors associated with this phenomenon included a higher IOP and longer SO tamponade duration.

Design, synthesis, structure-activity relationship and mechanism of action studies of a series of 4-chloro-1-phthalazinyl hydrazones as a potent agent against Leishmania braziliensis.

With the aim to identify a potential drug candidate to treat cutaneous leishmaniasis, a series of 1-phthalazinyl hydrazones were synthesized and tested against Leishmania braziliensis parasite, one of the main responsible of this disease in the world. A structure-activity relationship permitted to identify two phthalazines containing nitroheterocyclic moiety 3l and 3m as promising new lead compounds. These compounds showed a significant antileishmanial activity against promastigote form of L. braziliensis, with EC50 values in sub-micromolar and nanomolar ranges. The phthalazine 3l also displayed a selective and excellent activity against the clinically relevant intracellular amastigotes form, with a EC50 value in sub-micromolar range (0.59 µM), without affecting the viability of the host cells. Oxidative stress was identified as the possible mode of action of the most active phthalazine. Considering their significant antileishmanial activity and ease synthesis, the phthalazine containing nitroheterocyclic represents a promising agent against Leishmania braziliensis for the rational design of new leads.

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. .

Outcomes of cataract surgery in diabetic patients: results of the 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.

Anti-leishmanial evaluation of C2-aryl quinolines: mechanistic insight on bioenergetics and sterol biosynthetic pathway of Leishmania braziliensis.

A series of diverse simple C2-aryl quinolines was synthesized de novo via a straightforward synthesis based on the acid-catalyzed multicomponent imino Diels-Alder reactions. Seven selected quinolines were evaluated at different stages of Leishmania braziliensis parasite. Among them, the 6-ethyl-2-phenylquinoline 5f was able to inhibit the growth of promastigotes of this parasite without affecting the mammalian cells viability and decreasing the number of intracellular L. braziliensis amastigotes on BMDM macrophages. The mechanism of action studied for the selected compound consisted in: (1) alteration of parasite bioenergetics, by disrupting mitochondrial electrochemical potential and alkalinization of acidocalcisomes, and (2) inhibition of ergosterol biosynthetic pathway in promastigote forms. These results validate the efficiency of quinoline molecules as leishmanicide compounds.

Intravitreal bevacizumab for inflammatory choroidal neovascularization: results from the Pan-American Collaborative Retina Study Group at 24 months.

BACKGROUND: To evaluate the anatomical and functional outcomes of intravitreal bevacizumab (1.25 or 2.5 mg) in the treatment of inflammatory choroidal neovascularization at 24 months. METHODS: We reviewed the clinical records of 22 consecutive patients (23 eyes) with choroidal neovascularization secondary to chorioretinal inflammatory disease in this interventional retrospective multicenter case series. Sixteen eyes (63.6%) received a dose of 1.25 mg of intravitreal bevacizumab, and 7 eyes (36.4%) received a dose of 2.5 mg of intravitreal bevacizumab. RESULTS: At baseline, the mean best-corrected visual acuity was 0.68 logarithm of minimum angle of resolution (Early Treatment Diabetic Retinopathy Study chart = 20/100). After intravitreal bevacizumab, best-corrected visual acuity improved significantly to 0.41 logarithm of minimum angle of resolution (20/51), 0.42 logarithm of minimum angle of resolution (20/53), and 0.40 logarithm of minimum angle of resolution (20/50) at 6, 12, and 24 months, respectively (P < 0.05). Fourteen eyes (60.8%) received 1 injection. Central macular thickness by optical coherence tomography decreased from 375.3 µm (range: 240-634 µm) at baseline to 241.6 µm (range: 189-306 µm) at 24 months of follow-up (P < 0.0001). CONCLUSION: Intravitreal bevacizumab at doses of 1.25 mg and 2.5 mg seems to provide stability or improvement in best-corrected visual acuity, optical coherence tomography, and fluorescein angiogram in inflammatory choroidal neovascularization at 24 months. All patients were treated after the underlying uveitic condition was controlled.

Amiodarone and miltefosine act synergistically against Leishmania mexicana and can induce parasitological cure in a murine model of cutaneous leishmaniasis.

Leishmaniasis is parasitic disease that is an important problem of public health worldwide. Intramuscularly administered glucantime and pentostam are the most common drugs used for treatment of this disease, but they have significant limitations due to toxicity and increasing resistance. A recent breakthrough has been the introduction of orally administered miltefosine for the treatment of visceral, cutaneous, and mucocutaneous leishmaniasis, but the relative high cost and concerns about teratogenicity have limited the use of this drug. Searching for alternative drugs, we previously demonstrated that the antiarrhythmic drug amiodarone is active against Leishmania mexicana promastigotes and intracellular amastigotes, acting via disruption of intracellular Ca(2+) homeostasis (specifically at the mitochondrion and the acidocalcisomes of these parasites) and through inhibition of the parasite's de novo sterol biosynthesis (X. Serrano-Martín, Y. García-Marchan, A. Fernandez, N. Rodriguez, H. Rojas, G. Visbal, and G. Benaim, Antimicrob. Agents Chemother. 53:1403-1410, 2009). In the present work, we found that miltefosine also disrupts the parasite's intracellular Ca(2+) homeostasis, in this case by inducing a large increase in intracellular Ca(2+) levels, probably through the activation of a plasma membrane Ca(2+) channel. We also investigated the in vitro and in vivo activities of amiodarone and miltefosine, used alone or in combination, on L. mexicana. It was found that the drug combination had synergistic effects on the proliferation of intracellular amastigotes growing inside macrophages and led 90% of parasitological cures in a murine model of leishmaniasis, as revealed by a PCR assay using a novel DNA sequence specific for L. mexicana.

Amiodarone destabilizes intracellular Ca2+ homeostasis and biosynthesis of sterols in Leishmania mexicana.

Leishmaniasis represents a serious public health problem worldwide. The first line of treatment is based on glucantime and pentostan, which generate toxic effects in treated patients. We have recently shown that amiodarone, frequently used as an antiarrhythmic, possesses activity against Trypanosoma cruzi through the disruption of mitochondrial Ca(2+) homeostasis and the inhibition of parasite ergosterol biosynthesis, specifically at the level of oxidosqualene cyclase activity (G. Benaim, J. Sanders, Y. Garcia-Marchan, C. Colina, R. Lira, A. Caldera, G. Payares, C. Sanoja, J. Burgos, A. Leon-Rossell, J. Concepcion, A. Schijman, M. Levin, E. Oldfield, and J. Urbina, J. Med. Chem. 49:892-899, 2006). Here we show that at therapeutic concentrations, amiodarone has a profound effect on the viability of Leishmania mexicana promastigotes. Additionally, its effect on the viability of the parasite was greater against intracellular amastigotes than against promastigotes, and it did not affect the host cell. Using fluorimetric and confocal microscopy techniques, we also demonstrated that the mechanism of action of amiodarone was related to the disruption of intracellular Ca(2+) homeostasis through a direct action not only on the mitochondria but also on the acidocalcisomes. On the other hand, analysis of the free sterols in promastigotes incubated with amiodarone showed that this drug also affected the biosynthesis of 5-dehydroepisterol, which results in squalene accumulation, thus suggesting that amiodarone inhibits the squalene epoxidase activity of the parasite. Taken together, the results obtained in the present work point to a more general effect of amiodarone in trypanosomatids, opening potential therapeutic possibilities for this infectious disease.

Anti-VSG antibodies induce an increase in Trypanosoma evansi intracellular Ca2+ concentration.

Trypanosoma evansi and Trypanosoma vivax have shown a very high immunological cross-reactivity. Anti-T. vivax antibodies were used to monitor changes in the T. evansi intracellular Ca2+ concentration ([Ca2+]i) by fluorometric ratio imaging from single parasites. A short-time exposure of T. evansi parasites to sera from T. vivax-infected bovines induced an increase in [Ca2+]i, which generated their complete lysis. The parasite [Ca2+]i boost was reduced but not eliminated in the absence of extracellular Ca2+ or following serum decomplementation. Decomplemented anti-T. evansi VSG antibodies also produced an increase in the parasite [Ca2+]i, in the presence of extracellular Ca2+. Furthermore, this Ca2+ signal was reduced following blockage with Ni2+ or in the absence of extracellular Ca2+, suggesting that this response was a combination of an influx of Ca2+ throughout membrane channels and a release of this ion from intracellular stores. The observed Ca2+ signal was specific since (i) it was completely eliminated following pre-incubation of the anti-VSG antibodies with the purified soluble VSG, and (ii) affinity-purified anti-VSG antibodies also generated an increase in [Ca2+]i by measurements on single cells or parasite populations. We also showed that an increase of the T. evansi [Ca2+]i by the calcium A-23187 ionophore led to VSG release from the parasite surface. In addition, in vivo immunofluorescence labelling revealed that anti-VSG antibodies induced the formation of raft patches of VSG on the parasite surface. This is the first study to identify a ligand that is coupled to calcium flux in salivarian trypanosomes.

Glibenclamide, a blocker of K+(ATP) channels, shows antileishmanial activity in experimental murine cutaneous leishmaniasis.

Glibenclamide reduced the rate of lesion growth in BALB/c mice infected with Leishmania (Leishmania) mexicana, the effect was dose dependent, and the highest dose proved more effective than glucantime. Cross-resistance to glucantime was found in animals infected with a glibenclamide-resistant line, but combined therapy reduced lesion progression even in the glibenclamide-resistant strain.