Antiproliferative, Ultrastructural, and Physiological Effects of Amiodarone on Promastigote and Amastigote Forms of Leishmania amazonensis

Amiodarone (AMIO), the most frequently antiarrhythmic drug used for the symptomatic treatment of chronic Chagas' disease patients with cardiac compromise, has recently been shown to have also specific activity against fungi, Trypanosoma cruzi and Leishmania. In this work, we characterized the effects of AMIO on proliferation, mitochondrial physiology, and ultrastructure of Leishmania amazonensis promastigotes and intracellular amastigotes. The IC50 values were 4.21 and 0.46 μM against promastigotes and intracellular amastigotes, respectively, indicating high selectivity for the clinically relevant stage. We also found that treatment with AMIO leads to a collapse of the mitochondrial membrane potential (ΔΨm) and to an increase in the production of reactive oxygen species, in a dose-dependent manner. Fluorescence microscopy of cells labeled with JC-1, a marker for mitochondrial energization, and transmission electron microscopy confirmed severe alterations of the mitochondrion, including intense swelling and modification of its membranes. Other ultrastructural alterations included (1) presence of numerous lipid-storage bodies, (2) presence of large autophagosomes containing part of the cytoplasm and membrane profiles, sometimes in close association with the mitochondrion and endoplasmic reticulum, and (3) alterations in the chromatin condensation and plasma membrane integrity. Taken together, our results indicate that AMIO is a potent inhibitor of L. amazonensis growth, acting through irreversible alterations in the mitochondrial structure and function, which lead to cell death by necrosis, apoptosis and/or autophagy

Conhecendo a síndrome de autofermentação: etiopatogenia, apresentação e abordagem

Revisar os dados sobre síndrome da autofermentação disponíveis na literatura e reforçar a possibilidade dessa condição como hipótese durante as avaliações diagnósticas. Revisão de literatura de caráter exploratório com estudos selecionados nas plataformas PubMED e Google Scholar, no período de 2015 a 2024. Foram elegidos, após a aplicação dos critérios de seleção e exclusão, 20 artigos para a leitura completa e adicionados 4 materiais extras de valor para o estudo.  A síndrome da autofermentação é uma intoxicação alcoólica de origem endógena, causada, principalmente, por fungos fermentadores após um processo de disbiose intestinal. Suas principais manifestações incluem desorientação, descoordenação motora, marcha atáxica e desinibição social. O diagnóstico é realizado por anamnese detalhada, detecção de altos níveis séricos de álcool e teste do desafio dos carboidratos positivo. O manejo da condição consiste em evitar fatores que prejudiquem o microbioma intestinal e tratar os agentes causadores com uso de antifúngicos principalmente. A síndrome da autofermentação pode ter impacto nos contextos médico, legal e social. É necessário que ela seja mais disseminada entre a comunidade médica e leiga com intuito de permitir que o paciente possa ter um diagnóstico e tratamento adequados

Impact of safety-related dose reductions or discontinuations on sustained virologic response in HCV-infected patients: Results from the GUARD-C Cohort

BACKGROUND: Despite the introduction of direct-acting antiviral agents for chronic hepatitis C virus (HCV) infection, peginterferon alfa/ribavirin remains relevant in many resource-constrained settings. The non-randomized GUARD-C cohort investigated baseline predictors of safety-related dose reductions or discontinuations (sr-RD) and their impact on sustained virologic response (SVR) in patients receiving peginterferon alfa/ribavirin in routine practice. METHODS: A total of 3181 HCV-mono-infected treatment-naive patients were assigned to 24 or 48 weeks of peginterferon alfa/ribavirin by their physician. Patients were categorized by time-to-first sr-RD (Week 4/12). Detailed analyses of the impact of sr-RD on SVR24 (HCV RNA <50 IU/mL) were conducted in 951 Caucasian, noncirrhotic genotype (G)1 patients assigned to peginterferon alfa-2a/ribavirin for 48 weeks. The probability of SVR24 was identified by a baseline scoring system (range: 0-9 points) on which scores of 5 to 9 and <5 represent high and low probability of SVR24, respectively. RESULTS: SVR24 rates were 46.1% (754/1634), 77.1% (279/362), 68.0% (514/756), and 51.3% (203/396), respectively, in G1, 2, 3, and 4 patients. Overall, 16.9% and 21.8% patients experienced 651 sr-RD for peginterferon alfa and ribavirin, respectively. Among Caucasian noncirrhotic G1 patients: female sex, lower body mass index, pre-existing cardiovascular/pulmonary disease, and low hematological indices were prognostic factors of sr-RD; SVR24 was lower in patients with 651 vs. no sr-RD by Week 4 (37.9% vs. 54.4%; P = 0.0046) and Week 12 (41.7% vs. 55.3%; P = 0.0016); sr-RD by Week 4/12 significantly reduced SVR24 in patients with scores <5 but not 655. CONCLUSIONS: In conclusion, sr-RD to peginterferon alfa-2a/ribavirin significantly impacts on SVR24 rates in treatment-naive G1 noncirrhotic Caucasian patients. Baseline characteristics can help select patients with a high probability of SVR24 and a low probability of sr-RD with peginterferon alfa-2a/ribavirin

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

In vitro activity of the antifungal azoles itraconazole and posaconazole against Leishmania amazonensis.

Leishmaniasis, caused by protozoan parasites of the Leishmania genus, is one of the most prevalent neglected tropical diseases. It is endemic in 98 countries, causing considerable morbidity and mortality. Pentavalent antimonials are the first line of treatment for leishmaniasis except in India. In resistant cases, miltefosine, amphotericin B and pentamidine are used. These treatments are unsatisfactory due to toxicity, limited efficacy, high cost and difficult administration. Thus, there is an urgent need to develop drugs that are efficacious, safe, and more accessible to patients. Trypanosomatids, including Leishmania spp. and Trypanosoma cruzi, have an essential requirement for ergosterol and other 24-alkyl sterols, which are absent in mammalian cells. Inhibition of ergosterol biosynthesis is increasingly recognized as a promising target for the development of new chemotherapeutic agents. The aim of this work was to investigate the antiproliferative, physiological and ultrastructural effects against Leishmania amazonensis of itraconazole (ITZ) and posaconazole (POSA), two azole antifungal agents that inhibit sterol C14α-demethylase (CYP51). Antiproliferative studies demonstrated potent activity of POSA and ITZ: for promastigotes, the IC50 values were 2.74 µM and 0.44 µM for POSA and ITZ, respectively, and for intracellular amastigotes, the corresponding values were 1.63 µM and 0.08 µM, for both stages after 72 h of treatment. Physiological studies revealed that both inhibitors induced a collapse of the mitochondrial membrane potential (ΔΨm), which was consistent with ultrastructural alterations in the mitochondrion. Intense mitochondrial swelling, disorganization and rupture of mitochondrial membranes were observed by transmission electron microscopy. In addition, accumulation of lipid bodies, appearance of autophagosome-like structures and alterations in the kinetoplast were also observed. In conclusion, our results indicate that ITZ and POSA are potent inhibitors of L. amazonensis and suggest that these drugs could represent novel therapies for the treatment of leishmaniasis, either alone or in combination with other agents

Light microscopy of murine macrophages infected with <i>L. amazonensis</i> amastigotes.

<p>(A) Control culture with many amastigotes inside parasitophorous vacuoles. (B–H) After 72 h of treatment with different concentrations of POSA and ITZ, a significant reduction in the number of parasites and the presence of several empty parasitophorous vacuoles was observed.</p

Analysis of lipid body accumulation and plasma membrane integrity in <i>L. amazonensis</i> promastigotes.

<p>(A–B) Quantitative fluorimetric analysis using Nile Red (A) and Sytox Blue (B). Fluorescence intensity is expressed as arbitrary units (A.U.). The results were plotted as mean of three independent experiments and the bars represent the standard deviation. *p<0.01; **p<0.05; ***p<0.0001. (C–H) Differential interference contrast (DIC) microscopy (C, E, G) and fluorescence microscopy using Nile Red (D, F, H) of control <i>L. amazonensis</i> promastigotes and promastigotes treated with 1 µM POSA or ITZ for 48 h. The images demonstrate an accumulation of lipid bodies that are randomly distributed throughout the cytoplasm, confirming the increase in the fluorescence intensity observed in Fig. 4A.</p

Ultrathin sections of <i>L. amazonensis</i> promastigotes treated with different concentrations of ITZ and POSA.

<p>(A, B) 1 µM ITZ; (C, D) 1 µM POSA; (E) 3 µM POSA for 48 h; (F) 5 µM POSA for 72 h. Several alterations were observed in the mitochondrion-kinetoplast complex such as: intense disorganization and swelling (A, B, D); alterations in the mitochondrion membranes and the appearance of circular cristae (B, C, arrows); changes in the structure of the kinetoplast (B, D, E, F); and the presence of autophagosomes (A, C, D). In Fig. 7E, two large vacuoles containing membranes and portions of the cytoplasm were observed (asterisks). FP, flagellar pocket; GC, Golgi complex; k: kinetoplast; m, mitochondrion; N: nucleus, A: autophagosome.</p

Ultrathin sections of <i>L. amazonensis</i> intracellular amastigotes.

<p>Control intracellular amastigotes (A) and treated amastigotes with ITZ and POSA (B–G) were observed. (B, C) 500 nM ITZ; (D) 1 µM ITZ; (E–G) 6 µM POSA. Different ultrastructural alterations were observed: mitochondrial swelling (B, D); detachment of the plasma membrane (B, arrowhead); presence of a large megasome (C, black asterisk), lipid bodies (D, E, G, white asterisks) and many vacuoles in the cytoplasm (D, E, G); changes in kinetoplast structure (C, E); and a cell with an empty parasitophorous vacuole (PV) (G). f, flagellum; m, mitochondrion; N: nucleus; PV: parasitophorous vacuole, A: autophagosome, k: kinetoplast.</p