Public's perspective on COVID-19 adenovirus vector vaccines after thrombosis with thrombocytopenia syndrome (TTS) reports and associated regulatory actions - A cross-sectional study in six EU member states.

OBJECTIVE: In 2021, thrombosis with thrombocytopenia syndrome (TTS) was confirmed by the European Medicines Agency (EMA) as a rare side effect of the COVID-19 adenovirus vector vaccines Vaxzevria® and Jcovden®. This study aimed to describe the public's knowledge of TTS and how it affected the willingness to be vaccinated with COVID-19 vaccines and other vaccines in six European countries. METHODS: From June to October of 2022, a multi-country cross-sectional online survey was conducted in Denmark, Greece, Latvia, Netherlands, Portugal, and Slovenia. The minimum target of participants to be recruited was based on the size of the country's population. The results were analysed descriptively. RESULTS: In total, 3794 respondents were included in the analysis; across the six countries, 33.3 %-68.3 % reported being familiar with signs and symptoms of TTS, although 3.1-61.4 % of those were able to identify the symptoms correctly. The reported changes in willingness to be vaccinated against COVID-19 and with other vaccines varied per country. The largest reported change in the willingness to be vaccinated with Vaxzevria® and Jcovden® was observed in Denmark (61.2 %), while the willingness to be vaccinated with other COVID-19 vaccines changed most in Slovenia (30.4 %). The smallest decrease in willingness towards future vaccination against COVID-19 was reported in the Netherlands (20.9 %) contrasting with the largest decrease observed in Latvia (69.1 %). CONCLUSION: Knowledge about TTS seemed to have influenced the public's opinion in Europe resulting in less willingness to be vaccinated with Vaxzevria® and Jcovden®. Willingness for vaccination against COVID-19 with other vaccines and widespread use of vaccines to prevent other diseases also differed and seemed to be determined by the approaches taken by national health authorities when reacting to and communicating about COVID-19 vaccination risks. Further investigation of optimal risk communication strategies is warranted.

Motivation and Knowledge of Portuguese Community Pharmacists Towards the Reporting of Suspected Adverse Reactions to Medicines: A Cross-Sectional Survey.

The close contact between patients and community pharmacists, along with the extensive geographical distribution of pharmacies in Portugal, offer exceptional conditions to detect and report adverse drug reactions (ADR). This study aimed to evaluate the motivation and knowledge of spontaneous reporting of ADR by community pharmacists of Porto, Portugal. Secondly, we aimed to generate real-world evidence on the main factors determining ADR report and at raising potential alternatives to the current reporting procedure in community pharmacy. We performed a descriptive, cross-sectional, observational, anonymous web survey-based study. Between April and July 2021, a web survey was implemented, targeting community pharmacists in the Porto district, Portugal. We validated 217 surveys from pharmacists. Regular notifiers seem to be more familiarised than non-regular notifiers with the Portuguese Pharmacovigilance System (PPS), with the Portal RAM for reporting suspected ADR, and with the update of the concept of ADR. Moreover, regular notifiers seem to be more proactive with their care in questioning patients about ADR and have more self-knowledge to identify suspected ADR. Conversely, non-regular notifiers, seem to be more reluctant to be judged by their ADR reporting activities. Respondents suggested to simplify and optimise the reporting process (31% of the suggestions), or to integrate a reporting platform into the pharmacy's software (27%). This study identified opportunities to promote the ADR reporting process by community pharmacists, namely receiving feedback from the PPS on the reported case and its regulatory implications, implementing training programs in pharmacovigilance, and creating solutions to simplify the reporting process.

ß-heavy-spectrin stabilizes the constricting contractile ring during cytokinesis.

Cytokinesis requires the constriction of an actomyosin-based contractile ring and involves multiple F-actin crosslinkers. We show that partial depletion of the C. elegans cytokinetic formin generates contractile rings with low F-actin levels that constrict but are structurally fragile, and we use this background to investigate the roles of the crosslinkers plastin/PLST-1 and ß-heavy-spectrin/SMA-1 during ring constriction. We show that the removal of PLST-1 or SMA-1 has opposite effects on the structural integrity of fragile rings. PLST-1 loss reduces cortical tension that resists ring constriction and makes fragile rings less prone to ruptures and regressions, whereas SMA-1 loss exacerbates structural defects, leading to frequent ruptures and cytokinesis failure. Fragile rings without SMA-1 or containing a shorter SMA-1, repeatedly rupture at the same site, and SMA-1::GFP accumulates at repair sites in fragile rings and in rings cut by laser microsurgery. These results establish that ß-heavy-spectrin stabilizes the constricting ring and reveals the importance of ß-heavy-spectrin size for network connectivity at low F-actin density.

[Looking back on 20 years of work at the Porto Pharmacovigilance Centre, Portugal]. / Retrospectiva de 20 anos de atividade da Unidade de Farmacovigilância do Porto, Portugal.

Since 1963, the World Health Organization has acknowledged pharmacovigilance as a priority area in global public health, guaranteeing permanent monitoring of drug safety. This study aimed to characterize the reports of adverse drug reactions received by the Porto Pharmacovigilance Centre (UFPorto), Portugal, in the unit's two decades of work. The analysis included all reports of suspected adverse drug reactions received from January 2001 to December 2019. We calculated the annual reporting rates and distribution by origin, type of notifier and place of work, severity, prior knowledge, and causality of the reported adverse drug reactions. During the study period, UFPorto received 9,711 notifications of suspected adverse drug reactions. Hospital institutions reported the most suspected adverse drug reactions (n = 6,003; 64%), as did physicians among healthcare workers (n = 5,284; 54.4%). The most frequently reported adverse events were severe (n = 6,275; 72%) and are described in the respective Summary of Product Characteristics (n = 6,978; 72%). Most of the reports received by UFPorto were evaluated as having "probable" causality (n = 7,473; 77%), independently of the type of notifier. The results are consistent with other data previously reported in the international medical literature and official national reports. However, the underreporting rates are still higher than expected. In approximately 20 years, UFPorto has experienced an increase in its activity in various areas of drug safety.

Desde 1963 que a farmacovigilância foi reconhecida como uma área prioritária na saúde pública a nível global pela Organização Mundial da Saúde, garantindo a monitorização permanente da segurança dos medicamentos. O objetivo deste trabalho foi caracterizar as reações adversas a medicamentos recebidas pela Unidade de Farmacovigilância do Porto (UFPorto), Portugal, ao longo de duas décadas de atividade. Foram consideradas todas as notificações de suspeitas de reações adversas a medicamentos recebidas entre janeiro de 2001 e dezembro de 2019. Foram calculadas as taxas de notificação anuais, assim como a sua distribuição por origem, tipo de notificador e local de exercício da atividade, gravidade, conhecimento prévio e causalidade das reações adversas a medicamentos notificadas. No período em estudo, a UFPorto recebeu 9.711 notificações de suspeitas de reações adversas a medicamentos. As instituições hospitalares são aquelas que mais notificam (n = 6.003; 64%), assim como o médico entre os profissionais de saúde (n = 5.284; 54,4%). Os eventos adversos mais frequentemente reportados são graves (n = 6.275; 72%) e encontram-se descritos no respectivo Resumo das Características do Medicamento (n = 6.978; 72%). À maioria das notificações avaliadas pela UFPorto foi atribuído o grau de causalidade "provável" (n = 7.473; 77%), independentemente do tipo de notificador. Os resultados obtidos são concordantes com outros dados previamente reportados na literatura médica internacional e em relatórios oficiais nacionais. Contudo, continua-se a verificar taxas de subnotificação acentuadas, face ao esperado. Ao longo de aproximadamente 20 anos de atividade da UFPorto, tem-se verificado um aumento da sua atividade nas diversas vertentes da segurança do medicamento.

Desde 1963 la farmacovigilancia fue reconocida por la Organización Mundial de la Salud, como un área prioritaria en la salud pública a nivel global, garantizando la monitorización permanente de la seguridad de los medicamentos. El objetivo de este trabajo fue caracterizar las reacciones adversas a medicamentos recibidas por la Unidad de Farmacovigilancia de Oporto (UFPorto), Portugal, a lo largo de dos décadas de actividad. Se consideraron todas las notificaciones de sospechas de reacciones adversas a medicamentos, recibidas entre enero de 2001 y diciembre de 2019. Se calcularon las tasas de notificación anuales, así como su distribución por origen, tipo de notificador y lugar de ejercicio de la actividad, gravedad, conocimiento previo y causalidad de las reacciones adversas a medicamentos notificadas. En el período de estudio, la UFPorto recibió 9.711 notificaciones de sospechas reacciones adversas a medicamentos. Las instituciones hospitalarias son quienes más notifican (n = 6.003; 64%), así como el médico entre los profesionales de salud (n = 5.284; 54,4%). Los eventos adversos más frecuentemente reportados son graves (n = 6.275; 72%) y se encuentran descritos en lo respectivo Resumen de las Características del Medicamento (n = 6 978; 72%). A la mayoría de las notificaciones evaluadas por la UFPorto se le atribuyó el grado de causalidad "probable" (n = 7.473; 77%), independientemente del tipo de notificador. Los resultados obtenidos son concordantes con otros datos previamente reportados en la literatura médica internacional y en informes oficiales nacionales. No obstante, se continúa verificando tasas de subregistro acentuadas, frente a lo esperado. A lo largo de aproximadamente 20 años de actividad de la UFPorto, se ha verificado un aumento de su actividad en las diversas vertientes de la seguridad del medicamento.

Assessment of 17α-ethinylestradiol effects in Daphnia magna: life-history traits, biochemical and genotoxic parameters.

The occurrence of pharmaceuticals in aquatic ecosystems and the need to study them have increased over the years since they enter continuously the environment. Besides, these compounds are not intended for applications with environmental purposes, and therefore, little is known about their ecological effects, particularly in non-target organisms, as invertebrate species. Inside these substances, endocrine disrupting compounds (EDCs) have recently come into the limelight, due to environmental concentrations and consequently their detrimental effects on different organisms. 17α-ethinylestradiol (EE2) has been detected in the aquatic environment in various locations around the globe since it is the main synthetic hormone used as a female oral contraceptive and is also applied in veterinary medicine and animal production. The present study was intended to assess the chronic effects of EE2, in the non-target organism as Daphnia magna. Thus, to analyze the individual and subindividual impact, this aquatic organism was chronically exposed (21 days) to 0.00 (control group), 0.10, 1.00, 10.0, and 100 µg/L of EE2. Results here obtained demonstrated that D. magna exposed to the EE2 concentrations had significant effects in individual (life-history) and sub-individual (biochemical levels) parameters. Alterations as anticipation in the age at first reproduction, a decrease of the growth rate, oxidative stress, and lipid peroxidation were detected, as well as genotoxic damage. Therefore, it was possible to infer that EE2 can disrupt several metabolic pathways and physiological functions of D. magna, since EE2 demonstrated ecotoxicity, at environmentally relevant concentrations. This work reinforces the importance of examining the effects of more relevant exposures (more prolonged and with ecologically pertinent concentrations) of potential endocrine disruptors like EE2, to the freshwater organisms and ecosystem.

Retrospectiva de 20 anos de atividade da Unidade de Farmacovigilância do Porto, Portugal / Looking back on 20 years of work at the Porto Pharmacovigilance Centre, Portugal / Retrospectiva de 20 años de actividad de la Unidad de Farmacovigilancia de Oporto, Portugal

Resumo: Desde 1963 que a farmacovigilância foi reconhecida como uma área prioritária na saúde pública a nível global pela Organização Mundial da Saúde, garantindo a monitorização permanente da segurança dos medicamentos. O objetivo deste trabalho foi caracterizar as reações adversas a medicamentos recebidas pela Unidade de Farmacovigilância do Porto (UFPorto), Portugal, ao longo de duas décadas de atividade. Foram consideradas todas as notificações de suspeitas de reações adversas a medicamentos recebidas entre janeiro de 2001 e dezembro de 2019. Foram calculadas as taxas de notificação anuais, assim como a sua distribuição por origem, tipo de notificador e local de exercício da atividade, gravidade, conhecimento prévio e causalidade das reações adversas a medicamentos notificadas. No período em estudo, a UFPorto recebeu 9.711 notificações de suspeitas de reações adversas a medicamentos. As instituições hospitalares são aquelas que mais notificam (n = 6.003; 64%), assim como o médico entre os profissionais de saúde (n = 5.284; 54,4%). Os eventos adversos mais frequentemente reportados são graves (n = 6.275; 72%) e encontram-se descritos no respectivo Resumo das Características do Medicamento (n = 6.978; 72%). À maioria das notificações avaliadas pela UFPorto foi atribuído o grau de causalidade "provável" (n = 7.473; 77%), independentemente do tipo de notificador. Os resultados obtidos são concordantes com outros dados previamente reportados na literatura médica internacional e em relatórios oficiais nacionais. Contudo, continua-se a verificar taxas de subnotificação acentuadas, face ao esperado. Ao longo de aproximadamente 20 anos de atividade da UFPorto, tem-se verificado um aumento da sua atividade nas diversas vertentes da segurança do medicamento.

Abstract: Since 1963, the World Health Organization has acknowledged pharmacovigilance as a priority area in global public health, guaranteeing permanent monitoring of drug safety. This study aimed to characterize the reports of adverse drug reactions received by the Porto Pharmacovigilance Centre (UFPorto), Portugal, in the unit's two decades of work. The analysis included all reports of suspected adverse drug reactions received from January 2001 to December 2019. We calculated the annual reporting rates and distribution by origin, type of notifier and place of work, severity, prior knowledge, and causality of the reported adverse drug reactions. During the study period, UFPorto received 9,711 notifications of suspected adverse drug reactions. Hospital institutions reported the most suspected adverse drug reactions (n = 6,003; 64%), as did physicians among healthcare workers (n = 5,284; 54.4%). The most frequently reported adverse events were severe (n = 6,275; 72%) and are described in the respective Summary of Product Characteristics (n = 6,978; 72%). Most of the reports received by UFPorto were evaluated as having "probable" causality (n = 7,473; 77%), independently of the type of notifier. The results are consistent with other data previously reported in the international medical literature and official national reports. However, the underreporting rates are still higher than expected. In approximately 20 years, UFPorto has experienced an increase in its activity in various areas of drug safety.

Resumen: Desde 1963 la farmacovigilancia fue reconocida por la Organización Mundial de la Salud, como un área prioritaria en la salud pública a nivel global, garantizando la monitorización permanente de la seguridad de los medicamentos. El objetivo de este trabajo fue caracterizar las reacciones adversas a medicamentos recibidas por la Unidad de Farmacovigilancia de Oporto (UFPorto), Portugal, a lo largo de dos décadas de actividad. Se consideraron todas las notificaciones de sospechas de reacciones adversas a medicamentos, recibidas entre enero de 2001 y diciembre de 2019. Se calcularon las tasas de notificación anuales, así como su distribución por origen, tipo de notificador y lugar de ejercicio de la actividad, gravedad, conocimiento previo y causalidad de las reacciones adversas a medicamentos notificadas. En el período de estudio, la UFPorto recibió 9.711 notificaciones de sospechas reacciones adversas a medicamentos. Las instituciones hospitalarias son quienes más notifican (n = 6.003; 64%), así como el médico entre los profesionales de salud (n = 5.284; 54,4%). Los eventos adversos más frecuentemente reportados son graves (n = 6.275; 72%) y se encuentran descritos en lo respectivo Resumen de las Características del Medicamento (n = 6 978; 72%). A la mayoría de las notificaciones evaluadas por la UFPorto se le atribuyó el grado de causalidad "probable" (n = 7.473; 77%), independientemente del tipo de notificador. Los resultados obtenidos son concordantes con otros datos previamente reportados en la literatura médica internacional y en informes oficiales nacionales. No obstante, se continúa verificando tasas de subregistro acentuadas, frente a lo esperado. A lo largo de aproximadamente 20 años de actividad de la UFPorto, se ha verificado un aumento de su actividad en las diversas vertientes de la seguridad del medicamento.

Using the Four-Cell C. elegans Embryo to Study Contractile Ring Dynamics During Cytokinesis.

Cytokinesis is the process that completes cell division by partitioning the contents of the mother cell between the two daughter cells. It involves the highly regulated assembly and constriction of an actomyosin contractile ring, whose function is to pinch the mother cell in two. Research on the contractile ring has particularly focused on the signaling mechanisms that dictate when and where the ring is formed. In vivo studies of ring constriction are however scarce and its mechanistic understanding is therefore limited. Here we present several experimental approaches for monitoring ring constriction in vivo, using the four-cell C. elegans embryo as model. These approaches allow for the ring to be perturbed only after it forms and include the combination of live imaging with acute drug treatments, temperature-sensitive mutants and rapid temperature shifts, as well as laser microsurgery. In addition, we explain how to combine these with RNAi-mediated depletion of specific components of the cytokinetic machinery.

Network Contractility During Cytokinesis-from Molecular to Global Views.

Cytokinesis is the last stage of cell division, which partitions the mother cell into two daughter cells. It requires the assembly and constriction of a contractile ring that consists of a filamentous contractile network of actin and myosin. Network contractility depends on network architecture, level of connectivity and myosin motor activity, but how exactly is the contractile ring network organized or interconnected and how much it depends on motor activity remains unclear. Moreover, the contractile ring is not an isolated entity; rather, it is integrated into the surrounding cortex. Therefore, the mechanical properties of the cell cortex and cortical behaviors are expected to impact contractile ring functioning. Due to the complexity of the process, experimental approaches have been coupled to theoretical modeling in order to advance its global understanding. While earlier coarse-grained descriptions attempted to provide an integrated view of the process, recent models have mostly focused on understanding the behavior of an isolated contractile ring. Here we provide an overview of the organization and dynamics of the actomyosin network during cytokinesis and discuss existing theoretical models in light of cortical behaviors and experimental evidence from several systems. Our view on what is missing in current models and should be tested in the future is provided.

Involvement of mitochondrial dysfunction in nefazodone-induced hepatotoxicity.

Nefazodone (NEF) is an antidepressive agent that was widely used in the treatment of depression until its withdrawal from the market, due to reports of liver injury and failure. NEF hepatotoxicity has been associated with mitochondrial impairment due to interference with the OXPHOS enzymatic activities, increased ROS generation and decreased antioxidant defenses. However, the mechanisms by which NEF induces mitochondrial dysfunction in hepatocytes are not completely understood. Here, we investigated the mitochondrial mechanisms affected upon NEF exposure and whether these might be linked to drug hepatotoxicity, in order to infer liabilities of future drug candidates. Two moderately hepatotoxic NEF concentrations (20 and 50 µM) were selected from dose-response growth curves performed in HepG2 cells. Cell viability, caspase activity, nuclear morphology, mitochondrial transmembrane potential, mitochondrial superoxide levels, and the expression of genes associated with different cellular pathways were evaluated at different time points. NEF treatment led to an increase in the expression of genes associated with DNA-damage response, antioxidant defense and apoptosis and a decreased expression of genes encoding proteins involved in oxidative phosphorylation, DNA repair, cell proliferation and cell cycle progression, which seem to constitute mechanisms underlying the observed mitochondrial and cell function impairment.

How to promote adverse drug reaction reports using information systems - a systematic review and meta-analysis.

BACKGROUND: Adverse drug reactions (ADRs) are a well-recognized public health problem and a major cause of death and hospitalization in developed countries. The safety of a new drug cannot be established until it has been on the market for several years. Keeping drug reactions under surveillance through pharmacovigilance systems is indispensable. However, underreporting is a major issue that undermines the effectiveness of spontaneous reports. Our work presents a systematic review on the use of information systems for the promotion of ADR reporting. The aim of this work is to describe the state of the art information systems used to promote adverse drug reaction reporting. METHODS: A systematic review was performed with quantitative analysis of studies describing or evaluating the use of information systems to promote adverse drug reaction reporting. Studies with data related to the number of ADRs reported before and after each intervention and the follow-up period were included in the quantitative analysis. RESULTS: From a total of 3865 articles, 33 articles were included in the analysis; these articles described 29 different projects. Most of the projects were on a regional scale (62 %) and were performed in a hospital context (52 %). A total of 76 % performed passive promotion of ADR reporting and used web-based software (55 %). A total of 72 % targeted healthcare professionals and 24 % were oriented to patient ADR reporting. We performed a meta-analysis of 7 of the 29 projects to calculate the aggregated measure of the ADR reporting increase, which had an overall measure of 2.1 (indicating that the interventions doubled the number of ADRs reported). CONCLUSIONS: We found that most of the projects performed passive promotion of ADR reporting (i.e., facilitating the process). They were developed in hospitals and were tailored to healthcare professionals. These interventions doubled the number of ADR reports. We believe that it would be useful to develop systems to assist healthcare professionals with completing ADR reporting within electronic health records because this approach seems to be an efficient method to increase the ADR reporting rate. When this approach is not possible, it is essential to have a tool that is easily accessible on the web to report ADRs. This tool can be promoted by sending emails or through the inclusion of direct hyperlinks on healthcare professionals' desktops.

Ketogenic diets: from cancer to mitochondrial diseases and beyond.

BACKGROUND: The employment of dietary strategies such as ketogenic diets, which force cells to alter their energy source, has shown efficacy in the treatment of several diseases. Ketogenic diets are composed of high fat, moderate protein and low carbohydrates, which favour mitochondrial respiration rather than glycolysis for energy metabolism. DESIGN: This review focuses on how oncological, neurological and mitochondrial disorders have been targeted by ketogenic diets, their metabolic effects, and the possible mechanisms of action on mitochondrial energy homeostasis. The beneficial and adverse effects of the ketogenic diets are also highlighted. RESULTS AND CONCLUSIONS: Although the full mechanism by which ketogenic diets improve oncological and neurological conditions still remains to be elucidated, their clinical efficacy has attracted many new followers, and ketogenic diets can be a good option as a co-adjuvant therapy, depending on the situation and the extent of the disease.

Metabolomic Analyses of Leishmania Reveal Multiple Species Differences and Large Differences in Amino Acid Metabolism.

Comparative genomic analyses of Leishmania species have revealed relatively minor heterogeneity amongst recognised housekeeping genes and yet the species cause distinct infections and pathogenesis in their mammalian hosts. To gain greater information on the biochemical variation between species, and insights into possible metabolic mechanisms underpinning visceral and cutaneous leishmaniasis, we have undertaken in this study a comparative analysis of the metabolomes of promastigotes of L. donovani, L. major and L. mexicana. The analysis revealed 64 metabolites with confirmed identity differing 3-fold or more between the cell extracts of species, with 161 putatively identified metabolites differing similarly. Analysis of the media from cultures revealed an at least 3-fold difference in use or excretion of 43 metabolites of confirmed identity and 87 putatively identified metabolites that differed to a similar extent. Strikingly large differences were detected in their extent of amino acid use and metabolism, especially for tryptophan, aspartate, arginine and proline. Major pathways of tryptophan and arginine catabolism were shown to be to indole-3-lactate and arginic acid, respectively, which were excreted. The data presented provide clear evidence on the value of global metabolomic analyses in detecting species-specific metabolic features, thus application of this technology should be a major contributor to gaining greater understanding of how pathogens are adapted to infecting their hosts.

Leishmania TDR1 structure, a unique trimeric glutathione transferase capable of deglutathionylation and antimonial prodrug activation.

Thiol-dependent reductase I (TDR1), an enzyme found in parasitic Leishmania species and Trypanosoma cruzi, is implicated in deglutathionylation and activation of antimonial prodrugs used to treat leishmaniasis. The 2.3 Å resolution structure of TDR1 reveals a unique trimer of subunits each containing two glutathione-S-transferase (GST) domains. The similarities of individual domains and comparisons with GST classes suggest that TDR1 evolved by gene duplication, diversification, and gene fusion; a combination of events previously unknown in the GST protein superfamily and potentially explaining the distinctive enzyme properties of TDR1. The deglutathionylation activity of TDR1 implies that glutathione itself has regulatory intracellular roles in addition to being a precursor for trypanothione, the major low mass thiol present in trypanosomatids. We propose that activation of antiparasite Sb(V)-drugs is a legacy of the deglutathionylation activity of TDR1 and involves processing glutathione adducts with concomitant reduction of the metalloid to active Sb(III) species.

Impact of continuous axenic cultivation in Leishmania infantum virulence.

Experimental infections with visceral Leishmania spp. are frequently performed referring to stationary parasite cultures that are comprised of a mixture of metacyclic and non-metacyclic parasites often with little regard to time of culture and metacyclic purification. This may lead to misleading or irreproducible experimental data. It is known that the maintenance of Leishmania spp. in vitro results in a progressive loss of virulence that can be reverted by passage in a mammalian host. In the present study, we aimed to characterize the loss of virulence in culture comparing the in vitro and in vivo infection and immunological profile of L. infantum stationary promastigotes submitted to successive periods of in vitro cultivation. To evaluate the effect of axenic in vitro culture in parasite virulence, we submitted L. infantum promastigotes to 4, 21 or 31 successive in vitro passages. Our results demonstrated a rapid and significant loss of parasite virulence when parasites are sustained in axenic culture. Strikingly, the parasite capacity to modulate macrophage activation decreased significantly with the augmentation of the number of in vitro passages. We validated these in vitro observations using an experimental murine model of infection. A significant correlation was found between higher parasite burdens and lower number of in vitro passages in infected Balb/c mice. Furthermore, we have demonstrated that the virulence deficit caused by successive in vitro passages results from an inadequate capacity to differentiate into amastigote forms. In conclusion, our data demonstrated that the use of parasites with distinct periods of axenic in vitro culture induce distinct infection rates and immunological responses and correlated this phenotype with a rapid loss of promastigote differentiation capacity. These results highlight the need for a standard operating protocol (SOP) when studying Leishmania species.

Anti-leishmanial activity of the bisnaphthalimidopropyl derivatives.

Bisnaphthalimidopropyl (BNIP) derivatives were recently identified as inhibitors of the Leishmania Silent Information Regulator 2 (SIR2) NAD(+)-dependent deacetylase. In this report we have for the first time, determined the potential of these compounds to treat visceral leishmaniasis using BALB/c mice chronically infected with Leishmania infantum as a model. These experiments led to the identification of BNIPdiaminooctane (BNIPDaoct) as an effective compound able to induce significant reduction of the parasite load in the spleen and in the liver. Indeed, at a dose of 1mg/kg, BNIPDaoct was more effective to treat leishmaniasis in a short course treatment (3 or 6 drug administrations) than the standard amphotericin B. Moreover, no indications of hematological toxicity were detected as evaluated by the hemoglobin, hematocrit, white and red blood cell counts, hence making BNIPDaoct a potential therapeutic agent against leishmaniasis.