Topically Applied Therapies for the Treatment of Skin Disease: Past, Present, and Future.

The purpose of this review is to summarize essential biological, pharmaceutical, and clinical aspects in the field of topically applied medicines that may help scientists when trying to develop new topical medicines. After a brief history of topical drug delivery, a review of the structure and function of the skin and routes of drug absorption and their limitations is provided. The most prevalent diseases and current topical treatment approaches are then detailed, the organization of which reflects the key disease categories of autoimmune and inflammatory diseases, microbial infections, skin cancers, and genetic skin diseases. The complexity of topical product development through to large-scale manufacturing along with recommended risk mitigation approaches are then highlighted. As such topical treatments are applied externally, patient preferences along with the challenges they invoke are then described, and finally the future of this field of drug delivery is discussed, with an emphasis on areas that are more likely to yield significant improvements over the topical medicines in current use or would expand the range of medicines and diseases treatable by this route of administration. SIGNIFICANCE STATEMENT: This review of the key aspects of the skin and its associated diseases and current treatments along with the intricacies of topical formulation development should be helpful in making judicious decisions about the development of new or improved topical medicines. These aspects include the choices of the active ingredients, formulations, the target patient population's preferences, limitations, and the future with regard to new skin diseases and topical medicine approaches.

Cisplatin Toxicity Causes Neutrophil-Mediated Inflammation in Zebrafish Larvae.

Cisplatin is an antineoplastic agent used to treat various tumors. In mammals, it can cause nephrotoxicity, tissue damage, and inflammation. The release of inflammatory mediators leads to the recruitment and infiltration of immune cells, particularly neutrophils, at the site of inflammation. Cisplatin is often used as an inducer of acute kidney injury (AKI) in experimental models, including zebrafish (Danio rerio), due to its accumulation in kidney cells. Current protocols in larval zebrafish focus on studying its effect as an AKI inducer but ignore other systematic outcomes. In this study, cisplatin was added directly to the embryonic medium to assess its toxicity and impact on systemic inflammation using locomotor activity analysis, qPCR, microscopy, and flow cytometry. Our data showed that larvae exposed to cisplatin at 7 days post-fertilization (dpf) displayed dose-dependent mortality and morphological changes, leading to a decrease in locomotion speed at 9 dpf. The expression of pro-inflammatory cytokines such as interleukin (il)-12, il6, and il8 increased after 48 h of cisplatin exposure. Furthermore, while a decrease in the number of neutrophils was observed in the glomerular region of the pronephros, there was an increase in neutrophils throughout the entire animal after 48 h of cisplatin exposure. We demonstrate that cisplatin can have systemic effects in zebrafish larvae, including morphological and locomotory defects, increased inflammatory cytokines, and migration of neutrophils from the hematopoietic niche to other parts of the body. Therefore, this protocol can be used to induce systemic inflammation in zebrafish larvae for studying new therapies or mechanisms of action involving neutrophils.

Pentoxifylline reduces inflammation and prevents myocardial perfusion derangements in experimental chronic Chagas' cardiomyopathy.

BACKGROUND: Myocardial perfusion defect (MPD) is common in chronic Chagas cardiomyopathy (CCC) and is associated with inflammation and development of left ventricular systolic dysfunction. We tested the hypothesis that pentoxifylline (PTX) could reduce inflammation and prevent the development of MPD in a model of CCC in hamsters. METHODS AND RESULTS: We investigated with echocardiogram and rest myocardial perfusion scintigraphy at baseline (6-months after T. cruzi infection/saline) and post-treatment (after additional 2-months of PTX/saline administration), female Syrian hamsters assigned to 3 groups: T. cruzi-infected animals treated with PTX (CH + PTX) or saline (CH + SLN); and uninfected control animals (CO). At the baseline, all groups showed similar left ventricular ejection fraction (LVEF) and MPD areas. At post-treatment evaluation, there was a significant increase of MPD in CH + SLN group (0.8 ± 1.6 to 9.4 ± 9.7%), but not in CH + PTX (1.9 ± 3.0% to 2.7 ± 2.7%) that exhibited MPD area similar to CO (0.0 ± 0.0% to 0.0 ± 0.0%). The LVEF decreased in both infected groups. Histological analysis showed a reduced inflammatory infiltrate in CH + PTX group (395.7 ± 88.3 cell/mm2), as compared to CH + SLN (515.1 ± 133.0 cell/mm2), but larger than CO (193.0 ± 25.7 cell/mm2). The fibrosis and TNF-α expression was higher in both infected groups. CONCLUSIONS: The prolonged use of PTX is associated with positive effects, including prevention of MPD development and reduction of inflammation in the chronic hamster model of CCC.

Embryonic zebrafish response to a commercial formulation of azoxystrobin at environmental concentrations.

Azoxystrobin is a broad-spectrum strobilurin fungicide for use on a wide range of crops available to end-users as formulated products. Due to its extensive application, it has been detected in aquatic ecosystems, raising concerns about its environmental impact, which is still poorly explored. The objective of this work was to study the effects of a commercial formulation of azoxystrobin in the zebrafish embryo model. Sublethal and lethal effects were monitored during the exposure period from 2 h post fertilisation (hpf) to 96 hpf after exposure to azoxystrobin concentrations (1, 10 and 100 µg L-1). The responses of antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR)) as well as detoxifying enzymes (glutathione-s-transferase (GST) and carboxylesterase (CarE)) were evaluated at 96 hpf. Similarly, glutathione levels (reduced (GSH) and oxidised (GSSG) glutathione), neurotransmission (acetylcholinesterase (AChE)) and anaerobic respiration (lactate dehydrogenase (LDH)) -related enzymes were assayed. At 120 hpf, larvae from each group were used for behaviour analysis. Results from this study showed concentration-dependent teratogenic effects, particularly by increasing the number of malformations (yolk and eye), with a higher prevalence at the highest concentration. However, it was found that the lowest concentration induced a high generation of reactive oxygen species (ROS) and increased activity of SOD, GST, and CarE. In addition, GR and GSSG levels were decreased by the lowest concentration, suggesting an adaptive response to oxidative stress, which is also supported by the increased AChE activity and absence of behavioural changes. These findings advance the knowledge of the azoxystrobin developmental and environmental impacts, which may impose ecotoxicological risks to non-target species.

Host dysbiosis negatively impacts IL-9-producing T-cell differentiation and antitumour immunity.

BACKGROUND: Host-microbiota interactions shape T-cell differentiation and promote tumour immunity. Although IL-9-producing T cells have been described as potent antitumour effectors, their role in microbiota-mediated tumour control remains unclear. METHODS: We analysed the impact of the intestinal microbiota on the differentiation of colonic lamina propria IL-9-producing T cells in germ-free and dysbiotic mice. Systemic effects of the intestinal microbiota on IL-9-producing T cells and the antitumour role of IL-9 were analysed in a model of melanoma-challenged dysbiotic mice. RESULTS: We show that germ-free mice have lower frequency of colonic lamina propria IL-9-producing T cells when compared with conventional mice, and that intestinal microbiota reconstitution restores cell frequencies. Long-term antibiotic treatment promotes host dysbiosis, diminishes intestinal IL-4 and TGF-ß gene expression, decreases the frequency of colonic lamina propria IL-9-producing T cells, increases the susceptibility to tumour development and reduces the frequency of IL-9-producing T cells in the tumour microenvironment. Faecal transplant restores intestinal microbiota diversity, and the frequency of IL-9-producing T cells in the lungs of dysbiotic animals, restraining tumour burden. Finally, recombinant IL-9 injection enhances tumour control in dysbiotic mice. CONCLUSIONS: Host-microbiota interactions are required for adequate differentiation and antitumour function of IL-9-producing T cells.

Sucupira Oil-Loaded Nanostructured Lipid Carriers (NLC): Lipid Screening, Factorial Design, Release Profile, and Cytotoxicity.

Essential oils are odorant liquid oily products consisting of a complex mixture of volatile compounds obtained from a plant raw material. They have been increasingly proven to act as potential natural agents in the treatment of several human conditions, including diabetes mellitus (DM). DM is a metabolic disorder characterized by chronic hyperglycemia closely related to carbohydrate, protein and fat metabolism disturbances. In order to explore novel approaches for the management of DM our group proposes the encapsulation of sucupira essential oil, obtained from the fruits of the Brazilian plants of the genus Pterodon, in nanostructured lipid carriers (NLCs), a second generation of lipid nanoparticles which act as new controlled drug delivery system (DDS). Encapsulation was performed by hot high-pressure homogenization (HPH) technique and the samples were then analyzed by dynamic light scattering (DLS) for mean average size and polydispersity index (PI) and by electrophoretic light scattering (ELS) for zeta potential (ZP), immediately after production and after 24 h of storage at 4 °C. An optimal sucupira-loaded NLC was found to consist of 0.5% (m/V) sucupira oil, 4.5% (m/V) of Kollivax® GMS II and 1.425% (m/V) of TPGS (formulation no. 6) characterized by a mean particle size ranging from 148.1 ± 0.9815 nm (0 h) to 159.3 ± 9.539 nm (at 24 h), a PI from 0.274 ± 0.029 (0 h) to 0.305 ± 0.028 (24 h) and a ZP from -0.00236 ± 0.147 mV (at 0 h) to 0.125 ± 0.162 (at 24 h). The encapsulation efficiency and loading capacity were 99.98% and 9.6%, respectively. The optimized formulation followed a modified release profile fitting the first order kinetics, over a period of 8 h. In vitro cytotoxicity studies were performed against Caco-2 cell lines, for which the cell viability above 90% confirmed the non-cytotoxic profile of both blank and sucupira oil-loaded NLC.

Determination of 17α-ethinylestradiol and toxic metals in surface waters, and estimation of daily intake.

The presence of substances such as hormones and toxic metal in aquatic ecosystem is interesting to the scientific community due to their adverse effects. We quantified 17α-ethynylestradiol (EE2) and toxic metals in the surface waters from Sorocaba and Pirajibu Rivers, in São Paulo State, and we estimated the daily intake for hormone, based on the amount of water consumed. EE2, Cd, Hg, As, Pb, and Mn were seasonally quantified in six different locations along the rivers. EE2 was evaluated by high-performance liquid chromatography. Toxic metals were determined by inductively coupled plasma mass spectrometer. Considering the entire sample year, EE2 concentrations ranged from 4.5 to 48.2 µg L-1. Comparing Sorocaba and Pirajibu rivers, the sample point in the entrance of the Pirajibu River through the city of Itu, São Paulo State, had higher amounts of EE2. Regarding metals, all results are according to the Brazilian and World Health Organization guidelines for drinking-water quality, except for Mn levels, which were higher than the limits in Autumn season in two locations. The estimated daily intake ranged from 13.45 to 40.9 µg/day/person. In conclusion, concentrations of EE2 in the Sorocaba and Pirajibu Rivers were higher than in other countries. The levels were as high as an intake of one pill for each person every day (considering an oral contraceptive has 0.03 mg of ethinylestradiol). Even though concentrations of toxic elements are in accordance with the Brazilian Regulation and World Health Organization, legislation for hormones and drugs needs to advance.

Association between left ventricular mass, androgens, adiposity and insulin resistance in girls with precocious pubarche: a case-control study.

OBJECTIVE: Precocious pubarche (PP) has been linked to higher prevalence of metabolic disturbances and polycystic ovary syndrome (PCOS). The aim of the study was to assess echocardiographic parameters in PP girls and to analyse their relationship with androgens and insulin resistance (IR). DESIGN: Case-control study. PATIENTS: Thirty-five PP girls and 35 healthy age-matched controls. MEASUREMENTS: Clinical, hormonal and metabolic profiles, echocardiography, body composition and oral glucose tolerance test. RESULTS: Chronological age (10·04 ± 2·6 years in PP vs 10·13 ± 2·56 years in controls, P = 0·227), and pubertal stage at the time of the study were similar between the groups. PP girls had higher free androgen index (FAI) [1·39 (0·48-3·64) vs 1·06 (0·39-1·7), P = 0·005] and QUICKI (0·58 ± 0·08 vs 0·63 ± 0·12, P = 0·021). However, HOMA-IR was not significantly different between the groups [2·79 (1·84-4·05) vs 2·15 (1·09-3·23), P = 0·085]. After adjusting for total body fat, left ventricular mass (LVM) was higher in the PP group (97·31 ± 33·37 vs 81·25 ± 19·06 g, P = 0·017) as well as A' wave (5·66 ± 1·34 vs 5·09 ± 0·98 cm/s, P = 0·025), a measurement of diastolic function. FAI and total body fat were independent predictors of higher LVM and together with HOMA-IR contributed 72% of LVM variability in the PP group. CONCLUSION: In this study with PP girls, greater LVM, associated with higher androgen levels, IR and total body fat, occurred early in pubertal development.

Cortisol Quantification for Assessing Stress-Induced Changes in Zebrafish Larvae.

The stress response, mainly mediated by cortisol, plays a critical role in the regulation of physiological and behavioral homeostasis through a variety of mechanisms. Different aquatic animal models have been widely employed to understand the pathobiology of stress and stress-related brain disorders. The early life stress can affect the hypothalamic-pituitary-interrenal (HPI) axis and induce cellular and molecular impairments that impact the brain functioning later in life. However, these alterations have been poorly explored mainly due to the lack of suitable models. In this chapter, the vortex flow stimulation, an acute stress that causes a forced swimming and activates the HPI axis, is described and its correlations with behavioral outputs reported. To this end, the early life stages of zebrafish are used as animal models for modeling stress disorders experimentally. The behavioral despair model can be employed as an initial screening tool for assessing neural circuit activation and motor alterations. Taken together, the implementation of this strategy in this animal model allows the analysis of stress responses in a simple manner and its correlation with neural circuitries and motor alterations.

HCV Core-antigen Assay as an Effective Alternative to HCV RNA Quantification in Patients With Hepatitis C.

BACKGROUND/AIM: Hepatitis C virus (HCV) core antigen (Ag) test has been increasingly applied as an effective alternative to conventional molecular tests allowing rapid and affordable diagnosis, which is of paramount relevance to achieve global elimination of HCV infection. MATERIALS AND METHODS: ARCHITECT® HCV Ag test was evaluated in comparison with HCV RNA quantification test (CAP/CTM) to calculate its sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and to determine their correlation level. Its performance, according to low and high viral load values and in different treatment stages [during treatment (T), at the end of the therapeutic protocol (EOT) and when sustained virological response (SVR) was evaluated]. RESULTS: In total, 145 samples were included. Considering CAP/CTM, the sensitivity, specificity, PPV and NPV of the HCV-Ag test were 88.9%, 99.1%, 97.0% and 96.4%, respectively, and the correlation among tests was high (r=0.890), with only five discordant results. A decrease in sensitivity was found for low viral load values (<1,000 IU/ml), but the opposite was verified for high viral concentrations (≥1,000 IU/ml). A good agreement was verified for the T and EOT groups (k=0.789 and k=0.638) and an excellent agreement in the SVR group (k=1.000). CONCLUSION: HCV-Ag seems to be an effective alternative that can be routinely combined with other faster and more accessible tests (e.g., HCV antibody tests) for the identification of new HCV infections in suspected patients, eventually reserving the molecular techniques for samples with discordant results.

Behavioural impairment and oxidative stress by acute exposure of zebrafish to a commercial formulation of tebuconazole.

Tebuconazole is a systemic follicular fungicide known to cause diverse problems in non-target organisms namely associated to the pure active ingredient. As such, the objective of this work was to evaluate developmental changes induced by a tebuconazole commercial formulation to a non-target animal model. Zebrafish embryos at ± 2 h post-fertilization were exposed to tebuconazole wettable powder concentrations (0.05, 0.5 and 5 mg L-1) for 96 h with developmental toxicity assessed throughout the exposure period and biochemical parameters evaluated at the end of the exposure. Behavioural assessment (spatial exploration and response to stimuli) was conducted 24 h after the end of the exposure. While no developmental and physiological alterations were observed, exposure to tebuconazole resulted in an increased generation of reactive oxidative species at the 0.05 and 0.5 mg L-1 concentrations and a decreased GPx activity at the 0.5 mg L-1 concentration suggesting a potential protection mechanism. There was also a change in the avoidance-escape behaviour supporting an anxiolytic effect suggesting possible alterations in the central nervous system development demanding further studies.

Biochar from fungiculture waste for adsorption of endocrine disruptors in water.

The agricultural residues are ecofriendly alternatives for removing contaminants from water. In this way, a novel biochar from the spent mushroom substrate (SMS) was produced and assessed to remove endocrine disruptor from water in batch and fixed-bed method. SMS were dried, ground, and pyrolyzed. Pyrolysis was carried out in three different conditions at 250 and 450 °C, with a residence time of 1 h, and at 600 °C with a residence time of 20 min. The biochar was firstly tested in a pilot batch with 17α-ethinylestradiol (EE2) and progesterone. The residual concentrations of the endocrine disruptors were determined by HPLC. The biochar obtained at 600 °C showed the best removal efficiency results. Then, adsorption parameters (isotherm and kinetics), fixed bed tests and biochar characterization were carried out. The Langmuir model fits better to progesterone while the Freundlich model fits better to EE2. The Langmuir model isotherm indicated a maximum adsorption capacity of 232.64 mg progesterone/g biochar, and 138.98 mg EE2/g biochar. Images from scanning electrons microscopy showed that the 600 °C biochar presented higher porosity than others. In the fixed bed test the removal capacity was more than 80% for both endocrine disruptors. Thus, the biochar showed a good and viable option for removal of contaminants, such as hormones.

The Effect of Weaning and Slaughter Age on the Physicochemical and Sensory Characteristics of Arouquesa Beef-A PDO Portuguese Meat.

(1) Background: Autochthonous breeds meat is well accepted due to its sensory characteristics, perceived low environmental impact, and animal welfare. We aimed to evaluate the effect of weaning and slaughter age on the physicochemical and sensory characteristics of Arouquesa, a Portuguese Protected Designation of Origin (PDO) meat and to evaluate the psychological effect of knowing the weaning age on the consumer's hedonic evaluation. (2) Methods: Meat from 26 animals was assigned to 4 groups, with combinations of weaning (W) at 9 or 5 months and slaughter (S) at 9 or 12 months: W9-S9, W9-S12, W5-S9, and W5-S12. The meat was analysed for pH24h, colour (L*a*b*), cooking losses and shear force. A Check All that Apply test was made with 70 consumers; they were also asked to punctuate the hedonic appreciation of anonymous and weaning age-identified meat. (3) Results: W9-S9 were more tender, had lower shear force, and was juicier than W5-S9. When animals were slaughtered at 12 months, there were no differences in the physicochemical and sensory characteristics between the weaning ages. The effect of information about the weaning age influences the consumer's hedonic evaluation, as revealed by the comparison between the anonymous and identified samples. (4) Later weaning resulted in more tender meat when the slaughter was at 9 months and positively impacted consumer perception.

Growth Performance, Carcass and Meat Traits of Autochthonous Arouquesa Weaners Raised on Traditional and Improved Feeding Systems.

Arouquesa is an autochthonous bovine breed known for its Arouquesa PDO beef labeling. There are several production systems under the definition of PDO labeling. This study aimed to compare the effect of different production systems on carcass and meat traits for the Arouquesa breed. Two trials differing in diet and weaning age were conducted. The first trial included a TF group fed the traditional way and weaned at 9 months; a TF + S1 group, equal to TF, but with a starter supplement; and finally, a S1 + S2 group that was fed with a starter and a growth supplement and weaned at 5 months. The second trial was composed of a TF + S3 group fed like the TF + S1 group but reared until 12 months with a finishing supplement, and finally, the S3 group fed like the S1 + S2 group but reared until 12 months. In the first trial, the TF + S1 and S1 + S2 groups showed higher final live weight and average daily gain. In the second trial, we observed differences in the subcutaneous fat that was higher in the S3 group. Regarding meat traits, we observed differences in exudative and cooking losses in the first trial. In general, supplementation improved meat production without affecting meat quality parameters.

[Brazilian technological output in the area of nursing: advances and challenges]. / Produção tecnológica brasileira na área de enfermagem: avanços e desafios.

This article aims to analyze the patents registered in the nursing area, since these patents may be used as an indicator of the technological development in the area. It presents and discusses national technological productions, tracked through the "nursing" keyword, patented in the period from 1990-2009. This is a retrospective documental research, using, as a source, data from the National Industrial Property Institute (INPI). The information gathered is discussed in relation to the appropriation of the technologies, the incentive to develop them and register them as a source of knowledge in the nursing field, aiming the practice of care. Light and light hard technology productions are increasing in the nursing field. However, these are not registered and patented. The technological advance in the nursing field is emergent and needs policies for its development.

Teratogenic, Oxidative Stress and Behavioural Outcomes of Three Fungicides of Natural Origin (Equisetum arvense, Mimosa tenuiflora, Thymol) on Zebrafish (Danio rerio).

The improper use of synthetic fungicides has raised public concerns related to environmental pollution and animal health. Over the years, plant-derived antifungals have been investigated as safer alternatives, although little scientific evidence of its neurodevelopmental effects exist. The main objective of this study was to explore the effects of three alternative natural extracts (Equisetum arvense, Mimosa tenuiflora, Thymol) with antifungal properties during the early development of zebrafish by evaluating different teratogenic, oxidative stress and behavioural outcomes. Following the determination of the 96 h-LC50, exposure to sublethal concentrations showed the safety profile of both E. arvense and M. tenuiflora. However, following 96-h exposure to Thymol, increased lethality, pericardial oedema, yolk and eye deformations, and decreased body length were observed. The reduced and oxidized glutathione (GSH:GSSG) ratio was increased, and the glutathione-s-transferase activity in the group exposed to the highest Thymol concentration. Overall, these results support a more reducing environment associated with possible effects at the cellular proliferation level. In addition, the disruption of behavioural states (fear- and anxiety-like disorders) were noted, pointing to alterations in the c-Jun N-terminal kinase developmental signalling pathway, although further studies are required to explore this rationale. Notwithstanding, the results provide direct evidence of the teratogenic effects of Thymol, which might have consequences for non-target species.

STING Signaling Drives Production of Innate Cytokines, Generation of CD8+ T Cells and Enhanced Protection Against Trypanosoma cruzi Infection.

A variety of signaling pathways are involved in the induction of innate cytokines and CD8+ T cells, which are major players in protection against acute Trypanosoma cruzi infection. Previous data have demonstrated that a TBK-1/IRF3-dependent signaling pathway promotes IFN-ß production in response to Trypanosoma cruzi, but the role for STING, a main interactor of these proteins, remained to be addressed. Here, we demonstrated that STING signaling is required for production of IFN-ß, IL-6, and IL-12 in response to Trypanosoma cruzi infection and that STING absence negatively impacts activation of IRF-dependent pathways in response to the parasite. We reported no significant activation of IRF-dependent pathways and cytokine expression in RAW264.7 macrophages in response to heat-killed trypomastigotes. In addition, we showed that STING is essential for T. cruzi DNA-mediated induction of IFN-ß, IL-6, and IL-12 gene expression in RAW264.7 macrophages. We demonstrated that STING-knockout mice have significantly higher parasitemia from days 5 to 8 of infection and higher heart parasitism at day 13 after infection. Although we observed similar heart inflammatory infiltrates at day 13 after infection, IFN-ß, IL-12, CXCL9, IFN-γ, and perforin gene expression were lower in the absence of STING. We also showed an inverse correlation between parasite DNA and the expression of CXCL9, IFN-γ, and perforin genes in the hearts of infected animals at day 13 after infection. Finally, we reported that STING signaling is required for splenic IFN-ß and IL-6 expression early after infection and that STING deficiency results in lower numbers of splenic parasite-specific IFN-γ and IFN-γ/perforin-producing CD8+ T cells, indicating a pivotal role for STING signaling in immunity to Trypanosoma cruzi.

Co-Exposure of Cardiomyocytes to IFN-γ and TNF-α Induces Mitochondrial Dysfunction and Nitro-Oxidative Stress: Implications for the Pathogenesis of Chronic Chagas Disease Cardiomyopathy.

Infection by the protozoan Trypanosoma cruzi causes Chagas disease cardiomyopathy (CCC) and can lead to arrhythmia, heart failure and death. Chagas disease affects 8 million people worldwide, and chronic production of the cytokines IFN-γ and TNF-α by T cells together with mitochondrial dysfunction are important players for the poor prognosis of the disease. Mitochondria occupy 40% of the cardiomyocytes volume and produce 95% of cellular ATP that sustain the life-long cycles of heart contraction. As IFN-γ and TNF-α have been described to affect mitochondrial function, we hypothesized that IFN-γ and TNF-α are involved in the myocardial mitochondrial dysfunction observed in CCC patients. In this study, we quantified markers of mitochondrial dysfunction and nitro-oxidative stress in CCC heart tissue and in IFN-γ/TNF-α-stimulated AC-16 human cardiomyocytes. We found that CCC myocardium displayed increased levels of nitro-oxidative stress and reduced mitochondrial DNA as compared with myocardial tissue from patients with dilated cardiomyopathy (DCM). IFN-γ/TNF-α treatment of AC-16 cardiomyocytes induced increased nitro-oxidative stress and decreased the mitochondrial membrane potential (ΔΨm). We found that the STAT1/NF-κB/NOS2 axis is involved in the IFN-γ/TNF-α-induced decrease of ΔΨm in AC-16 cardiomyocytes. Furthermore, treatment with mitochondria-sparing agonists of AMPK, NRF2 and SIRT1 rescues ΔΨm in IFN-γ/TNF-α-stimulated cells. Proteomic and gene expression analyses revealed that IFN-γ/TNF-α-treated cells corroborate mitochondrial dysfunction, transmembrane potential of mitochondria, altered fatty acid metabolism and cardiac necrosis/cell death. Functional assays conducted on Seahorse respirometer showed that cytokine-stimulated cells display decreased glycolytic and mitochondrial ATP production, dependency of fatty acid oxidation as well as increased proton leak and non-mitochondrial oxygen consumption. Together, our results suggest that IFN-γ and TNF-α cause direct damage to cardiomyocytes' mitochondria by promoting oxidative and nitrosative stress and impairing energy production pathways. We hypothesize that treatment with agonists of AMPK, NRF2 and SIRT1 might be an approach to ameliorate the progression of Chagas disease cardiomyopathy.

Toxic effects of a mancozeb-containing commercial formulation at environmental relevant concentrations on zebrafish embryonic development.

The toxicological knowledge of mancozeb (MZ)-containing commercial formulations on non-target species is scarce and limited. Therefore, the objective of this work was to represent a realistic application scenario by evaluating the toxicity of environmental relevant and higher concentrations of a commercial formulation of MZ using zebrafish embryos. Following determination of the 96-h LC50 value, the embryos at the blastula stage (~ 2 h post-fertilisation, hpf) were exposed to 0.5, 5, and 50 µg L-1 of the active ingredient (~ 40× lower than the 96-h LC50). During the exposure period (96 h), lethal, sublethal, and teratogenic parameters, as well as behaviour analysis, at 120 hpf, were assayed. Biochemical parameters such as oxidative stress-linked enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR)), reactive oxygen species (ROS) levels, and glutathione levels (GSH and GSSG), as well as the activity of degradation (glutathione S-transferase (GST) and carboxylesterase (CarE)), neurotransmission (acetylcholinesterase (AChE)), and anaerobic respiration (lactate dehydrogenase (LDH))-related enzymes, were analysed at the end of the exposure period. Exposed embryos showed a marked decrease in the hatching rate and many malformations (cardiac and yolk sac oedema and spinal torsions), with a higher prevalence at the highest concentration. A dose-dependent decreased locomotor activity and a response to an aversive stimulus, as well as a light-dark transition decline, were observed at environmental relevant concentrations. Furthermore, the activities of SOD and GR increased while the activity of GST, AChE, and MDA contents decreased. Taken together, the involvement of mancozeb metabolites and the generation of ROS are suggested as responsible for the developmental phenotypes. While further studies are needed to fully support the hypothesis presented, the potential cumulative effects of mancozeb-containing formulations and its metabolites could represent an environmental risk which should not be disregarded.

Leprosy and Kaposi sarcoma presenting as an immune reconstitution inflammatory syndrome in a patient with AIDS.

The simultaneous presence of infectious organisms within cutaneous lesions of Kaposi sarcoma in persons with AIDS has been demonstrated. We describe a patient with concurrent leprosy and Kaposi sarcoma presenting as an immune reconstitution inflammatory syndrome in the setting of AIDS.