Identification of Immunodominant Proteins of the Leishmania (Viannia) naiffi SubProteome as Pan-Specific Vaccine Targets against Leishmaniasis.

Leishmaniasis is a wide-spectrum disease caused by parasites from Leishmania genus. A well-modulated immune response that is established after the long-lasting clinical cure of leishmaniasis can represent a standard requirement for a vaccine. Previous studies demonstrated that Leishmania (Viannia) naiffi causes benign disease and its antigens induce well-modulated immune responses in vitro. In this work we aimed to identify the immunodominant proteins present in the soluble extract of L. naiffi (sLnAg) as candidates for composing a pan-specific anti-leishmaniasis vaccine. After immunoblotting using cured patients of cutaneous leishmaniasis sera and proteomics approaches, we identified a group of antigenic proteins from the sLnAg. In silico analyses allowed us to select mildly similar proteins to the host; in addition, we evaluated the binding potential and degree of promiscuity of the protein epitopes to HLA molecules and to B-cell receptors. We selected 24 immunodominant proteins from a sub-proteome with 328 proteins. Homology analysis allowed the identification of 13 proteins with the most orthologues among seven Leishmania species. This work demonstrated the potential of these proteins as promising vaccine targets capable of inducing humoral and cellular pan-specific immune responses in humans, which may in the future contribute to the control of leishmaniasis.

In-Depth Quantitative Proteomics Characterization of In Vitro Selected Miltefosine Resistance in Leishmania infantum.

Visceral leishmaniasis (VL) is a neglected disease caused by Leishmania parasites. Although significant morbidity and mortality in tropical and subtropical regions of the world are associated with VL, the low investment for developing new treatment measures is chronic. Moreover, resistance and treatment failure are increasing for the main medications, but the emergence of resistance phenotypes is poorly understood at the protein level. Here, we analyzed the development of resistance to miltefosine upon experimental selection in a L. infantum strain. Time to miltefosine resistance emergence was ~six months and label-free quantitative mass-spectrometry-based proteomics analyses revealed that this process involves a remodeling of components of the membrane and mitochondrion, with significant increase in oxidative phosphorylation complexes, particularly on complex IV and ATP synthase, accompanied by increased energy metabolism mainly dependent on ß-oxidation of fatty acids. Proteins canonically involved in ROS detoxification did not contribute to the resistant process whereas sterol biosynthesis enzymes could have a role in this development. Furthermore, changes in the abundance of proteins known to be involved in miltefosine resistance such as ABC transporters and phospholipid transport ATPase were detected. Together, our data show a more complete picture of the elements that make up the miltefosine resistance phenotype in L. infantum.

Nitric Oxide Resistance in Leishmania (Viannia) braziliensis Involves Regulation of Glucose Consumption, Glutathione Metabolism and Abundance of Pentose Phosphate Pathway Enzymes.

In American Tegumentary Leishmaniasis production of cytokines, reactive oxygen species and nitric oxide (NO) by host macrophages normally lead to parasite death. However, some Leishmania braziliensis strains exhibit natural NO resistance. NO-resistant strains cause more lesions and are frequently more resistant to antimonial treatment than NO-susceptible ones, suggesting that NO-resistant parasites are endowed with specific mechanisms of survival and persistence. To tests this, we analyzed the effect of pro- and antioxidant molecules on the infectivity in vitro of L. braziliensis strains exhibiting polar phenotypes of resistance or susceptibility to NO. In addition, we conducted a comprehensive quantitative mass spectrometry-based proteomics analysis of those parasites. NO-resistant parasites were more infective to peritoneal macrophages, even in the presence of high levels of reactive species. Principal component analysis of protein concentration values clearly differentiated NO-resistant from NO-susceptible parasites, suggesting that there are natural intrinsic differences at molecular level among those strains. Upon NO exposure, NO-resistant parasites rapidly modulated their proteome, increasing their total protein content and glutathione (GSH) metabolism. Furthermore, NO-resistant parasites showed increased glucose analogue uptake, and increased abundance of phosphotransferase and G6PDH after nitrosative challenge, which can contribute to NADPH pool maintenance and fuel the reducing conditions for the recovery of GSH upon NO exposure. Thus, increased glucose consumption and GSH-mediated redox capability may explain the natural resistance of L. braziliensis against NO.

Malnutrition Aggravates Alterations Observed in the Gut Structure and Immune Response of Mice Infected with Leishmania infantum.

Malnutrition is a risk factor for developing visceral leishmaniasis and its severe forms. Our group demonstrated that malnourished animals infected with Leishmania infantum had severe atrophies in lymphoid organs and T cell subpopulations as well as altered levels of thymic and splenic chemotactic factors, all of which resulted in dysfunctional lymphoid microenvironments that promoted parasite proliferation. Here, we hypothesize that malnutrition preceding parasite infection leads to structural and immunological changes in the gut mucosae, resulting in a failure in the immune response sensed in the intestine. To evaluate this, we analyzed the immunopathological events resulting from protein malnutrition in the guts of BALB/c mice infected with L. infantum. We observed lymphocytic/lymphoplasmacytic inflammatory infiltrates and lymphoid hyperplasia in the duodenum of well-nourished-infected mice; such alterations were worsened when malnutrition preceded infection. Parasite infection induced a significant increase of duodenal immunoglobulin A (IgA) of well-nourished animals, but those levels were significantly decreased in malnourished-infected mice. In addition, increased levels of Th17-related cytokines in duodenums of malnourished animals supported local inflammation. Together, our results suggest that the gut plays a potential role in responses to L. infantum infection-and that such responses are impaired in malnourished individuals.

Starvation and pH stress conditions induced mitochondrial dysfunction, ROS production and autophagy in Trypanosoma cruzi epimastigotes.

Chagas disease is a neglected illness endemic in Latin America that mainly affects rural populations. The etiological agent of Chagas disease is the protozoan Trypanosoma cruzi, which has three different parasite stages and a dixenous life cycle that includes colonization of the vertebrate and invertebrate hosts. During its life cycle, T. cruzi is subjected to stress conditions, including variations in nutrient availability and pH, which impact parasite survival and differentiation. The plasticity of mitochondrial function in trypanosomatids has been defined as mitochondrial activity related to substrate availability. Thus, mitochondrial remodeling and autophagy, which is a constitutive cellular process of turnover and recycling of cellular components, may constitute a response to the nutritional and pH stress in the host. To assess these processes, epimastigotes were subjected to acidic, alkaline, and nutritional stress conditions, and mitochondrial function and its influence on the autophagic process were evaluated. Our data demonstrated that the three stress conditions affected the mitochondrial structure, inducing organelle swelling and impaired oxidative phosphorylation. Stressed epimastigotes produced increased ROS levels and overexpressed antioxidant enzymes. The stress conditions resulted in an increase in the number of autophagosomes and exacerbated the expression of different autophagy-related genes (Atgs). A correlation between mitochondrial dysfunction and autophagic phenotypes was also observed. After 24 h, acid stress and nutritional deprivation induced metacyclogenesis phenotypes (mitochondrial remodeling and autophagy). On the other hand, alkaline stress was transient due to insect blood feeding and culminated in an increase in autophagic flux as a survival mechanism.

Factors associated with non-invasive mechanical ventilation failure in patients with hematological neoplasia and their association with outcomes.

BACKGROUND: The usefulness of non-invasive mechanical ventilation (NIMV) in oncohematological patients is still a matter of debate. AIM: To analyze the rate of noninvasive ventilation failure and the main characteristics associated with this endpoint in oncohematological patients with acute respiratory failure (ARF). METHODS: A ventilatory support protocol was developed and implemented before the onset of the study. According to the PaO2/FiO2 (P/F) ratio and clinical judgment, patients received supplementary oxygen therapy, NIMV, or invasive mechanical ventilation (IMV). RESULTS: Eighty-two patients were included, average age between 52.1 ± 16 years old; 44 (53.6%) were male. The tested protocol was followed in 95.1% of cases. Six patients (7.3%) received IMV, 59 (89.7%) received NIMV, and 17 (20.7%) received oxygen therapy. ICU mortality rates were significantly higher in the IMV (83.3%) than in the NIMV (49.2%) and oxygen therapy (5.9%) groups (P < 0.001). Among the 59 patients who initially received NIMV, 30 (50.8%) had to eventually be intubated. Higher SOFA score at baseline (1.35 [95% CI = 1.12-2.10], P = 0.007), higher respiratory rate (RR) (1.10 [95% CI = 1.00-1.22], P = 0.048), and sepsis on admission (16.9 [95% CI = 1.93-149.26], P = 0.011) were independently associated with the need of orotracheal intubation among patients initially treated with NIMV. Moreover, NIMV failure was independently associated with ICU (P < 0.001) and hospital mortality (P = 0.049), and mortality between 6 months and 1 year (P < 0.001). CONCLUSION: The implementation of a NIMV protocol is feasible in patients with hematological neoplasia admitted to the ICU, even though its benefits still remain to be demonstrated. NIMV failure was associated with higher SOFA and RR and more frequent sepsis, and it was also related to poor prognosis.

In-depth quantitative proteomics uncovers specie-specific metabolic programs in Leishmania (Viannia) species.

Leishmania species are responsible for a broad spectrum of diseases, denominated Leishmaniasis, affecting over 12 million people worldwide. During the last decade, there have been impressive efforts for sequencing the genome of most of the pathogenic Leishmania spp. as well as hundreds of strains, but large-scale proteomics analyses did not follow these achievements and the Leishmania proteome remained mostly uncharacterized. Here, we report a comprehensive comparative study of the proteomes of strains representing L. braziliensis, L. panamensis and L. guyanensis species. Proteins extracted by SDS-mediated lysis were processed following the multi-enzyme digestion-filter aided sample preparation (FASP) procedure and analysed by high accuracy mass spectrometry. "Total Protein Approach" and "Proteomic Ruler" were applied for absolute quantification of proteins. Principal component analysis demonstrated very high reproducibility among biological replicates and a very clear differentiation of the three species. Our dataset comprises near 7000 proteins, representing the most complete Leishmania proteome yet known, and provides a comprehensive quantitative picture of the proteomes of the three species in terms of protein concentration and copy numbers. Analysis of the abundance of proteins from the major energy metabolic processes allow us to highlight remarkably differences among the species and suggest that these parasites depend on distinct energy substrates to obtain ATP. Whereas L. braziliensis relies the more on glycolysis, L. panamensis and L. guyanensis seem to depend mainly on mitochondrial respiration. These results were confirmed by biochemical assays showing opposite profiles for glucose uptake and O2 consumption in these species. In addition, we provide quantitative data about different membrane proteins, transporters, and lipids, all of which contribute for significant species-specific differences and provide rich substrate for explore new molecules for diagnosing purposes. Data are available via ProteomeXchange with identifier PXD017696.

Papel do enfermeiro no período perioperatório para prevenção da trombose venosa profunda / Rol del enfermero en el período perioperatorio para la prevención de la trombosis venosa profunda / The role of the nurse along the perioperative period in order to prevent deep vein thrombosis

Objetivos: Conhecer como os enfermeiros realizam a prevenção da Trombose Venosa Profunda (TVP) em pacientes submetidos a cirurgias de grande porte no período perioperatório; e levantar os fatores de risco para o desenvolvimento de TVP identificados pelos enfermeiros. Método: Estudo de caso com abordagem qualitativa. Amostra composta por 12 enfermeiros da unidade de internação, centro cirúrgico e sala de recuperação pós-anestésica de um hospital especializado em trauma de Porto Alegre (RS). Utilizou-se entrevista semiestruturada com 10 questões norteadoras. Para interpretação dos dados utilizou-se Análise de Conteúdo de Bardin. Resultados: Emergiram três categorias: Fatores de Risco para TVP; Medidas Preventivas de TVP e Dificuldades na Execução da Sistematização da Assistência de Enfermagem Perioperatória. Conclusão: Os enfermeiros realizam prevenção de TVP com massagem, observação, exame físico/avaliação, medidas protetivas e mudança de posição, entretanto falta autonomia para aplicar algumas medidas preventivas. Apontaram vários fatores de risco para a ocorrência de TVP conforme literatura, evidenciando conhecimento sobre a patologia.

Objetivos: Conocer como los enfermeros realizan la prevención de la trombosis de la vena en los pacientes sometidos a las cirugías de porte grande en el período perioperatorio; y colectar los factores de riesgo para el desarrollo de la trombosis de la vena que fueron identificados por los enfermeros. Método: Estudio del caso con el enfoque cualitativo. La muestra fue compuesta por 12 enfermeros de la unidad de hospitalización, quirófano y sala de recuperación post-anestesia de un hospital especializado en traumas en Porto Alegre, Rio Grande do Sul, Brasil. Se aplicó una entrevista semiestructurada con 10 preguntas principales. Se utilizó, para la interpretación de los datos, el análisis del contenido de Bardin. Resultados: Se emergieron tres categorías: los factores del riesgo para la trombosis de la vena; las medidas preventivas de la trombosis de la vena y las dificultades en la ejecución de del SAEP. Conclusión: Los enfermeros realizan la prevención de la trombosis de la vena con el masaje, la observación, el examen / evaluación física, las medidas de protección y el cambio de posición, pero carecen de autonomía para aplicar algunas medidas preventivas. Ellos mostraron varios factores de riesgo para la aparición de la trombosis de la vena de acuerdo a la literatura, mostrándose el conocimiento sobre la enfermedad.

Objectives: Determining how nurses implement Deep Vein Thrombosis (DVT) prevention during the perioperative period in patients submitted to large surgeries, and detecting the risk factors to the occurences of DVT previously traced by nurses. Methodology: Case study with qualitative approach. A sample of 12 nurses in the inpatient unit, operating room and post-anesthesia recovery room of a hospital specializing in trauma Porto Alegre (RS), Brazil. We used semi-structured interviews with 10 leading questions. For data interpretation, the Content Analysis Method introduced by Bardin was adopted. Results: Three final categories came up from the content analysis: Risk Factors to DVT, Preventive Measures to DVT and Adversities in Implementing the Systematization of Perioperative Nursing Care. Conclusion: Nurses perform prevention of DVT with massage, observation, physical examination/assessment, protective measures and change of position, but lack autonomy to apply some preventive measures. They showed several risk factors for the occurrence of DVT as literature, showing knowledge about the disease.