Mecanismos de resposta inflamatória em modelos murinos de Plasmodium berghei ANKA, suscetível ou não à malária cerebral

Tese (doutorado)—Universidade de Brasília, Faculdade de Medicina, Programa de Pós-Graduação em Patologia Molecular, 2013.A malária ainda é um importante problema de saúde global, acometendo milhões de pessoas e resultando em aproximadamente 1 milhão de mortes por ano. A forma cerebral é uma das complicações mais grave da infecção pelo Plasmodium falciparum em seres humanos, e sua patogênese não está ainda totalmente esclarecida. A hiperativação do sistema imunitário tem sido considerada um importante determinante da evolução para as formas graves. O desequilíbrio da resposta imunitária com polarização para a resposta inflamatória tem um papel central na hiperativação do endotélio vascular, no sequestro de eritrócitos parasitados na microvasculatura cerebral e nas lesões das células cerebrais. Os mediadores inflamatórios são liberados em resposta aos antígenos plasmodiais, às próprias citocinas, aos eicosanóides e a outros produtos como a hemozoína e heme livre, que estimulam diferentes vias de ativação e regulação intracelular. O objetivo deste trabalho foi avaliar os mecanismos da resposta inflamatória envolvidos nas alterações cerebrais em camundongos suscetíveis (CBA e C57BL/6) ou resistentes (BALB/c) à malária cerebral para melhor esclarecer os possíveis mecanismos imunopatogênicos determinantes das formas graves da doença e melhor caracterizar os modelos experimentais da malária. Foram utilizados para este estudo camundongos machos de 8 a 12 semanas de idade, infectados ou não com 106 eritrócitos parasitados pelo Plasmodium berghei ANKA. Para cada linhagem, a morte espontânea foi avaliada dia a dia e a parasitemia foi comparada entre eles no terceiro, quinto e oitavo dia da infecção. A produção de óxido nítrico foi determinada pela reação de Griess. O peróxido de hidrogênio foi determinado pelo teste de oxidação do vermelho de fenol na presença de peroxidase. As citocinas IL-1β, IL-4, IL-10, IL-12, IL-17 e o FNT-α foram quantificadas nos homogenatos do cérebro e cerebelo por teste imunoenzimático. A presença de COX-2 e 5-LOX foi avaliada nos macrófagos peritoneais e micróglia por citometria de fluxo, e nos vasos cerebrais e células cerebrais por imunohistoquímica. A ativação do metabolismo lipídico foi avaliada pela quantificação dos corpúsculos lipídicos no citoplasma dos macrófagos peritoneais pela técnica do Oil red e Bodipy. As vias de ativação do NF?B, o inflamassoma e a regulação pelo PPAR- ? foram avaliados no citoplasma e no núcleo dos macrófagos peritoneais. Cortes histológicos do cérebro também foram avaliados utilizando o método clássico de coloração pela hematoxilina-eosina e também por microscopia eletrônica de varredura. A permeabilidade vascular foi avaliada pela quantificação do extravasamento do corante de azul de Evans para o tecido cerebral. Os camundongos C57BL/6 começaram a morrer no sexto dia após a infecção, os camundongos CBA, dois dias depois, enquanto 55% dos camundongos BALB/c ainda estavam vivos no décimo quinto dia da infecção. A parasitemia nos camundongos C57BL/6 foi maior do que na linhagem BALB/c no terceiro e quinto dias após a infecção e no oitavo dia também foi maior do que nos camundongos CBA. Nos camundongos C57BL/6 houve ativação da via canônica do NF?B, e diminuição da produção de NO e aumento do H2O2, FNT-α e IL-10; não houve alteração na produção de IL-4, IL-12 e IL17; e houve aumento na formação de corpúsculos lipídicos e predominância da expressão de 5-LOX. Observamos também ativação do inflamassoma NALPR1, porém diminuição da produção IL-1β. Nos camundongos CBA houve ativação preponderantemente da via não-canônica do NF?B, diminuição da produção do FNT-α e da IL-10, não houve alteração da produção de IL-12, NO e H2O2, mas observamos um padrão de IL-4 e IL-17 maior do que o das outras linhagens. Houve também ativação do inflamassoma, mas não houve alteração na produção de IL-1β. Nos camundongos BALB/c houve ativação preponderantemente da via canônica do NF?B, mas não houve alteração na produção de IL-12, NO e H2O2, que são estimulados pela atividade desta via. Ainda observamos diminuição na produção de FNT-α e IL-1β. Observamos que houve aumento da formação de corpúsculos lipídicos, mas não houve aumento paralelo na expressão de COX-2 e 5-LOX. Entretanto, a linhagem BALB/c foi a única que apresentou uma translocação importante do regulador PPAR-γ para o núcleo. O perfil de resposta foi seguido de alterações histopatológicas características para cada linhagem, sendo os camundongos C57BL/6 os que apresentaram a maior permeabilidade e sequestro de leucócitos. Os camundongos CBA também apresentaram aumento de permeabilidade, porém menor do que os camundongos C57BL/6, e também apresentaram maior sequestro de eritrócitos do que leucócitos. Poucas alterações foram observadas nos camundongos BALB/c. Os resultados desse trabalho evidenciam diferentes mecanismos de respostas celulares que devem em conjunto ter contribuído para a evolução clínica da malária e mostram que a escolha do modelo experimental para pesquisas em malária deve levar em consideração a linhagem do camundongo e suas capacidades individuais de resposta. Além disso, indicam que a forma cerebral pode ser resultado de diferentes mecanismos fisiopatológicos. Provavelmente, esses resultados também expliquem as diferentes evoluções clínicas observadas da malária humana. _________________________________________________________________________________ ABSTRACTMalaria is one of the most important problems of public health, affecting millions people and resulting in almost one million death every year. Cerebral disease is one of the most severe complications of Plasmodium falciparum malaria and its immunopathogenesis is not well established. It has been considered that the excessive activation of the immune system by some parasite components is critical to the pathogenesis of severe malaria. The imbalance of immune response with polarization towards the inflammatory mechanisms plays a key role in the activation of endothelial vessels, in the sequestration of parasitized erythrocytes within the small vessels of brain and in cerebral cells lesions. The inflammatory mediators are released in response to plasmodial antigens, cytokines, eicosanoids and other products as hemozoin and free heme, which stimulates differently the activation and regulation of intracellular pathways. This work aimed at evaluating the inflammatory response which is involved in cerebral alterations in susceptible (CBA and C57BL/6) and resistant (BALB/c) mice to cerebral malaria, to better understanding of the immunopathological mechanisms and to better characterize the experimental murine models of malaria. In this work, 8-12 weeks-old male mice were infected with 106 Plasmodium berghei ANKA parasitized erythrocytes. For each strain, the survival rate and parasitemia were assessed. The nitric oxide production was evaluated by the Griess reaction and the hydrogen peroxide production was assessed by the Pick´s method. IL-1β, IL-4, IL-10, IL-12, IL-17 and TNF-α production in brain tissue were quantified by an ELISA test. The COX-2 and 5-LOX expression in microglia and peritoneal macrophages were analyzed in a flow cytometer. COX-2 and 5-LOX expression were evaluated in cerebral cells and vessels by immunohistochemistry. The lipid body formation is a hallmark of the activation of lipid metabolism and it was quantified in peritoneal macrophages by oil red O staining and bodipy fluorescent probe. The activation of NF?B pathway, inflammasome and PPAR-? were evaluated in the nuclei and cytoplasm of peritoneal macrophages. Histopathological evaluation of the brain was performed by hematoxilin-eosin staining and by scanning electron microscopy. Vascular permeability was measured by Evans blue dye extraction. The C57BL/6 mice began to die on the sixth day post-infection, CBA mice, two days later, while 55% of BALB/c mice still remained alive 15 days after infection. C57BL/6 mice showed the highest parasitemia compared with the two other strains. Only the canonical NF?B pathway was activated in C57BL/6 mice, there was a decrease in NO production, and an increase in H2O2, TNF-α e IL-10 levels after Plasmodium infection, but no alteration was observed in IL-4, IL-12 and IL-17 production; the lipid bodies formation was enhanced and 5-LOX was predominantly expressed. It was also observed the NALPR1 inflammasome activation, but the IL-1β production was decreased. The non-canonical NF?B pathway was more activated than the canonical one in CBA mice. They showed a decline in TNF-α and IL-10 productions, while no alteration in IL-12, NO and H2O2 was observed; the IL-4 and IL-17 pattern production was higher in these mice than in the other strains. The inflammasome was activated, but no production of IL-1β was observed. The BALB/c mice showed predominantly activation of the canonical NF?B pathway, but the products stimulated by the activity of this pathway, such as, IL-12, NO e H2O2 was not changed in infected animals, and even a decreased production of TNF-α and IL-1β was observed. There was an increased formation of lipid bodies, however, COX-2 and 5-LOX were not significantly expressed. In addition, higher translocation of PPAR-γ into the nuclei was observed in this resistant model. These profiles of responses were followed by histopathological alterations that were characteristics for each strain. The C57BL/6 mice showed the highest vascular permeability and leukocyte sequestration; the CBA mice also showed increased vascular permeability but lower than C57BL/6 mice, and they showed more erythrocyte than leukocyte sequestered. Few changes were observed in BALB/c mice. The results presented in this work suggest that different mechanisms of cellular responses may have contributed to determinate clinical malaria and showed that the choice of the experimental model for research on malaria should take into consideration the strain of mice and their individual capacities of responses. They also showed that the cerebral form of the disease may result from different pathophysiological mechanisms. Probably, these results may also explain the different clinical outcomes observed in human malaria

Phylloseptin-1 is leishmanicidal for amastigotes of Leishmania amazonensis inside infected macrophages

Leishmania protozoans are the causal agents of neglected diseases that represent an important public health issue worldwide. The growing occurrence of drug-resistant strains of Leishmania and severe side effects of available treatments represent an important challenge for the leishmaniases treatment. We have previously reported the leishmanicidal activity of phylloseptin-1 (PSN-1), a peptide found in the skin secretion of Phyllomedusa azurea (=Pithecopus azureus), against Leishmania amazonensis promastigotes. However, its impact on the amastigote form of L. amazonensis and its impact on infected macrophages are unknown. In this work, we evaluated the effects of PSN-1 on amastigotes of L. amazonensis inside macrophages infected in vitro. We assessed the production of hydrogen peroxide and nitric oxide, as well as the levels of inflammatory and immunomodulatory markers (TGF-β, TNF-α and IL-12), in infected and non-infected macrophages treated with PSN-1. Treatment with PSN-1 decreased the number of infected cells and the number of ingested amastigotes per cell when compared with the untreated cells. At 32 µM (64 µg/mL), PSN-1 reduced hydrogen peroxide levels in both infected and uninfected macrophages, whereas it had little effect on NO production or TGF-β release. The effect of PSN-1 on IL-12 and TNF-α secretion depended on its concentration, but, in general, their levels tended to increase as PSN-1 concentration increased. Further in vitro and in vivo studies are needed to clarify the mechanisms of action of PSN-1 and its interaction with the immune system aiming to develop pharmacological applications

Catálogo Taxonômico da Fauna do Brasil: setting the baseline knowledge on the animal diversity in Brazil

The limited temporal completeness and taxonomic accuracy of species lists, made available in a traditional manner in scientific publications, has always represented a problem. These lists are invariably limited to a few taxonomic groups and do not represent up-to-date knowledge of all species and classifications. In this context, the Brazilian megadiverse fauna is no exception, and the Catálogo Taxonômico da Fauna do Brasil (CTFB) (http://fauna.jbrj.gov.br/), made public in 2015, represents a database on biodiversity anchored on a list of valid and expertly recognized scientific names of animals in Brazil. The CTFB is updated in near real time by a team of more than 800 specialists. By January 1, 2024, the CTFB compiled 133,691 nominal species, with 125,138 that were considered valid. Most of the valid species were arthropods (82.3%, with more than 102,000 species) and chordates (7.69%, with over 11,000 species). These taxa were followed by a cluster composed of Mollusca (3,567 species), Platyhelminthes (2,292 species), Annelida (1,833 species), and Nematoda (1,447 species). All remaining groups had less than 1,000 species reported in Brazil, with Cnidaria (831 species), Porifera (628 species), Rotifera (606 species), and Bryozoa (520 species) representing those with more than 500 species. Analysis of the CTFB database can facilitate and direct efforts towards the discovery of new species in Brazil, but it is also fundamental in providing the best available list of valid nominal species to users, including those in science, health, conservation efforts, and any initiative involving animals. The importance of the CTFB is evidenced by the elevated number of citations in the scientific literature in diverse areas of biology, law, anthropology, education, forensic science, and veterinary science, among others

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Components of Banisteriopsis caapi, a Plant Used in the Preparation of the Psychoactive Ayahuasca, Induce Anti-Inflammatory Effects in Microglial Cells

Banisteriopsis caapi is used to prepare the psychoactive beverage ayahuasca, and both have therapeutic potential for the treatment of many central nervous system (CNS) conditions. This study aimed to isolate new bioactive compounds from B. caapi extract and evaluate their biological activity, and that of the known &beta;-carboline components of the plant (harmine, harmaline, and tetrahydroharmine), in BV-2 microglial cells, the in vivo activation of which is implicated in the physiopathology of CNS disorders. B. caapi extract was fractionated using semipreparative liquid chromatography (HPLC-DAD) and the exact masses ([M + H]+m/z) of the compounds in the 5 isolated fractions were determined by high-resolution LC-MS/MS: F1 (174.0918 and 233.1289), F2 (353.1722), F3 (304.3001), F4 (188.1081), and F5 (205.0785). Harmine (75.5&ndash;302 &micro;M) significantly decreased cell viability after 2 h of treatment and increased the number of necrotic cells and production of reactive oxygen species at equal or lower concentrations after 24 h. F4 did not impact viability but was also cytotoxic after 24 h. Most treatments reduced proinflammatory cytokine production (IL-2, IL-6, IL-17, and/or TNF), especially harmaline and F5 at 2.5 &micro;M and higher concentrations, tetrahydroharmine (9.3 &micro;M and higher), and F5 (10.7 &micro;M and higher). The results suggest that the compounds found in B. caapi extract have anti-inflammatory potential that could be explored for the development of treatments for neurodegenerative diseases

Impact of polyphenols in phagocyte functions

Polyphenols are a broad group of substances with potential health benefits found in plant species. Several of these compounds are capable of influencing the activation of intracellular signaling pathways, such as NF-kB, MAPK and JAK-STAT, responsible for the production of various inflammatory mediators such as tumor necrosis factor α (TNF-α) and interleukin 1 beta (IL-1β) and 12 (IL-12), enzymes involved in the production of reactive species such as inducible nitric oxide synthase (iNOS) and superoxide dehydrogenase (SOD), as well as enzymes involved in the production of eicosanoids, such as cyclooxygenase (COX) and lipoxygenase (LO). There is increased interest in the use of polyphenol-rich foods because of their immunomodulatory effect; however, the mechanisms used during macrophage responses are extremely complex and little is known about the effects of polyphenols on these cells. As such, this review summarizes the current view of polyphenol influences on macrophages

Components of Banisteriopsis caapi, a plant used in the preparation of the psychoactive ayahuasca, induce anti-inflammatory effects in microglial cells

Banisteriopsis caapi is used to prepare the psychoactive beverage ayahuasca, and both have therapeutic potential for the treatment of many central nervous system (CNS) conditions. This study aimed to isolate new bioactive compounds from B. caapi extract and evaluate their biological activity, and that of the known β-carboline components of the plant (harmine, harmaline, and tetrahydroharmine), in BV-2 microglial cells, the in vivo activation of which is implicated in the physiopathology of CNS disorders. B. caapi extract was fractionated using semipreparative liquid chromatography (HPLC-DAD) and the exact masses ([M + H]+m/z) of the compounds in the 5 isolated fractions were determined by high-resolution LC-MS/MS: F1 (174.0918 and 233.1289), F2 (353.1722), F3 (304.3001), F4 (188.1081), and F5 (205.0785). Harmine (75.5–302 µM) significantly decreased cell viability after 2 h of treatment and increased the number of necrotic cells and production of reactive oxygen species at equal or lower concentrations after 24 h. F4 did not impact viability but was also cytotoxic after 24 h. Most treatments reduced proinflammatory cytokine production (IL-2, IL-6, IL-17, and/or TNF), especially harmaline and F5 at 2.5 µM and higher concentrations, tetrahydroharmine (9.3 µM and higher), and F5 (10.7 µM and higher). The results suggest that the compounds found in B. caapi extract have anti-inflammatory potential that could be explored for the development of treatments for neurodegenerative diseases

Acute Effects of the Consumption of Passiflora setacea Juice on Metabolic Risk Factors and Gene Expression Profile in Humans

International audienceBackground: Passiflora setacea (PS) is a passionfruit variety of the Brazilian savannah and is a rich source of plant food bioactives with potential anti-inflammatory activity. This study aimed to investigate the effect of an acute intake of PS juice upon inflammation, metabolic parameters, and gene expression on circulating immune cells in humans. Methods: Overweight male volunteers (n = 12) were enrolled in two double-blind placebo-controlled studies. Blood samples were collected from fasting volunteers 3 h after the consumption of 250 mL of PS juice or placebo (PB). Metabolic parameters (insulin, glucose, total cholesterol, high-density lipoprotein (LDL), high-density lipoprotein (HDL), and total triglycerides) and circulating cytokines were evaluated (study 1). Peripheral blood mononuclear cell (PBMC) from the same subjects were isolated and RNA was extracted for transcriptomic analyses using microarrays (study 2). Results: Insulin and homeostatic model assessment for insulin resistance (HOMA-IR) levels decreased statistically after the PS juice intake, whereas HDL level increased significantly. Interleukin (IL)-17A level increased after placebo consumption, whereas its level remained unchanged after PS juice consumption. Nutrigenomic analyses revealed 1327 differentially expressed genes after PS consumption, with modulated genes involved in processes such as inflammation, cell adhesion, or cytokine-cytokine receptor. Conclusion: Taken together, these clinical results support the hypothesis that PS consumption may help the prevention of cardiometabolic diseases

A panel of markers for identification of malignant and non-malignant cells in culture from effusions

The aim of the present study was to identify cell types in primary culture from malignant and non-malignant effusions. Effusion samples were subjected to cytology and culture. Immunocytochemistry was performed in cytological slides to evaluate malignancy (positivity for malignancy markers) and in culture slides for identification of cell types in growth. A total of 143 effusion samples (pleural n=76; peritoneal n=37; pericardial n=4; and peritoneal lavage n=26) were analyzed. Cell growth was observed in 34.9% of all samples and immunocytochemistry for identification of cell types in culture slides was conclusive in 90% of them. In non-malignant samples (n=28), growth of mesothelial cells, macrophages and of both cell types was identified in 82.14, 10.71 and 7.14%, respectively. In malignant samples (n=17, all carcinomas), growth of malignant epithelial cells and of both malignant epithelial and mesothelial cells was identified in 41.17 and 23.52%, respectively. In the remaining 35.29% of malignant samples, the only cells in growth were mesothelial and/or macrophages instead of malignant epithelial cells. In conclusion, in culture of malignant effusions, mesothelial cells may be simultaneously identified with malignant epithelial cells. Besides, mesothelial cells and macrophages may be the only cells identified in malignant effusion culture. Therefore, a broad panel of cell markers should be used for unmistakable identification of cells in studies of effusion primary culture. The ideal malignant effusion sample to obtain culture of neoplastic cells should be that without the presence of mesothelial cells and macrophages