Interaction of malaria parasites with host late endocytic and autophagic pathways is essential for Plasmodium liver stage development

RESUMO: A Malária é causada por parasitas do género Plasmodium, sendo a doença parasitária mais fatal para o ser humano. Apesar de, durante o século passado, o desenvolvimento económico e a implementação de diversas medidas de controlo, tenham permitido erradicar a doença em muitos países, a Malária continua a ser um problema de saúde grave, em particular nos países em desenvolvimento. A Malária é transmitida através da picada de uma fêmea de mosquito do género Anopheles. Durante a picada, os esporozoítos são injetados na pele do hospedeiro, seguindo-se a fase hepática e obrigatória do ciclo de vida. No fígado, os esporozoítos infetam os hepatócitos onde se replicam, dentro de um vacúolo parasitário (VP) e de uma forma imunitária silenciosa, em centenas de merozoitos. Estas novas formas do parasita são as responsáveis por infetar os eritrócitos, iniciando a fase sanguínea da doença, onde se os primeiros sintomas se manifestam, tais como a característica febre cíclica. A fase hepática da doença é a menos estudada e compreendida. Mais ainda, as interações entre o VP e os organelos da células hospedeira estão ainda pouco caracterizados. Assim, neste estudo, as interações entre os organelos endocíticos e autofágicos da célula hospedeira e o VP foram dissecados, observando-se que os anfisomas, que são organelos resultantes da intersecção do dois processos de tráfego intracelular, interagem com o parasita. Descobrimos que a autofagia tem também uma importante função imunitária durante a fase hepática inicial, ao passo, que durante o desenvolvimento do parasita, já numa fase mais tardia, o parasita depende da interação com os endossomas tardios e anfisomas para crescer. Vesiculas de BSA, EGF e LC3, foram, também, observadas dentro do VP, sugerindo que os parasitas são capazes de internalizar material endocítico e autofágico do hospedeiro. Mais ainda, mostramos que esta interação depende da cinase PIKfyve, responsável pela conversão do fosfoinositidio-3-fosfato no fosfoinositidio-3,5-bifosfato, uma vez que inibindo esta cinase o parasita não é capaz de crescer normalmente. Finalmente, mostramos que a proteína TRPML1, uma proteína efetora do fosfoinositidio-3,5-bifosfato, e envolvida no processo de fusão das membranas dos organelos endocíticos e autofágicos, também é necessária para o crescimento do parasita. Desta forma, o nosso estudo sugere que a membrana do VP funde com vesiculas endocíticas e autofágicas tardias, de uma forma dependente do fositidio-3,5-bifosfato e do seu effetor TRPML1, permitindo a troca de material com a célula hospedeira. Concluindo, os nossos resultados evidenciam que o processo autofágico que ocorre na célula hospedeira tem um papel duplo durante a fase hepática da malaria. Enquanto numa fase inicial os hepatócitos usam o processo autofágico como forma de defesa contra o parasita, já durante a fase de replicação o VP funde com vesiculas autofágicas e endocíticas de forma a obter os nutrientes necessários ao seu desenvolvimento.--------- ABSTRACT: Malaria, which is caused by parasites of the genus Plasmodium, is the most deadly parasitic infection in humans. Although economic development and the implementation of control measures during the last century have erradicated the disease from many areas of the world, it remains a serious human health issue, particularly in developing countries. Malaria is transmitted by female mosquitoes of the genus Anopheles. During the mosquito blood meal, Plasmodium spp. sporozoites are injected into the skin dermis of the vertebrate host, followed by an obligatory liver stage. Upon entering the liver, Plasmodium parasites infect hepatocytes and silently replicate inside a host cell-derived parasitophorous vacuole (PV) into thousands of merozoites. These new parasite forms can infect red blood cells initiating the the blood stage of the disease which shows the characteristic febrile malaria episodes. The liver stage is the least characterized step of the malaria infection. Moreover, the interactions between the Plasmodium spp. PV and the host cell trafficking pathways are poorly understood. We dissected the interaction between Plasmodium parasites and the host cell endocytic and autophagic pathways and we found that both pathways intersect and interconnect in the close vicinity of the parasite PV, where amphisomes are formed and accumulate. Interestingly, we observed a clearance function for autophagy in hepatocytes infected with Plasmodium berghei parasites at early infection times, whereas during late liver stage development late endosomes and amphisomes are required for parasite growth. Moreover, we found the presence of internalized BSA, EGF and LC3 inside parasite vacuoles, suggesting that the parasites uptake endocytic and autophagic cargo. Furthermore, we showed that the interaction between the PV and host traffic pathways is dependent on the kinase PIKfyve, which converts the phosphoinositide PI(3)P into PI(3,5)P2, since PIKfyve inhibition caused a reduction in parasite growth. Finally, we showed that the PI(3,5)P2 effector protein TRPML1, which is involved in late endocytic and autophagic membrane fusion, is also required for parasite development. Thus, our studies suggest that the parasite parasitophorous vacuole membrane (PVM) is able to fuse with late endocytic and autophagic vesicles in a PI(3,5)P2- and TRPML1-dependent manner, allowing the exchange of material between the host cell and the parasites, necessary for the rapid development of the latter that is seen during the liver stage of infection. In conclusion, we present evidence supporting a specific and essential dual role of host autophagy during the course of Plasmodium liver infection. Whereas in the initial hours of infection the host cell uses autophagy as a cell survival mechanism to fight the infection, during the replicative phase the PV fuses with host autophagic and endocytic vesicles to obtain nutrients required for parasite growth.

O sal na alimentação dos jovens : avaliação e perceção do sal das refeições

RESUMO: Considerando que a pressão arterial elevada constitui um dos maiores fatores de risco para as doenças cardiovasculares, a sua associação ao consumo elevado de sal, e o facto das escolas constituírem ambientes de excelência para a aquisição de bons hábitos alimentes e promoção da saúde, o objetivo deste estudo foi avaliar o conteúdo de sal presente nas refeições escolares e a perceção dos consumidores sobre o sabor salgado. A quantificação de sal foi realizada com um medidor portátil. Para avaliar a perceção dos consumidores foi desenvolvido e aplicado um questionário a alunos das escolas preparatórias e secundárias e aos responsáveis pela preparação e confeção das refeições. Foram analisadas um total de 898 componentes de refeições, incluindo refeições escolares e de restauração padronizada. Em média, as refeições escolares disponibilizam entre 2,83 a 3,82 g de sal por porção servida (p=0,05), o que representa entre duas a cinco vezes mais as necessidades das crianças e jovens. Os componentes das refeições padronizadas apresentam um valor médio de sal que varia entre 0,8 e 2,57 g por porção (p=0,05), o que pode contribuir para um valor total de sal por refeição mais elevado comparativamente com as refeições escolares. O sabor das refeições é percecionado como sendo nem salgado nem insonso para a maioria dos estudantes, o que parece demonstrar habituação à intensidade/ quantidade de sal consumida. Os responsáveis pelas refeições, apesar de apresentarem conhecimentos sobre sal e a necessidade da sua limitação, demonstram barreiras e limitações e perceções à sua redução. A realização de escolhas alimentares saudáveis e adequadas só é possível se suportada por um ambiente facilitador dessas mesmas escolas. O impacto que o consumo de sal tem na saúde, em particular nas doenças crónicas, torna imperativa a implementação de estratégias de redução de sal ao nível da indústria e dos serviços de catering e restauração, em particular direcionadas para o público mais jovem.------------ABSTRACT Considering the fact that high blood pressure is a major rick factor for cardiovascular disease and its association to salt intake and the fact that schools are considered ideal environments to promote health and proper eating habits, the objective of this study was to evaluate the amount of salt in meals served in school canteens and consumers perceptions about salt. Quantification of salt was performed using a portable salt meter - PAL ES2. For food perception we constructed a questionnaire that was applied to students from high schools. A total of 898 food samples were analysed. Bread presents the highest value with a mean of 1.35 (SD=0.12). Salt in soups ranges from 0.72 g/100 g to 0.80 g/100 g (p=0.05) and main courses from 0.71 g/100 to 0.97 g/100g (p=0.05). Salt in school meals is high with a mean value from 2.83 to 3.82 g of salt per meal, which is between 2 and 5 times more than the RDA for children. The components of standardized meals have an average value of salt ranging from 0.8 to 2.57 g per serving, which may contribute to a higher intake of salt per meal compared to school meals. Moreover, a high percentage of students consider meals neither salty nor lacking in salt, which shows they are used to the intensity/amount of salt consumed. Despite the knowledge and perceived necessity about salt reduction, those responsible for cooking and preparing meals, still demonstrate barriers and limitation in doing so. Making healthy choices is only possible if backed up by an environment where such choices are accessible. Therefore salt reduction strategies, aimed at the food industry and catering services should be implemented, with children and young people targeted as a major priority.

Blood feeding patterns of Nyssomyia intermedia and Nyssomyia neivai (Diptera, Psychodidae) in a cutaneous leishmaniasis endemic area of the Ribeira Valley, State of São Paulo, Brazil

Introduction The aim of this study was to identify the blood feeding sources of Nyssomyia intermedia (Ny. intermedia) and Nyssomyia neivai (Ny. neivai), which are Leishmania vectors and the predominant sandfly species in the Ribeira Valley, State of São Paulo, Brazil, an endemic area for cutaneous leishmaniasis. Methods Specimens were captured monthly between February 2001 and December 2003 on a smallholding and a small farm situated in the Serra district in the Iporanga municipality. The blood meals of 988 engorged females were tested using the avidin-biotin immunoenzymatic enzyme-linked immunosorbent assay (ELISA). Seven blood meal sources were investigated: human, dog, chicken, bovine, pig, horse and rat. Results The results showed that among the females that fed on one or more blood sources, the respective percentages for Ny. intermedia and Ny. neivai, respectively, were as follows: human (23% and 36.8%), pig (47.4% and 26.4%), chicken (25.7% and 36.8%) and dog (3.9% and 0%), and the differences in the blood sources between the two species were statistically significant (p = 0.043). Conclusions Both species had predominant reactivity for one or two blood sources, and few showed reactivity indicating three or four sources. Many different combinations were observed among the females that showed reactivity for more than one source, which indicated their opportunistic habits and eclecticism regarding anthropic environmental conditions.

Eclectic feeding behavior of Lutzomyia (Nyssomyia) intermedia (Diptera, Psychodidae, Phlebotominae) in the transmission area of American cutaneous leishmaniasis, state of Paraná, Brazil

Introduction The blood meal source of sandflies provides valuable information about the vector/host interaction and allows for an understanding of American cutaneous leishmaniasis (ACL) transmission mechanisms. The aim of this study was to identify the blood meal sources of Lutzomyia (Nyssomyia) intermedia in an endemic area of leishmaniasis in Brazil's State of Paraná using a precipitin test. Methods Sandflies were collected in the rural locality of Epitácio Pessoa within the City of Adrianópolis, State of Paraná, in southern Brazil. A total of 864 female sandflies were captured, and 862 (99.8%) were identified as L. intermedia species. However, two unidentified specimens were considered to be part of the genus Lutzomyia. Results Among the females examined, 396 specimens presented reactions to a certain type of tested antiserum, and most (67.9%) reacted to the simple type. These sandflies fed mainly on the blood of birds, opossums, and rodents, but specimens that fed on the blood of humans, dogs, horses, cattle, and cats were also found. Among the cross-reactions found (32.1%), bird/rodent, bird/opossum, bird/dog, bird/human, and horse/dog cross-reactions were the most common. Conclusions These results demonstrate a tendency in the eclectic feeding behavior of L. intermedia and support its potential role as a vector for ACL in the study area.

Biology of Triatoma carcavalloi Jurberg, Rocha & Lent, 1998 under laboratory conditions

Introduction Triatoma carcavalloi is a wild species that is found in sympatry with Triatoma rubrovaria and Triatoma circummaculata, which are vectors of Trypanosoma cruzi currently found in rural areas of Rio Grande do Sul, Brazil. Methods Fertility was assessed and to determine the incubation period, the eggs were observed until hatching. The first meal was offered to 1st stage nymphs. The intermolt period was also determined. The number of blood meals was quantified at each nymphal stage and the resistance to fasting as the period between ecdysis and death. Mortality was assessed and longevity was determined by recording the time that elapsed from molting to the adult stage and until death. The developmental cycle was assessed by recording the length in days of each stage from molting to adult hood. Results The average incubation period was 22.7 days. The average first meal occurred 3.1 days after hatching. The 5th stage nymph to adult intermolting period was the longest at 193.4 days. The average number of feedings during nymphal development was 13.4. The resistance to fasting assay indicated that the 3rd, 4th and 5th stage nymphs presented higher resistance than did adults. The highest mortality rate was observed in the 3rd stage nymphs (22.2%). The average length of adult survival was 25.6 weeks, and the average total life cycle lasted 503.4 days. Conclusions This study is the first report on the biology of T. carcavalloi that fed on mice. The presented findings expand the bionomic knowledge of these species.

Effect of feeding frequency on the reproductive efficiency of two species of Triatoma with different epidemiological importance

Introduction In Triatominae, reproductive efficiency is an important factor influencing population dynamics, and a useful parameter in measuring a species' epidemiological significance as a vector of Trypanosoma cruzi (Chagas, 1909). The reproductive efficiency of triatomines is affected by food availability; hence, we measured and compared the effects of feeding frequency on the reproductive parameters of Triatoma patagonica (Del Ponte, 1929) and Triatoma infestans (Klug, 1934), and the effects of starvation on T. patagonica. Methods Couples from both species were fed weekly, or every 3 weeks; in addition, females in couples of T. patagonica were not fed. Each couple was observed weekly and reproductive efficiency was assessed on the following parameters: fecundity (eggs/female), fertility (eggs hatched/eggs laid), initiation and end of oviposition, initiation of mating, number of matings/week, and number of reproductive weeks. Relative meal size index (RMS), blood consumption index (CI), and E values (eggs/mg blood) were also calculated. Results Changes in feeding frequency affected the reproductive parameters of T. patagonica only, with a decrease in fecundity and number of reproductive weeks for those fed every 3 weeks, or not fed. The reproductive period, RMS index, and CI were lower for T. patagonica than T. infestans. However, despite the lower fecundity of T. patagonica, this species required less blood to produce eggs, with an E values of 2 compared to 2.94 for T. infestans. Conclusions Our results suggest that the differences in fecundity observed between species reflect the availability of food in their natural ecotopes.

Epidemiological factors associated with seropositivity for toxoplasmosis in pregnant women from Gurupi, State of Tocantins, Brazil

Introduction Knowledge of the prevalence and risk factors for Toxoplasma gondii dissemination among pregnant women is relevant because the parasite can be spread from mother to infant. The objective of this study was to assess the epidemiology and risk factors of toxoplasmosis in pregnant women from Gurupi, State of Tocantins, Brazil, from February 2012 to June 2013. Methods The study population included 487 pregnant women. Sociodemographic, dietary and cultural data were collected using a standardized and validated form. Peripheral blood was collected for serologic testing using the ELISA test (IgM/IgG antibodies). The data were analyzed by comparing seropositivity with risk factors using crude and adjusted odds ratios. Results The prevalence rate for IgG and IgM antibodies was 68.7% and 5.7%, respectively. Sociodemographic characteristics associated with toxoplasmosis risk included the following: education level ≤ 8 years (OR: 6.612; CI: 1.450-30.144), age ≥ 30 years (OR: 5.273; CI: 1.166-23.844), working outside the home (OR: 1.604; CI: 1.015-2.536), and family income of two minimum wages or lower (OR: 2.700; CI: 1.891-8.182). Regarding dietary habits, there was a significant association of seropositivity with meat intake (OR: 1.78; CI: 1.149-4.080), cutting vegetables without washing the cutting board beforehand (OR: 2.051; CI: 1.165-3.614), frequent intake of vegetables (OR: 2.051; CI: 1.368-3.006) and in natura milk intake (OR: 2.422; CI: 1.014-5.785). Conclusions The high prevalence rates of toxoplasmosis in Gurupi are related to age, raw meat and in natura milk intake, as well as education level, working outside the home, and poor hygienic habits during meal preparation.

Nutritional status and food intake at workplace as health indicators for industrial workers

This study aimed to verify the correlation among the nutritional composition of the food consumed in the work environment, the energy expenditure and the nutritional status of workers from different sectors (administration and production) in different industries. The anthropometric data, in addition to the energy expenditure and food intake at lunch were evaluated for 292 workers, all of them included in the Brazilian Worker Food Program (also called PAT). The food consumption was assessed from the direct observation of the meal, for five consecutive days. The obtained data were analyzed by Pearson correlation test and by a Principal Components Analysis. Prevalence of overweight was detected in the studied population, according to the Body Mass Index (BMI). A statistically significant difference was found in terms of the energy expenditure of physical activity and daily energy expenditure in relation to gender and the working sector. The obtained results indicate that there is significant positive correlation (p < 0.01) between the following variables: body weight and BMI (r = 0.84), weight and daily energy expenditure (DEE) (r = 0.52), BMI and DEE (r = 0.27), DEE and energy (r = 0.38), and energy and lipid intake (r = 0.50). These findings seems to indicate the importance of ensuring an adequate balance of nutrients at meals, due to the heterogeneity of workers, in particular in the case of those workers who perform tasks or functions requiring less energy expenditure.

Haematophagic behavior in laboratory of Lutzomyia cruzi (Mangabeira) (Diptera: Psychodidae) in relation to three mammalian blood sources in Manaus, Brazil

The sand fly Lutzomyia cruzi is considered as one of vectors of visceral leishmaniasis in Brazil. This work examined optimum feeding age, feeding time, host preference, fecundity rates, and female blood meal volume taken by single females from a closed colony of L. cruzi. Mean feeding time was longer on hamsters, 6.6 minutes, than on humans, 5.7 minutes. 49.1% of the 48h-old flies fed on humans and 43.3% of 72h-old flies fed on hamsters. Of a total of 120 females, 61% fed on humans and 25% fed on hamsters. Total fecundity was significantly higher in females fed on hamster than on human or opossum. Laboratory-reared L. cruzi females fed earlier, more promptly, and preferably on humans than on hamsters when offered these blood-meal sources simultaneously. The blood-meal volume is higher in females fed on hamsters than other hosts (human and opossum).

O perfil e as motivações do visitante gastronómico e do enoturista de Guimarães

Dissertação de mestrado em Geografia (área de especialização em Planeamento e Gestão do Território)

Lipid metabolism in rats fed diets containing different types of lipids

OBJECTIVE: To assess the effect of different types of lipid diets on the lipid metabolism of aging rats. METHODS: Fifty male Wistar rats were studied from the time of weaning to 12 and 18 months of age. Their diets were supplemented as follows: with soybean oil (S), canola oil (CA), lard and egg yolk (LE), and canola oil + lard and egg yolk (CA + LE). Blood pressure (BP) was measured every month, and the heart/body ratio (H/BR) was determined. The rats were euthanized at the age of 12 and 18 months, and blood samples were collected for lipid analysis as follows: total cholesterol (TC), LDL-C, VLDL-C, HDL-C, triglycerides (TG), and glucose. RESULTS: The type of oil ingested by the animals significantly altered BP, H/BR, and serum lipid levels in rats at 12 and 18 months. No difference was observed in the survival curve of the animals in the different groups. The LE group had the highest BP, and the CA group was the only one in which BP did not change with aging. A reduction in the H/BR was observed in the LE and CA+LE animals. At the age of 12 months, differences in TC, HDL-C, LDL-C, VLDL-C, TG, and glucose were observed. At the age of 18 months, a significant difference in TC, HDL-C, and glucose was observed. The highest TC value was found in the CA group and the lowest in the S group. CONCLUSION: No increase in BP occurred, and an improvement was evident in the lipid profile of rats fed a diet supplemented with CA, in which an elevation in HDL-C levels was observed, as compared with levels with the other types of diet.

Gorduras em laticínios, ovos, margarinas e óleos: implicações para a aterosclerose

FUNDAMENTO: Para o aconselhamento adequado de um planejamento alimentar, com vistas à prevenção da cardiopatia isquêmica, é necessário o conhecimento da composição química dos alimentos. ObJETIVO: Analisar a composição de gorduras, ácidos graxos e colesterol de alguns exemplos nacionais de óleos comestíveis, manteigas, margarinas, laticínios e ovos, utilizando os limites de uma dieta para prevenção de doença arterial coronariana. MÉTODOS: Foi analisada a composição de óleos comestíveis, manteigas, margarinas, laticínios e ovos. Os resultados foram empregados de acordo com o recomendado pela American Heart Association para uma dieta de 1.800 calorias. RESULTADOS: Na comparação entre os óleos comestíveis, o de canola apresentou-se como o melhor. Já entre os leites, o desnatado é o mais recomendável, porém não há vantagens em relação ao semidesnatado. No caso do ovo, não existiram diferenças nos tipos encontrados no mercado. Em relação aos queijos, o tipo minas é o que apresentou o menor conteúdo de colesterol e gordura saturada. Na comparação margarina com manteiga, a primeira levou vantagem quando os níveis de gorduras trans que não ultrapassam o recomendado. CONCLUSÃO: A composição de alimentos usados no Brasil indica que dietas com objetivo de prevenção primária e secundária da cardiopatia isquêmica podem priorizar o uso de leite semidesnatado, óleo de girassol, margarinas com baixo teor de gorduras trans e queijo do tipo minas.

Neural Mechanisms and Delayed Gastric Emptying of Liquid Induced Through Acute Myocardial Infarction in Rats

Background: In pathological situations, such as acute myocardial infarction, disorders of motility of the proximal gut can trigger symptoms like nausea and vomiting. Acute myocardial infarction delays gastric emptying (GE) of liquid in rats. Objective: Investigate the involvement of the vagus nerve, α 1-adrenoceptors, central nervous system GABAB receptors and also participation of paraventricular nucleus (PVN) of the hypothalamus in GE and gastric compliance (GC) in infarcted rats. Methods: Wistar rats, N = 8-15 in each group, were divided as INF group and sham (SH) group and subdivided. The infarction was performed through ligation of the left anterior descending coronary artery. GC was estimated with pressure-volume curves. Vagotomy was performed by sectioning the dorsal and ventral branches. To verify the action of GABAB receptors, baclofen was injected via icv (intracerebroventricular). Intravenous prazosin was used to produce chemical sympathectomy. The lesion in the PVN of the hypothalamus was performed using a 1mA/10s electrical current and GE was determined by measuring the percentage of gastric retention (% GR) of a saline meal. Results: No significant differences were observed regarding GC between groups; vagotomy significantly reduced % GR in INF group; icv treatment with baclofen significantly reduced %GR. GABAB receptors were not conclusively involved in delaying GE; intravenous treatment with prazosin significantly reduced GR% in INF group. PVN lesion abolished the effect of myocardial infarction on GE. Conclusion: Gastric emptying of liquids induced through acute myocardial infarction in rats showed the involvement of the vagus nerve, alpha1- adrenergic receptors and PVN.

Comparação de Proteínas: alimentação das aves I

Beginning an experiment on protein comparisons, at the Poultry and Rabbitry Departament of the Esc. Sup. de Agricultura "Luiz de Queiroz", University of S. Paulo, four groups of growing chicks were submitted during 40 days to the following rations: basal part - 50 corn meal and 30 wheat bran, variable part - R1 - 10 tankage and 10 peanut meal, R2 - 10 tankage and 10 cottonseed meal, R3 - 7 peanut meal, 7 cocoanut meal and 7 cottonseed meal, R4 - 5 tankage, 5 peanut, 5 cocoanut and 5 cottenseed meal, R2 and R3 gave results which may be considered as equal and inferior than those obtained with the others, R4 being the best one. The statistical analises showed no significant differences.

A influência dos pigmentos amarelo -laranja de vários alimentos na coloração da gema de ôvo de galinha

The effect of different feeds in comparison with that of maize grains on the egg yolk color was observed. It was found that deep orange and yellow orange maize give satisfactory coloration to the yolk, respectively orange and yellow. The most intense color was observed when green feed was used in combination with deep orange maize. Green feeds as chicory, alfafa, cabbage, welsh onion and banana leaves and alfafa or chicory meal proved to be good in giving orange color to the yolk. Yellow yolk was obtained when Guinea grass or carica fruit were used in the ration. Carrot and beet without leaves did not give satisfactory color to the egg yolk. The observations with other feeds are being continued.