Chloroquine inhibits pro-inflammatory effects of heme on macrophages and invivo.

BACKGROUND: Chloroquine has been used successfully to treat Malaria, including by chloroquine-resistant Plasmodium sp., indicating that it has effects on disease itself. Since heme has inflammatory effects and contributes to the pathogenesis of hemolytic diseases, we hypothesize that the anti-inflammatory effect of chloroquine is partially due to its inhibitory effect on heme-induced macrophage activation and on inflammatory tissue damage. METHODS: Bone marrow derived macrophages (BMDMs) were incubated with chloroquine before stimulation with heme, in different conditions, to evaluate cytokines secretion, ROS production, mitogen activated protein kinases (MAPK) or spleen tyrosine kinase (Syk) activation, alone or combined with LPS. The effects of chloroquine upon heme inflammation were also evaluated in vivo, through simultaneous i.p. injection of LPS and heme, intratracheal instillation of Poly-IC followed by heme injection, and in a rhabdomyolysis model. RESULTS: Chloroquine inhibited TNF secretion, mitochondrial ROS production, MAPK, and Syk activation induced by heme. Inhibition of TNF production could be mimicked by zinc ionophore quercetin, but not by primaquine, a chloroquine analog with low affinity for heme. IL-6 and IL-1ß secretions induced by heme in the presence of PRRs agonists were inhibited by chloroquine, but not by calcium chelator BAPTA or inhibitor of endosomal acidification concamycin B. Chloroquine also protected mice from heme inflammatory effects in vivo, inhibiting lethal synergism with PRR agonists, lung pathology caused by heme injection after intratracheal instillation of Poly-IC, and delaying death after rhabdomyolisis. CONCLUSION: Our data indicate that chloroquine might be used as a supportive therapy to control heme-induced deleterious inflammation in different hemolytic diseases.

Immune-related redox metabolism of embryonic cells of the tick Rhipicephalus microplus (BME26) in response to infection with Anaplasma marginale.

BACKGROUND: It is well known that reactive oxygen species (ROS) and reactive nitrogen species (RNS) are involved in the control of pathogens and microbiota in insects. However, the knowledge of the role of ROS and RNS in tick-pathogen and tick-microbiota interactions is limited. Here, we evaluated the immune-related redox metabolism of the embryonic cell line BME26 from the cattle tick Rhipicephalus microplus in response to Anaplasma marginale infection. METHODS: A high-throughput qPCR approach was used to determine the expression profile of 16 genes encoding proteins involved in either production or detoxification of ROS and RNS in response to different microbial challenges. In addition, the effect of RNAi-mediated gene silencing of catalase, glutathione peroxidase, thioredoxin and protein oxidation resistance 1 in the control of infection with A. marginale was evaluated. RESULTS: Infection with A. marginale resulted in downregulation of the genes encoding ROS-generating enzymes dual oxidase and endoplasmic reticulum oxidase. In contrast, the genes encoding the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, thioredoxin, thioredoxin reductase and peroxiredoxin were upregulated. The gene expression pattern in response to infection with Rickettsia rickettsii and exposure to heat-killed microorganisms, Micrococcus luteus, Enterobacter cloacae or S. cerevisiae was the opposite of that triggered by A. marginale challenge. The simultaneous silencing of three genes, catalase, glutathione peroxidase, and thioredoxin as well as the oxidation resistance 1 gene by RNAi apparently favoured the colonization of BME26 cells by A. marginale, suggesting that the antioxidant response might play a role in the control of infection. CONCLUSIONS: Taken together, our results suggest that a general response of tick cells upon microbial stimuli is to increase ROS/RNS production. In contrast, A. marginale infection triggers an opposite profile, suggesting that this pathogen might manipulate the tick redox metabolism to evade the deleterious effect of the oxidant-based innate immune response.

Acquisition of exogenous haem is essential for tick reproduction.

Haem and iron homeostasis in most eukaryotic cells is based on a balanced flux between haem biosynthesis and haem oxygenase-mediated degradation. Unlike most eukaryotes, ticks possess an incomplete haem biosynthetic pathway and, together with other (non-haematophagous) mites, lack a gene encoding haem oxygenase. We demonstrated, by membrane feeding, that ticks do not acquire bioavailable iron from haemoglobin-derived haem. However, ticks require dietary haemoglobin as an exogenous source of haem since, feeding with haemoglobin-depleted serum led to aborted embryogenesis. Supplementation of serum with haemoglobin fully restored egg fertility. Surprisingly, haemoglobin could be completely substituted by serum proteins for the provision of amino-acids in vitellogenesis. Acquired haem is distributed by haemolymph carrier protein(s) and sequestered by vitellins in the developing oocytes. This work extends, substantially, current knowledge of haem auxotrophy in ticks and underscores the importance of haem and iron metabolism as rational targets for anti-tick interventions.

Atividade da catalase no pulmão, rim e intestino delgado não isquemiado de ratos após reperfusão intestinal / Catalase activity in lung, kidney and small bowel non-ischemic in rats after intestinal reperfusion

OBJETIVO: Avaliar a atividade catalase, após lesão por isquemia e reperfusão intestinal e estudar as alterações deste antioxidante em órgãos situados à distância do insulto inicial. MÉTODOS: Utilizaram-se 18 ratos do tipo Wistar, aleatoriamente distribuídos em três grupos. 1-Controle, 2-Simulação e 3-Isquemia/Reperfusão. Neste último, realizou-se isquemia no íleo, por 60 minutos, seguida de reperfusão por 30 minutos. No grupo 2 efetuou-se apenas uma laparotomia. Foram retirados, de todos os animais, segmentos do intestino com e sem reperfusão, além do pulmão e rim direitos para exame com microscopia óptica. A atividade da catalase foi aferida em espectrofotômetro ajustado para 240 nm. Utilizaram-se os testes estatísticos Mann e Whitney e Kruskal Wallis. RESULTADOS: Observou-se aumento significante (p < 0.05), da atividade da catalase nas porções do intestino isquemiado e não isquemiado, além do pulmão. Houve redução da atividade enzimática no rim. No grupo com reperfusão observaram-se alteração nas vilosidades, infiltrado inflamatório em todas as vísceras, além de áreas de atelectasia pulmonar. CONCLUSÃO: O estresse oxidativo intestinal, em ratos, causa alterações bioquímicas à distância com mobilização dos mecanismos de defesa antioxidante pulmonar, em segmento intestinal não isquemiado e no rim, com esgotamento precoce das reservas deste último, no entanto, sem lesão celular relevante, destas vísceras.

OBJECTIVE: This study aimed to assess the catalase activity after ischemia and reperfusion and to study the changes of this antioxidant in organs located far from the initial insult. METHODS: Eighteen Wistar rats were randomly divided into three groups. 1-Control, 2-Simulation and 3-Ischemia and Reperrfusion. In the latter it was done an ischemia of the ileum for 60 minutes followed by reperfusion for 30 minutes. In group 2 only laparotomy was performed. From all animals it was taken segments of the reperfused and non reperfused intestine, as well of the right kidney and lung to be evaluated under light microscopy. Catalase activity was measured in spectrophotometer with a wavelength set to 240 nm. It was used Mann Whitney and Kruskal Wallis statistical tests. RESULTS: There was a significant increase (p <0.05) in the catalase activity not only at small bowel ischemic and non-ischemic segments but also at lungs. However the enzymatic activity decreases in the kidney. In all organs studied at reperfusion group it was found a slight villi derangement, mild congestion and infiltration with inflammatory cells, and areas of pulmonary atelectasis. CONCLUSION: The intestinal oxidative stress in rats causes biochemical changes at distance, with mobilization of antioxidant defense mechanisms in lung, non-ischemic intestinal segment and kidney, with early decrease in this last organ, however, with no relevant cellular damage.

Oxidized tissue proteins after intestinal reperfusion injury in rats

OBJETIVO: Analisar se a mensuração da proteína carbonilada pode ser validada como método capaz de permitir a identificação de um estresse oxidativo intestinal causado por lesões decorrentes da isquemia e reperfusão. MÉTODOS: Vinte e cinco ratos machos da linhagem Wistar, pesando entre 200 e 250g, foram divididos em três grupos. Grupo I – controle (n = 10). Grupo II – simulação (n = 5) e grupo III (n = 10) submetido a 60 minutos de isquemia intestinal e igual intervalo para reperfusão. Para este fim clampeou-se a artéria mesentérica superior no seu terço distal. Alterações histológicas e os níveis de proteínas carboniladas foram determinados em amostras obtidas em todos os grupos. No grupo III foram estudados segmentos ileais reperfundidos e normais. RESULTADOS: Em todos os segmentos reperfundidos houve edema da mucosa e submucosa, além de infiltrado inflamatório da lâmina própria. Os níveis de proteína carbonilada aumentaram no grupo III, inclusive nos segmentos não isquemiados. A sensibilidade e a especificidade da proteína carbonilada no tecido foram, respectivamente, de 94% e 88%. CONCLUSÃO: O procedimento da proteína carbonilada é útil como marcador biológico do estresse oxidativo após isquemia e reperfusão intestinal em ratos. Também foi relevante o efeito do estresse oxidativo, observado à distância do lócus da lesão primária.

Oxidized tissue proteins after intestinal reperfusion injury in rats.

PURPOSE: To analyse if the carbonyl proteins measurement could be validated as a method that allows the identification of an intestinal oxidative stress after ischemia and reperfusion injury. METHODS: Twenty-five male Wistar rats (n = 21) weighting 200 to 250 g were divided into three groups. Group I--control (n = 10). Group II--sham (n = 5) and Group III (n = 10) subjected to 60 minutes of intestinal ischemia and equal period of reperfusion. For this purpose it was clamped the superior mesenteric artery in its distal third. Histological changes and carbonyl protein levels were determined in the samples of all groups. In group III, samples of both normal and reperfused ileal segment were studied. RESULTS: All the reperfused segments showed mucosal and submucosal swelling and inflammatory infiltrate of the lamina propria. Levels of carbonyl protein rose in group III, including in the non-ischemic segments. The sensitivity and specificity of the carbonyl protein tissue levels were respectively 94% and 88%. CONCLUSION: The carbonyl protein method is a useful biologic marker of oxidative stress after the phenomenon of intestinal ischemia and reperfusion in rats. It was also noteworthy that the effects of oxidative stress could be seen far from the locus of the primary injury.

The roles of haemolymphatic lipoproteins in the oogenesis of Rhodnius prolixus

The fates of purified 32P-vitellin and 32P-lipophorin were followed in vitellogenic females of Rhodnius prolixus. While the radioactivity from 32P-vitellin 6 hours after injection was found almost exclusively in the ovary, the radioactivity from injected 32P-lipophorin was found distributed among several organs. In the ovary, the radioactivity from 32P-vitellin was associated with the contents of the yolk granules. 32P-lipophorin delivered a great amount of radioactive phospholipids to the ovary with no accumulation of its protein moiety, as observed after its iodination with 131I. The delivery of phospholipids was inhibited at 0ºC and by the metabolic inhibitors, sodium azide and sodium fluoride. Comparison of the radioactivity incorporation from 32P-lipophorin with that of 14C-inulin suggests that the 32P-phospholipids from lipophorin are not taken up by fluid phase endocytosis. The data presented here are compatible with the concept of lipophorin as a carrier of lipids in insects and provide evidence that lipophorin transports phospholipids as shown previously for other classes of lipids. The utilization by the oocytes of the phospholipids transported by lipophorin is discussed.