Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1, a novel neuronal protein that regulates energy balance

To identify genes involved in the central regulation of energy balance, we compared hypothalamic mRNA from lean and obese Psammomys obesus, a polygenic model of obesity, using differential display PCR. One mRNA transcript was observed to be elevated in obese, and obese diabetic, P. obesus compared with lean animals and was subsequently found to be increased 4-fold in the hypothalamus of lethal yellow agouti (Ay/a) mice, a murine model of obesity and diabetes. Intracerebroventricular infusion of antisense oligonucleotide targeted to this transcript selectively suppressed its hypothalamic mRNA levels and resulted in loss of body weight in both P. obesus and Sprague Dawley rats. Reductions in body weight were mediated by profoundly reduced food intake without a concomitant reduction in metabolic rate. Yeast two-hybrid screening, and confirmation in mammalian cells by bioluminescence resonance energy transfer analysis, demonstrated that the protein it encodes interacts with endophilins, mediators of synaptic vesicle recycling and receptor endocytosis in the brain. We therefore named this transcript Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1 (SGIP1). SGIP1 encodes a large proline-rich protein that is expressed predominantly in the brain and is highly conserved between species. Together these data suggest that SGIP1 is an important and novel member of the group of neuronal molecules required for the regulation of energy homeostasis.

Functional and metabolic remodelling in GLUT4-deficient hearts confers hyper-responsiveness to substrate intervention

Impaired glucose uptake is associated with both cardiac hypertrophy and contractile dysfunction, but whether there are common underlying  mechanisms linking these conditions is yet to be determined. Using a ‘gene dose’ Cre-Lox GLUT4-deficient murine model, we examined the effect of suppressed glucose availability on global myocardial gene expression and glycolysis substrate bypass on the function of isolated perfused hearts. Performance of hearts from 22- to 60-week-old male GLUT4 knockout (KO, > 95% reduction in GLUT4), GLUT4 knockdown (KD, 85% reduction in cardiac GLUT4) and C57Bl/6 wild-type (WT) controls was measured ex vivo in Langendorff mode perfusion. DNA microarray was used to profile mRNA expression differences between GLUT4-KO and GLUT4-KD hearts. At 22 weeks, GLUT4-KO hearts exhibited cardiac hypertrophy and impaired contractile function ex vivo, characterized by a 40% decrease in developed pressure. At 60 weeks, dysfunction was accentuated in GLUT4-KO hearts and evident in GLUT4-KD hearts. Exogenous pyruvate (5 mM) restored systolic pressure to a level equivalent to WT (GLUT4-KO, 176.8 ± 13.2 mmHg vs. WT, 146.4 ± 9.56 mmHg) in 22-week-old GLUT4-KO hearts but not in 60-week-old GLUT4-KO hearts. In GLUT4-KO, DNA microarray analysis detected downregulation of a number of genes centrally involved in mitochondrial oxidation and upregulation of other genes indicative of a shift to cytosolic β-oxidation of long chain fatty acids. A direct link between cardiomyocyte GLUT4 deficiency, hypertrophy and contractile dysfunction is demonstrated. These data provide mechanistic insight into the myocardial metabolic adaptations associated with short and long-term insulin resistance and indicate a window of opportunity for substrate intervention and functional ‘rescue’.

Generation of recombinant influenza A viruses lacking immunodominant CD8+ T cell epitopes

The induction of a cytotoxic T lymphocyte (CTL) response following influenza infection can lead to the formation of immunity capable of recognizing viruses of a different antigenicity. Our ability to exploit such broadly reactive responses in vaccination strategies is hampered by a lack of understanding on the regulation of CTL responses. In this report, we describe the utilization of reverse genetics to produce a range of recombinant viruses lacking immunodominant murine CTL epitopes. Recombinant viruses lacking the epitopes had indistinguishable growth properties in vitro and in vivo compared with the wild-type virus. Analysis of a primary immune response to these viruses showed that mutation of the anchor-binding residue leads to a loss of a response to that epitope, but no compensating increase in responses to other immunodominant epitopes. The utilization of reverse genetics and the murine model of influenza infection hold great promise for elucidating the factors regulating the CTL response.

Cigarette smoke-induced changes to alveolar macrophage phenotype and function are improved by treatment with procysteine

Defective efferocytosis may perpetuate inflammation in smokers with or without chronic obstructive pulmonary disease (COPD). Macrophages may phenotypically polarize to classically activated M1 (proinflammatory; regulation of antigen presentation) or alternatively activated M2 (poor antigen presentation; improved efferocytosis) markers. In bronchoalveolar lavage (BAL)–derived macrophages from control subjects and smoker/ex-smoker COPD subjects, we investigated M1 markers (antigen-presenting major histocompatibility complex [MHC] Classes I and II), complement receptors (CRs), the high-affinity Fc receptor involved with immunoglobulin binding for phagocytosis (Fc-gamma receptor, FcγR1), M2 markers (dendritic cell–specific intercellular adhesion molecule-grabbing nonintegrin [DC-SIGN] and arginase), and macrophage function (efferocytosis and proinflammatory cytokine production in response to LPS). The availability of glutathione (GSH) in BAL was assessed, because GSH is essential for both M1 function and efferocytosis. We used a murine model to investigate macrophage phenotype/function further in response to cigarette smoke. In lung tissue (disaggregated) and BAL, we investigated CRs, the available GSH, arginase, and efferocytosis. We further investigated the therapeutic effects of an oral administration of a GSH precursor, cysteine l-2-oxothiazolidine-4-carboxylic acid (procysteine). Significantly decreased efferocytosis, available GSH, and M1 antigen–presenting molecules were evident in both COPD groups, with increased DC-SIGN and production of proinflammatory cytokines. Increased CR-3 was evident in the current-smoker COPD group. In smoke-exposed mice, we found decreased efferocytosis (BAL and tissue) and available GSH, and increased arginase, CR-3, and CR-4. Treatment with procysteine significantly increased GSH, efferocytosis (BAL: control group, 26.2%; smoke-exposed group, 17.66%; procysteine + smoke-exposed group, 27.8%; tissue: control group, 35.9%; smoke-exposed group, 21.6%; procysteine + smoke-exposed group, 34.5%), and decreased CR-4 in lung tissue. Macrophages in COPD are of a mixed phenotype and function. The increased efferocytosis and availability of GSH in response to procysteine indicates that this treatment may be useful as adjunct therapy for improving macrophage function in COPD and in susceptible smokers.

Role of increased CD8/CD28null T cells and alternative co-stimulatory molecules in chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory lung disease; it is a leading cause of death and existing treatments have no proven disease-modifying effect. The mechanisms underlying this resistance are largely unknown, but suggest the presence of some self-maintaining pathogenic process, possibly initiated by cigarette smoking, that prevents the normal resolution of inflammation. We have previously reported increased production of proinflammatory cytokines and granzyme b by CD8⁺ T cells in COPD; costimulatory receptor/ligand interactions required include CD80:86/CD28, B7-1/CTLA4, 4-1BB/1BBL and OX40/OX40L. We hypothesized that a dysregulated expression/function of these molecules may play a role in inflammatory/autoimmune components of COPD. We analysed T cell co-stimulatory molecules in blood from 34 controls, 15 smokers and 48 COPD subjects. We assessed the potential functional relevance of CD8/CD28^null cells in COPD by measuring their production of proinflammatory cytokines, co-stimulatory molecules, granzyme and perforin. A smoke-exposed murine model was applied to investigate the relative expression of CD8/CD28^null T cells in blood, lung tissue and airway. CD8/CD28^null cells were increased in both current- and ex-smoker COPD groups; these cells expressed significantly more interferon (IFN)-γ, OX40, 4-1BB, CTLA4, granzyme and perforin when stimulated than CD8/CD28⁺ T cells. There were no changes in CD4/CD28^null T cells. In mice exposed to cigarette smoke for 12 weeks, CD8/CD28^null T cells were significantly increased in the airway with a trend for an increase in lung tissue and blood. Increased production of proinflammatory cytokines and expression of alternative co-stimulatory molecules by CD8/CD28^null T cells may play a role in inflammatory or autoimmune responses in COPD and identify therapeutic targets.

Zinc and zinc transporters in macrophages and their roles in efferocytosis in COPD

Our previous studies have shown that nutritional zinc restriction exacerbates airway inflammation accompanied by an increase in caspase-3 activation and an accumulation of apoptotic epithelial cells in the bronchioles of the mice. Normally, apoptotic cells are rapidly cleared by macrophage efferocytosis, limiting any secondary necrosis and inflammation. We therefore hypothesized that zinc deficiency is not only pro-apoptotic but also impairs macrophage efferocytosis. Impaired efferocytic clearance of apoptotic epithelial cells by alveolar macrophages occurs in chronic obstructive pulmonary disease (COPD), cigarette-smoking and other lung inflammatory diseases. We now show that zinc is a factor in impaired macrophage efferocytosis in COPD. Concentrations of zinc were significantly reduced in the supernatant of bronchoalveolar lavage fluid of patients with COPD who were current smokers, compared to healthy controls, smokers or COPD patients not actively smoking. Lavage zinc was positively correlated with AM efferocytosis and there was decreased efferocytosis in macrophages depleted of Zn in vitro by treatment with the membrane-permeable zinc chelator TPEN. Organ and cell Zn homeostasis are mediated by two families of membrane ZIP and ZnT proteins. Macrophages of mice null for ZIP1 had significantly lower intracellular zinc and efferocytosis capability, suggesting ZIP1 may play an important role. We investigated further using the human THP-1 derived macrophage cell line, with and without zinc chelation by TPEN to mimic zinc deficiency. There was no change in ZIP1 mRNA levels by TPEN but a significant 3-fold increase in expression of another influx transporter ZIP2, consistent with a role for ZIP2 in maintaining macrophage Zn levels. Both ZIP1 and ZIP2 proteins were localized to the plasma membrane and cytoplasm in normal human lung alveolar macrophages. We propose that zinc homeostasis in macrophages involves the coordinated action of ZIP1 and ZIP2 transporters responding differently to zinc deficiency signals and that these play important roles in macrophage efferocytosis.

Avaliação da atividade antimalárica de extratos obtidos de algas marinhas no litoral do Rio Grande do Norte

Malaria is a major parasitic disease worldwide, accounting for about 500 million cases and causing 2 million to 3 million deaths annually. Four species are responsible for transmitting this disease to humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale. The parasite resistance to antimalarial drugs and the usual limitations of the vector control implications are contributing to the spread of the disease. The most of significant advances in the search for new antimalarial drugs is based on natural components, the main ones being currently used antimalarial drugs derived from plants. Research on natural products of marine origin (particularly algae) show that some species possess antiplasmodial activity. Knowing that the coast of Rio Grande do Norte is home to several species of algae, the present study was to evaluate, for the first time, the antimalarial activity of ethanolic extracts of seaweed Spatoglossum schroederi, Gracilaria birdiae and Udotea flabellum against Plasmodium falciparum 3D7 strain tests and in vitro using the murine model (Plasmodium berghei) for evaluation in vivo. These species were ground, macerated with ethanol for 24 hours and the extracts concentrated in rotaevaporador (45 ° C ± 5 ° C). For in vitro tests, the extracts were diluted and tested at concentrations between 100 and 1.56 μg/ml (seven concentrations in triplicate), in order to obtain IC50 of each extract. The cytotoxicity tests with macrophages and BGM were performed using the MTT colorimetric assay. BGM macrophages and cells were distributed in 96 wells per plate (1x 105 to macrophages and 1x104 cells per well for BGM) and incubated for 24h at 37 ° C. The ethanol extracts were diluted and tested at concentrations of 100 to 1,56 μg/ml (seven concentrations in triplicate). After periods of 24 hours of incubation with the extracts, 100 μg of MTT was added to each well, and 3 hours elapsed, the supernatant was removed and added 200 μl of DMSO in each well. The absorbance of each well was obtained by reading on a spectrophotometer at 570 nm filter. To evaluate the acute toxicity in vivo, Swiss mice received a single dose (oral) 2000 mg/kg/animal of each extract tested. The parameters of acute toxicity were observed for 8 days. For in vivo tests, Swiss mice were inoculated with 1x105 erythrocytes infected with P. berghei. The treatment was given first to fourth day after infection with 0.2 ml of the extracts in doses of 1000 and 500 mg//g animal. The negative control group received 0.2 ml of 2% Tween-20, whereas the positive control group received sub-dose of chloroquine (5 mg/kg/animal). The assessment of antimalarial activity was done by suppressing suppressing the parasitemia at 5 and 7 days after infection. The growth inhibition of parasites was determined relative to negative control (% inhibition = parasitaemia in control - parasitemia in sample / parasitemia control x 100), the mortality of animals was monitored daily for 30 days The results showed that algae Spatoglossum schroederi and Udotea flabellum showed antimalarial activity in vitro, with reduced parasitemia of 70.54% and 54, respectively. The extracts of the three algae tested showed moderate to high cytotoxicity. Algae S. schroederi and U. flabellum were active against P. berghei only at doses of 500 mg / kg with reduction ranging from 54.58 to 52.65% for the fifth day and from 32.24 to 47.34% for the seventh day, respectively. No toxicity was observed in vivo at the dose tested, over the 8 days of observation. Although preliminary data, the bioactive components in those possible seaweed may be promising for the development of new anti-malarial drugs

Efeito antiinflamatório de fucana extraída da alga Parda spatoglossum schroederii em modelos experimentais de Peritonite, choque não séptico e colite

Fucans is a name used for sulfated polysaccharides, which is most characteristic structure of the presence of sulfated L-fucose, are found in brown seaweed (Phaeophyceae) and echinoderms (sea urchins and sea cucumbers). These polysaccharides have been reported to possess anticoagulant, antitumor, anti-viral, anti-proliferative and anti-inflammatory activities. Therefore, in the present study was evaluate the effect of the fucan from the brown seaweed Spatoglossum schroederii in models of peritonitis and non-septic shock induced by zymosan, as well as in a murine model of colitis induces by DSS. So, the mice treatment by intravenous route with the fucan was able to reduce the exudate formation and the cell migration in the model of acute peritonitis induced by zymosan during the kinetic of 6, 24 and 48 hours. Similarly, in the model of non-septic shock induced by zymosan the fucan demonstrated a protector effect to inhibited the cellular migration to the peritoneo, to decrease the levels of IL-6 in the serum and in the peritoneal exudate, to attenuate the lose of weight in the mice; beside to reduce the serum levels of hepatic transaminases and as well as the liver injury. In the model of murine colitis, the treatment with the fucan reduced the lose of weight of the animals, decreased the levels of IL-17 and IFN- produced in the gut and decrease the intestinal lesion induced by DSS. In conclusion, the fucan used in this study presented a significant protector effect in the murine models of inflammation

Efeitos do potencial probiótico Enterococcus faecium 32 e de sua associação com o extrato da alga marinha Caulerpa mexicana no tratamento de colite experimental murina

Ulcerative colitis comprising an inflammatory bowel disease, whose most severe consequence is the development of intestinal neoplasia. The drugs currently used to treat the disease trigger a variety of serious adverse effects and are not effective in many cases. Recent studies demonstrated the effectiveness of natural products for the treatment of inflammatory processes. Seaweed extracts and their purified products have shown protective effects in models of inflammation and the association of traditional therapies with probiotics has significantly improved the clinical symptoms of ulcerative colitis. Therefore, the aims of this study include evaluating the potential effects of the use of probiotic strain Enterococcus faecium 32 (Ef32), the methanolic extract of the green seaweed Caulerpa mexicana (M.E.) and their concomitant administration in a murine model of colitis induced by dextran sodium sulfate (DSS). Accordingly, C57BL /6 mice were pretreated orally with Ef32 (109 CFU/ml) for seven days. In the seven days following, the colitis was induced by administration of 3% DSS (w/v) diluted in the animals drinking water. During this period, animals were treated daily with Ef32 and the M.E. (2.0 mg/kg) every other day by intravenous route. The development of colitis was monitored by the disease activity index (DAI), which takes into account the loss of body weight, consistency and presence of blood in stools. After euthanasia, the colon was removed, its length measured and tissue samples were destined for histological analysis and culture for cytokine quantification. The levels of cytokines in the culture supernatant of the colon were measured by ELISA. The treatments with the probiotic Ef32 or the M.E. alone or the combination of these two substances provoked significant improvement as to weight loss and DAI, and prevented the shortening of the colon in response to DSS. The isolated treatments triggered a slight improvement in intestinal mucosal tissue damage. However, their combination was able to completely repair the injury triggered by DSS. The association was also able to reduce the levels of all the cytokines analyzed (IFN-γ, IL-4, IL-6, IL-12, IL-17A and TNF-α). On the other hand, the treatment with Ef32 did not interfere with the levels of TNF-α, whereas treatment with M.E. did not alter the levels of IL-6. Moreover, the treatment with Ef32 not interferes in TNF-α levels, whereas treatment with M.E. did not alter the levels of IL-6. Therefore, the potential probiotic Ef32 and M.E. and especially when these samples were associated proved promising alternatives in the treatment of ulcerative colitis as demonstrated in an experimental model because of its beneficial effects on morphological and clinical parameters, and by reducing the production of proinflammatory cytokines of Th1, Th2 and Th17

Experimental paracoccidioidomycosis in the mouse. III. Histopathological and immunological findings after intravenous infection in the presence or absence of previous immunization

Cinqüenta camundongos suíços, brancos, com quatro semanas de idade, foram inoculados com 5x10(5) formas leveduriformes, viáveis de Paracoccidiodes brasiliensis (cepa 18). Dez destes animais tinham sido previamente imunizados com antígeno particulado de P. brasiliensis, durante quatro semanas, por injeção intradérmica. Os controles consistiram de 10 animais que foram somente imunizados e 10 inoculados com solução salina estéril. Os animais foram sacrificados após 2, 4, 7, 11 e 16 semanas. Estudamos: 1) resposta de hipersensibilidade retardada medida pelo teste do coxim plantar, 24 horas antes do sacrifício; 2) anticorpo- gênese específica avaliada pelo teste de imunodifusão dupla em gel de ágar; 3) histopatologia dos pulmões, fígado, baço, supra-renal e rins. Observamos: 1) os animais imunizados desenvolveram resposta imunecelular mais intensa que os infectados; 2) a infecção deprimiu a resposta imunecelular dos animais imunizados; 3) a histopatologia da infecção endovenosa revelou inflamação granulomatosa sistêmica e progressiva. Os animais infectados após imunização prévia apresentaram inflamação pulmonar menos extensa, com granulomas menores e com reduzido número de fungos. O presente modelo murino de paracoccidioidomicose mimetiza alguns achados da forma humana subaguda da micose (doença sistêmica com depressão da imunidade celular). O esquema de imunização extrapulmonar utilizado foi capaz de induzir certo grau de proteção do pulmão contra um desafio infeccioso pelo P. brasiliensis.

Protective effects of Tacrolimus, a calcineurin inhibitor, in experimental periodontitis in rats

Objective: Periodontitis is a well-appreciated example of leukocyte-mediated bone loss and inflammation with pathogenic features similar to those observed in other inflammatory diseases, such as arthritis. Since Tacrolimus, is an immunomodulatory drug used for the treatment of some cases of arthritis, we hypothesized that it may modulate periodontal disease.Design: Using a murine model of ligature-induced periodontal disease, we assessed the effects of daily administrations of Tacrolimus (1 mg/kg body weight) on bone loss, enzymatic (myeloperoxidase) analysis, differential white blood cells counts, airpouch exudate and cytokine expression for 5-30 days.Results: Radiographic, enzymatic (myeloperoxidase) and histological analysis revealed that Tacrolimus reduced the severity of periodontitis. More specifically, Tacrolimus suppressed the expression of serum interleukin (IL-1 beta), tumour necrosis factor (TNF-alpha), IL-6, airpouch exudate PGE(2) and leukocytosis usually observed after the induction of periodontitis. Tacrolimus treatment in periodontitis-induced rats conferred protection against the inflammation-induced tissue and bone loss associated with periodontitis, through a mechanism involving IL-1 beta, TNF-alpha and IL-6.Conclusions: the effects of Tacrolimus on periodontal disease pathogenesis may provide clues to a novel approach to host modulation therapy in destructive periodontal disease. (C) 2007 Elsevier Ltd. All rights reserved.

Intermittent Therapy with 1,25 Vitamin D and Calcitonin Prevents Cyclosporin-Induced Alveolar Bone Loss in Rats

Bone loss associated with cyclosporin A (CsA) therapy can result in serious morbidity to patients. Intermittent administration of 1,25 Vitamin D and calcitonin reduces osteopenia in a murine model of postmenopausal osteoporosis. The purpose of this study was to evaluate the effects of this therapeutic approach on CsA-induced alveolar bone loss in rats. Forty male Wistar rats were allocated to four experimental groups according to the treatment received during 8 weeks: (1) CsA (10 mg/kg/day, s.c.); (2) 1,25 Vitamin D (2 mu g/kg, p.o.; in weeks 1, 3, 5, and 7) plus calcitonin (2 mu g/kg, i.p.; in weeks 2, 4, 6, and 8); (3) CsA concurrently with intermittent 1,25 Vitamin D and calcitonin administration; and (4) the control treatment group (vehicle). At the end of the 8-week treatment period, serum concentrations of bone-specific alkaline phosphatase, tartrate-resistant acid phosphatase (TRAP-5b), osteocalcin, interleukin (IL)-1 beta, IL-6, and tumor necrosis factor alpha (TNF-alpha) were measured and an analysis of bone volume, bone surface, number of osteoblasts, and osteoclasts was performed. CsA administration resulted in significant alveolar bone resorption, as assessed by a lower bone volume and an increased number of osteoclasts, and increased serum bone-specific alkaline phosphatase, TRAP-5b, IL-1 beta, IL-6, and TNF-alpha concentrations. The intermittent administration of calcitriol and calcitonin prevented the CsA-induced osteopenic changes and the increased serum concentrations of TRAP-5b and inflammatory cytokines. Intermittent calcitriol/calcitonin therapy prevents CsA-induced alveolar bone loss in rats and normalizes the production of associated inflammatory mediators.

Functional Characterization of an Aspergillus fumigatus Calcium Transporter (PmcA) that Is Essential for Fungal Infection

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Recent mouse and rat methods for the study of experimental oral candidiasis

The Candida genus expresses virulence factors that, when combined with immunosuppression and other risk factors, can cause different manifestations of oral candidiasis. The treatment of mucosal infections caused by Candida and the elucidation of the disease process have proven challenging. Therefore, the study of experimentally induced oral candidiasis in rats and mice is useful to clarify the etiopathology of this condition, improve diagnosis, and search for new therapeutic options because the disease process in these animals is similar to that of human candidiasis lesions. Here, we describe and discuss new studies involving rat and mouse models of oral candidiasis with respect to methods for inducing experimental infection, methods for evaluating the development of experimental candidiasis, and new treatment strategies for oral candidiasis. © 2013 Landes Bioscience.

Estudo in vivo dos efeitos da terapia fotodinâmica, mediada pelo Photothazine® e luz led, sobre Cândida Albicans resistente a Fluconazol

Pós-graduação em Reabilitação Oral - FOAR