Gabriela Freitas Pereira de Souza

Fractalkine (CX3CL1) Is Involved in the Early Activation of Hypothalamic Inflammation in Experimental Obesity

Hypothalamic inflammation is a common feature of experimental obesity. Dietary fats are important triggers of this process, inducing the activation of toll-like receptor-4 (TLR4) signaling and endoplasmic reticulum stress. Microglia cells, which are the cellular components of the innate immune system in the brain, are expected to play a role in the early activation of diet-induced hypothalamic inflammation. Here, we use bone marrow transplants to generate mice chimeras that express a functional TLR4 in the entire body except in bone marrow–derived cells or only in bone marrow–derived cells. We show that a functional TLR4 in bone marrow–derived cells is required for the complete expression of the diet-induced obese phenotype and for the perpetuation of inflammation in the hypothalamus. In an obesity-prone mouse strain, the chemokine CX3CL1 (fractalkine) is rapidly induced in the neurons of the hypothalamus after the introduction of a high-fat diet. The inhibition of hypothalamic fractalkine reduces diet-induced hypothalamic inflammation and the recruitment of bone marrow–derived monocytic cells to the hypothalamus; in addition, this inhibition reduces obesity and protects against diet-induced glucose intolerance. Thus, fractalkine is an important player in the early induction of diet-induced hypothalamic inflammation, and its inhibition impairs the induction of the obese and glucose intolerance phenotypes.

Antithrombogenic Polynitrosated Polyester/Poly(methyl methacrylate) Blend for the Coating of Blood‐Contacting Surfaces

A nitric oxide (NO) donor polyester containing multiple S-nitrosothiol (S-NO) groups covalently attached to the polymer backbone was synthesized through the esterification of poly(ethylene glycol) with mercaptosuccinic acid, followed by the nitrosation of the -SH moieties. The polynitrosated polyester (PNPE) obtained was blended with poly(methyl methacrylate) (PMMA), yielding solid films capable of releasing NO. Scanning electron microscopy analysis showed that acrylic plates and stainless steel intracoronary stents can be coated with continuous and adherent PNPE/PMMA films. After an initial NO burst, these films release NO spontaneously in dry condition or immersed in aqueous solution at constant rates of 1.8 and 180 nmol/g/h, respectively, for more than 24 h at physiological temperature. PNPE/PMMA coated surfaces were shown to inhibit platelet adhesion when in contact with whole blood. These results show that PNPE/PMMA blend can be used for the coating of blood-contacting surfaces, with potential to inhibit thrombosis and restenosis after stenting.

n-3 Fatty Acids Induce Neurogenesis of Predominantly POMC-Expressing Cells in the Hypothalamus.

Apoptosis of hypothalamic neurons is believed to play an important role in the development and perpetuation of obesity. Similar to the hippocampus, the hypothalamus presents constitutive and stimulated neurogenesis, suggesting that obesity-associated hypothalamic dysfunction can be repaired. Here, we explored the hypothesis that n-3 polyunsaturated fatty acids (PUFAs) induce hypothalamic neurogenesis. Both in the diet and injected directly into the hypothalamus, PUFAs were capable of increasing hypothalamic neurogenesis to levels similar or superior to the effect of brain-derived neurotrophic factor (BDNF). Most of the neurogenic activity induced by PUFAs resulted in increased numbers of proopiomelanocortin but not NPY neurons and was accompanied by increased expression of BDNF and G-protein–coupled receptor 40 (GPR40). The inhibition of GPR40 was capable of reducing the neurogenic effect of a PUFA, while the inhibition of BDNF resulted in the reduction of global hypothalamic cell. Thus, PUFAs emerge as a potential dietary approach to correct obesity-associated hypothalamic neuronal loss.

S-nitrosoglutathione decreases inflammation and bone resorption in experimental periodontitis in rats.

BACKGROUND S-nitrosoglutathione (GSNO) is a nitric oxide donor that may exert antioxidant, anti-inflammatory, and microbicidal actions and is thus a potential drug for the topical treatment of periodontitis. In this study, the effect of intragingival injections of GSNO-containing polyvinylpyrrolidone (PVP) formulations is evaluated in a rat model of periodontitis. METHODS Periodontal disease was induced by placing a sterilized nylon (000) thread ligature around the cervix of the second left upper molar of the animals, which received intragingival injections of PVP; saline; or PVP/GSNO solutions which corresponded to GSNO doses of 25, 100, and 500 nmol; 1 hour before periodontitis induction, and thereafter, daily for 11 days. RESULTS PVP/GSNO formulations at doses of 25 and/or 100, but not 500 nmol caused significant inhibition of alveolar bone loss, increase of bone alkaline phosphatase, decrease of myeloperoxidase activity, as well as significant reduction of inflammatory and oxidative stress markers when compared to saline and PVP groups. These effects were also associated with a decrease of matrix metalloproteinases 1 and 8, inducible nitric oxide synthase, and nuclear factor-κB immunostaining in the periodontium. CONCLUSION Local intragingival injections of GSNO reduces inflammation and bone loss in experimental periodontal disease.

S-nitrosoglutathione (GSNO) is cytotoxic to intracellular amastigotes and promotes healing of topically treated Leishmania major or Leishmania braziliensis skin lesions

We read with great interest the letter by Kö ser et al., 1 responding to our article on the characterization of the embB gene in the area of Barcelona. 2 The point that Kö ser et al. 1 raise is very interesting and deserves further comment. Codon 378 has indeed been described by several authors as a phylogenetic polymorphism not related to resistance. 3,4 However, in our opinion the role of this codon remains unclear, since some studies describe the existence of ethambutol-resistant isolates with a mutation only in this codon. 5,6 Even one work 7 cited by Kö ser et al. 1 includes two isolates with a single Glu378Ala substitution and a decreased susceptibility to ethambutol. The design of our microarray was based on the existing literature , taking into account all the possible embB codons that have been implicated in ethambutol resistance. We finally included the ones that were also found in our setting. The microarray system was not designed to determine the association of each mutation with the phenotypic resistance (allelic exchange experiments would be required for this purpose), but to reflect the variety of embB substitutions prevalent in our area. Moreover, the frequency of mutations in embB378 is low (,2% in our study), so we consider that the probability of misassigning a result of embB378 mutation to phenotypic resistance rather than to an epidemiological cause is negligible and does not compromise the effectiveness of the microarray. Regarding the isolates, we included a collection of Mycobacterium tuberculosis complex (MTBC) clinical isolates (not identified to the species level). Isolate 233R has now been analysed and identified as M. tuberculosis/Mycobacterium canettii. It contains the embB378Glu variant; therefore, a homoplastic event may be present (experimental error was ruled out). Likewise, the MIRU-VNTR genotyping was performed not for epidemiological purposes (i.e. for the identification of lineages), but to establish the real frequency of embB mutations among circulating MTBC isolates in our geographical area. Finally, we stress that the main objective of this study was to highlight the relevance of mutations in embB codons apart from embB306, focusing on codon 406, which represents 20% of the embB mutations in our area. Our results show that this target (embB406) should be included in any genotypic method for rapid ethambutol resistance detection. References 1 Kö ser CU, Bryant JM, Comas I et al. Comment on: Characterization of the embB gene in Mycobacterium tuberculosis isolates …

S-nitrosoglutathione inhibits inducible nitric oxide synthase upregulation by redox posttranslational modification in experimental diabetic retinopathy.

PURPOSE Diabetic retinopathy (DR) is associated with nitrosative stress. The purpose of this study was to evaluate the beneficial effects of S-nitrosoglutathione (GSNO) eye drop treatment on an experimental model of DR. METHODS Diabetes (DM) was induced in spontaneously hypertensive rats (SHR). Treated animals received GSNO eye drop (900 nM or 10 μM) twice daily in both eyes for 20 days. The mechanisms of GSNO effects were evaluated in human RPE cell line (ARPE-19). RESULTS In animals with DM, GSNO decreased inducible nitric oxide synthase (iNOS) expression and prevented tyrosine nitration formation, ameliorating glial dysfunction measured with glial fibrillary acidic protein, resulting in improved retinal function. In contrast, in nondiabetic animals, GSNO induced oxidative/nitrosative stress in tissue resulting in impaired retinal function. Nitrosative stress was present markedly in the RPE layer accompanied by c-wave dysfunction. In vitro study showed that treatment with GSNO under high glucose condition counteracted nitrosative stress due to iNOS downregulation by S-glutathionylation, and not by prevention of decreased GSNO and reduced glutathione levels. This posttranslational modification probably was promoted by the release of oxidized glutathione through GSNO denitrosylation via GSNO-R. In contrast, in the normal glucose condition, GSNO treatment promoted nitrosative stress by NO formation. CONCLUSIONS In this study, a new therapeutic modality (GSNO eye drop) targeting nitrosative stress by redox posttranslational modification of iNOS was efficient against early damage in the retina due to experimental DR. The present work showed the potential clinical implications of balancing the S-nitrosoglutathione/glutathione system in treating DR.

Defective regulation of POMC precedes hypothalamic inflammation in diet-induced obesity

Obesity is the result of a long-term positive energy balance in which caloric intake overrides energy expenditure. This anabolic state results from the defective activity of hypothalamic neurons involved in the sensing and response to adiposity. However, it is currently unknown what the earliest obesity-linked hypothalamic defect is and how it orchestrates the energy imbalance present in obesity. Using an outbred model of diet-induced obesity we show that defective regulation of hypothalamic POMC is the earliest marker distinguishing obesity-prone from obesity-resistant mice. The early inhibition of hypothalamic POMC was sufficient to transform obesity-resistant in obesity-prone mice. In addition, the post-prandial change in the blood level of β-endorphin, a POMC-derived peptide, correlates with body mass gain in rodents and humans. Taken together, these results suggest that defective regulation of POMC expression, which leads to a change of β-endorphin levels, is the earliest hypothalamic defect leading to obesity.

S-nitroso-N-acetylcysteine attenuates liver fibrosis in experimental nonalcoholic steatohepatitis

S-Nitroso-N-acetylcysteine (SNAC) is a water soluble primary S-nitrosothiol capable of transferring and releasing nitric oxide and inducing several biochemical activities, including modulation of hepatic stellate cell activation. In this study, we evaluated the antifibrotic activity of SNAC in an animal model of nonalcoholic steatohepatitis (NASH) induced in Sprague-Dawley rats fed with a choline-deficient, high trans fat diet and exposed to diethylnitrosamine for 8 weeks. The rats were divided into three groups: SNAC, which received oral SNAC solution daily; NASH, which received the vehicle; and control, which received standard diet and vehicle. Genes related to fibrosis (matrix metalloproteinases [MMP]-13, -9, and -2), transforming growth factor β-1 [TGFβ-1], collagen-1α, and tissue inhibitors of metalloproteinase [TIMP-1 and -2] and oxidative stress (heat-shock proteins [HSP]-60 and -90) were evaluated. SNAC led to a 34.4% reduction in the collagen occupied area associated with upregulation of MMP-13 and -9 and downregulation of HSP-60, TIMP-2, TGFβ-1, and collagen-1α. These results indicate that oral SNAC administration may represent a potential antifibrotic treatment for NASH.

Bactericidal effect of S-nitrosothiols against clinical isolates from keratitis

Background The purpose of this study was to evaluate the antimicrobial activity of two nitric oxide donors, ie, S-nitrosoglutathione (GSNO) and S-nitroso-N-acetylcysteine (SNAC), against clinical isolates from patients with infectious keratitis. Methods Reference broth microdilution assays were performed to determine the minimum inhibitory and bactericidal concentrations for GSNO and SNAC against four American Type Culture Collection strains and 52 clinical isolates from patients with infectious keratitis as follows: 14 (26.9%) Pseudomonas species; 13 (25.0%) coagulase-negative Staphylococci; 10 (19.2%) Staphylococcus aureus; nine (17.3%) Serratia marcescens; and six (11.5%) Enterobacter aerogenes. Sterility control and bacterial growth control were also performed. Results SNAC showed lower minimum inhibitory and bactericidal concentrations than GSNO for all clinical isolates from patients with infectious keratitis. For Gram-positive bacteria, mean minimum inhibitory and bactericidal concentrations were 2.1 ± 1.3 and 8.6 ± 3.8 mM for SNAC and 4.6 ± 3.2 and 21.5 ± 12.5 mM for GSNO (P < 0.01). For Gram-negative bacteria, mean minimum inhibitory and bactericidal concentrations were 3.3 ± 1.4 and 6.1 ± 3.4 mM for SNAC and 12.4 ± 5.4 and 26.5 ± 10.1 mM for GSNO (P < 0.01). The minimum bactericidal to inhibitory concentration ratio was ≤8 in 100% of all isolates tested for SNAC and in 94.2% tested for GSNO. Conclusions SNAC and GSNO had effective inhibitory and bactericidal effects against bacterial isolates from keratitis. SNAC showed greater antimicrobial activity than GSNO against all bacteria. Gram-positive bacteria were more susceptible to the inhibitory and bactericidal effects of the S-nitrosothiols.

In vitro inhibition of linoleic acid peroxidation by primary S-nitrosothiols

Nitric oxide (•NO) is an effective chain-breaking antioxidant in the inhibition of lipid peroxidation and circulates in vivo mainly as primary S-nitrosothiols (RSNOs). In this work, the in vitro peroxidation of linoleic acid-SDS comicelles (LA-SDS) catalyzed by soybean lipoxygenase (SLO) and FeII ions was monitored in the presence and absence of three primary RSNOs: S-nitrosocysteine, S-nitroso-N-acetylcysteyne and S-nitrosoglutathione. Kinetic measurements based on the formation of conjugated double bonds and fluorescent oxidized LA-lysine adducts, showed that RSNOs are more potent antioxidants than their corresponding free thiols (RSHs) in equimolar conditions. These results are consistent with the blocking of LA-SDS peroxidation by RSNOs through the inactivation of peroxyl/alkoxyl (LOO•/LO•) radicals, leading to nitrogen-containing products of oxidized LA, which release free •NO. These results indicate that endogenous RSNOs may play a major role in the blocking of lipid peroxidation in vivo, through the primary inactivation of alkoxyl/peroxyl radicals and also of preformed lipid hydroperoxides.