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Sepsis is a syndrome of physiological and biochemical abnormalities induced by an infection that represents a major public health concern. It involves the early activation of inflammatory responses. Crotoxin (CTX), the major toxin of the South American rattlesnake Crotalus durissus terrificus venom, presents longstanding anti-inflammatory properties. Since immune system modulation may be a strategic target in sepsis management, and macrophages' functional and secretory activities are related to the disease's progression, we evaluated the effects of CTX on macrophages from septic animals. Balb/c male mice submitted to cecal ligation and puncture (CLP) were treated with CTX (0.9 μg/animal, subcutaneously) 1 h after the procedure and euthanized after 6 h. We used plasma samples to quantify circulating cytokines and eicosanoids. Bone marrow differentiated macrophages (BMDM) were used to evaluate the CTX effect on macrophages' functions. Our data show that CTX administration increased the survival rate of the animals from 40% to 80%. Septic mice presented lower plasma concentrations of IL-6 and TNF-α after CTX treatment, and higher concentrations of LXA

Cardiomyopathy is a well-known complication of sepsis that may deteriorate when accompanied by obesity. To test this hypothesis we fed C57black/6 male mice for 6 week with a high fat diet (60% energy) and submitted them to endotoxemic shock using E. coli LPS (10 mg/kg). Inflammatory markers (cytokines and adhesion molecules) were determined in plasma and heart tissue, as well as heart mitochondrial biogenesis and function. Obesity markedly shortened the survival rate of mouse after LPS injection and induced a persistent systemic inflammation since TNFα, IL-1β, IL-6 and resistin plasma levels were higher 24 h after LPS injection. Heart tissue inflammation was significantly higher in obese mice, as detected by elevated mRNA expression of pro-inflammatory cytokines (IL-1β, IL-6 and TNFα). Obese animals presented reduced maximum respiratory rate after LPS injection, however fatty acid oxidation increased in both groups. LPS decreased mitochondrial DNA content and mitochondria biogenesis factors, such as PGC1α and PGC1β, in both groups, while NRF1 expression was significantly stimulated in obese mice hearts. Mitochondrial fusion/fission balance was only altered by obesity, with no influence of endotoxemia. Obesity accelerated endotoxemia death rate due to higher systemic inflammation and decreased heart mitochondrial respiratory capacity.

Amyloid-β oligomers (AβOs) toxicity causes mitochondrial dysfunction, leading to synaptic failure in Alzheimer’s disease (AD). Considering presynaptic high energy demand and tight Ca2+ regulation, impairment of mitochondrial function can lead to deteriorated neural activity and cell death. In this study, an AD mouse model induced by ICV (intracerebroventricular) injection of AβOs was used to investigate the toxicity of AβOs on presynaptic function. As a therapeutic approach, GUO (guanosine) was given by oral route to evaluate the neuroprotective effects on this AD model. Following 24 h and 48 h from the model induction, behavioral tasks and biochemical analyses were performed, respectively. AβOs impaired object recognition (OR) short-term memory and reduced glutamate uptake and oxidation in the hippocampus. Moreover, AβOs decreased spare respiratory capacity, reduced ATP levels, impaired Ca2+ handling, and caused mitochondrial swelling in hippocampal synaptosomes. Guanosine crossed the BBB, recovered OR short-term memory, reestablished glutamate uptake, recovered mitochondrial Ca2+ homeostasis, and partially prevented mitochondrial swelling. Therefore, this endogenous purine presented a neuroprotective effect on presynaptic mitochondria and should be considered for further studies in AD models.

We investigate the effect of the banana green peels extract (BPE) as a preventive treatment against NAFLD in high-fat diet fed mice. Mice received daily doses of 100 or 250 mg/kg of BPE for 12 weeks along with the high-fat diet. BPE reduced weight gain (p .0001), adipose tissue hypertrophy (p .0001), and improved glucose homeostasis (p .0001). Plasma levels of glucose-dependent insulinotropic polypeptide, triglycerides, total cholesterol, LDL-cholesterol, non-esterified fatty acids, aspartate and alanine transaminase, leptin, and resistin were decreased in BPE treated mice (p .05). BPE effects on lipid metabolism were associated with decreased gene expression of lipogenic enzymes and increased expression of enzymes related to fatty acid and cholesterol degradation (p .05). Plasma and liver bile acid (BA) profiles were modulated by BPE, with positive correlations between specific BA and UCP-1, CPT-1 and PGC-1β expression in brown adipose tissue (p .05). BPE reduced hepatic steatosis and inflammation, possibly due to reduced p65 NF-κB nuclear translocation (p .05) and modulation of oxidative stress (p .05). These data indicate that BPE is a source of phytochemical compounds with promising effects toward the prevention of metabolic disorders associated with obesity.

Musa cavendish, commonly known as banana, is a fruit with nutritional and therapeutic properties. We investigated the chemical composition and in vitro cytotoxic effect of M. cavendish green peel extract (MHE) on cancer cells for the first time. The compounds characterization was performed by HPLC-UV/Vis and FIA-ESI-IT-MSn. We investigated in vitro cytotoxic effect of Musa cavendish green peels extract (MHE) in HepG2, A-375, MCF-7 and Caco-2 cancer cells. We evaluated the effect of MHE on proliferation of different cell lines through apoptosis, necrosis, mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) content determination. We identified 12 compounds from different classes in the extract, including derivatives of phenolic acids, aglycone flavonoids, glycoside flavonoids and catecholamines. Our results indicate that MHE exerts, after 48 h treatment, an accentuated antiproliferative effect from the dose of 100 μg/mL in all cell lines tested. In HepG2 cells, these effects were related to the induction of cell death, both necrotic and apoptotic, and remarkable changes in cell morphology. Depolarization of MMP and high ROS content were also observed in the cells in a dose-dependent manner. Our results show that MHE may be used as a source of new drugs with anticancer activity.

Purpose hyperammonemic encephalopathy is a potentially fatal condition associated with fibrolamellar hepatocellular carcinoma. The mechanism involved in hyperammonemia in patients with fibrolamellar carcinoma was unclear until a possible physiopathological pathway was recently proposed. An ornithine transcarboxylase dysfunction was suggested as a result of increased ornithine decarboxylase activity induced by c-Myc overexpression. This c-Myc overexpression resulted from Aurora Kinase A overexpression derived from the activity of a chimeric kinase that is the final transcript of a deletion in chromosome 19, common to all fibrolamellar carcinomas. Methods we performed the analysis of the expression of all enzymes involved and tested for the mutation in chromosome 19 in fresh frozen samples of fibrolamellar hepatocellular carcinoma, non-tumor liver and hepatic adenomatosis. Results specific DNAJB-PRKACA fusion protein that results from the recurrent mutation on chromosome 19 common to all fibrolamellar carcinoma was detected only in the fibrolamellar carcinoma sample. Fibrolamellar carcinoma and adenomiomatosis samples presented increased expression of Aurora Kinase A, c-MYC and ornithine decarboxylase when compared to normal liver, while ornithine transcarbamylase was decreased. Conclusion The proposed physiopathological pathway is correct and that overexpression of c-Myc may also be responsible of hyperammonemia in patients with other types of rapidly growing hepatomas. This gives further evidence to apply new and adequate treatment to this severe complication.

The global emergence of Covid-19 has caused huge human casualties. Clinical manifestations of the disease vary from asymptomatic to lethal, and the symptomatic form can be associated with cytokine storm and non-homeostatic inflammatory response. In face of the urgent demand for effective drugs to treat Covid-19, we have searched for candidate compounds using a drug repurposing approach based on in silico analysis followed by biological validation. Here we identified celastrol, a pentacyclic triterpene isolated from Tripterygium wilfordii Hook F – a plant used in traditional Chinese medicine – as one of the best compounds out of 39 repurposed drug candidates. Celastrol reverted gene expression signature from SARS-CoV-2-infected cells; bound with high-affinity energy to viral molecular targets such as main protease (Mpro) and receptor-biding domain (RBD); inhibited SARS-CoV-2 replication in monkey (Vero and Vero-ACE2) and human (Caco-2 and Calu-3) cell lines; and decreased interleukin-6 (IL-6) secretion in SARS-CoV-2-infected human cell lines. Interestingly, celastrol acted in a concentration-dependent manner, with undetectable signs of cytotoxicity. Therefore, celastrol is a promising lead drug candidate to treat Covid-19 due to its ability to suppress SARS-CoV-2 replication and IL-6 production in infected cells, two critical events in the pathophysiology of this disease.

Made available in DSpace on 2021-06-25T11:02:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 The rapid spread of SARS-CoV-2 caused a global pandemic. Due to the lack of treatment and vaccines, safety strategies must be found to stop the virus dissemination. The objective of this study was to evaluate the virucidal activity of ozonated water, a powerful oxidizing agent, against SARS-CoV-2. A special faucet was the source of ozonated water at a low concentration (0.2–0.8 ppm). At this concentration, tests with SARS-Cov-2 in Vero CCL81 lineage showed two log10 reduction in virus infectivity upon 1 min exposure to ozonated water, in comparison to controls. It shows the potential as an efficient and rapid alternative for controlling viral spread in hospitals and other environments. Departamento de Biologia Celular Faculdade de Medicina de Ribeirão Preto Universidade de São Paulo (USP) Departamento de Doenças Dermatológicas Infecciosas e Parasitárias Faculdade de Medicina de São José do Rio Preto (FAMERP) Universidade Estadual Paulista (UNESP) Universidade Estadual Paulista (UNESP)

Background: Chronic lymphedema is a common complication of lymphatic obstruction, particularly after cancer treatment, characterized by an increased volume of the affected extremity, partly caused by the accumulation of excessive adipose tissue. The relationship between lymph vessels' obstruction and fat deposit is, however, poorly understood. Objective: Our central hypothesis was that the inflammatory process caused by lymph stasis precedes the adipocyte differentiation and fat deposition. Methods and Results: We used a modified mouse tail model to produce secondary lymphedema. Animals were treated with dexamethasone, or the procedure was performed in nitric oxide synthase 2 (NOS2)-deficient mice to evaluate the role of inflammation in lymphedema formation. Adipose tissue (Lipin) and inflammatory markers (IL-6, MCP-1, and F4-80) were analyzed in histological samples and by quantitative polymerase chain reaction. We observed an increased deposition of fat into the affected area that starts 3 weeks after lymph vessel ligation; it further increased after 6 weeks. Genes involved in the inflammatory process were upregulated before adipocyte maturation. Treatment with dexamethasone or the use of inducible nitric oxide synthase knockout mice blocked the inflammatory reaction and inhibited the accumulation of fat distal to the lymphatic obstruction. Conclusion: In the modified mouse tail lymphedema, inflammation precedes adipogenesis. Our data suggest that MCP-1 and nitric oxide may be potential targets for lymphedema management.

Although SARS-CoV-2 severe infection is associated with a hyperinflammatory state, lymphopenia is an immunological hallmark, and correlates with poor prognosis in COVID-19. However, it remains unknown if circulating human lymphocytes and monocytes are susceptible to SARS-CoV-2 infection. In this study, SARS-CoV-2 infection of human peripheral blood mononuclear cells (PBMCs) was investigated both in vitro and in vivo. We found that in vitro infection of whole PBMCs from healthy donors was productive of virus progeny. Results revealed that monocytes, as well as B and T lymphocytes, are susceptible to SARS-CoV-2 active infection and viral replication was indicated by detection of double-stranded RNA. Moreover, flow cytometry and immunofluorescence analysis revealed that SARS-CoV-2 was frequently detected in monocytes and B lymphocytes from COVID-19 patients, and less frequently in CD4+T lymphocytes. The rates of SARS-CoV-2-infected monocytes in PBMCs from COVID-19 patients increased over time from symptom onset. Additionally, SARS-CoV-2-positive monocytes and B and CD4+T lymphocytes were detected by immunohistochemistry in post mortem lung tissue. SARS-CoV-2 infection of blood circulating leukocytes in COVID-19 patients may have important implications for disease pathogenesis, immune dysfunction, and virus spread within the host.

Systemic inflammatory response, as observed in sepsis and severe COVID-19, may lead to endothelial damage. Therefore, we aim to compare the extent of endothelial injury and its relationship to inflammation in both diseases. We included patients diagnosed with sepsis (SEPSIS group, n = 21), mild COVID-19 (MILD group, n = 31), and severe COVID-19 (SEVERE group, n = 24). Clinical and routine laboratory data were obtained, circulating cytokines (INF-γ, TNF-α, and IL-10) and endothelial injury markers (E-Selectin, Tissue Factor (TF) and von Willebrand factor (vWF)) were measured. Compared to the SEPSIS group, patients with severe COVID-19 present similar clinical and laboratory data, except for lower circulating IL-10 and E-Selectin levels. Compared to the MILD group, patients in the SEVERE group showed higher levels of TNF-α, IL-10, and TF. There was no clear relationship between cytokines and endothelial injury markers among the three studied groups; however, in SEVERE COVID-19 patients, there is a positive relationship between INF-γ with TF and a negative relationship between IL-10 and vWF. In conclusion, COVID-19 and septic patients have a similar pattern of cytokines and endothelial dysfunction markers. These findings highlight the importance of endothelium dysfunction in COVID-19 and suggest that endothelium should be better evaluated as a therapeutic target for the disease.

Antimicrobial peptides possess a myriad of molecular properties including bacterial killing and the regulation of many aspects of innate immunity. Cathelicidins are a group of antimicrobial peptides widely investigated by the scientific community. Many studies have focused on the bactericidal and pro-inflammatory roles of cathelicidins. Because the role of endogenous cathelicidin expression remains obscure in deep-seated systemic infections, we induced sepsis in cathelicidin knockout and wild-type (WT) mice by cecal ligation and puncture, performing transcriptome screening by DNA microarray in conjunction with other immunologic assays. Cathelicidin-deficient mice showed increased survival compared to WT mice in this established experimental model of polymicrobial sepsis, in association with upregulation of certain key inflammatory response genes. Therefore, cathelicidins can exert both pro- and anti-inflammatory activities depending on the disease and cellular context.The role of cathelicidin in a CLP model is investigated using cathelicidin-KO mice. Cathelicidin-KO mice show an enhanced immune response and improved survival rates. An anti-inflammatory effect of cathelicidin is likely to be detrimental for CLP. Cathelicidin-KO mice show upregulation of genes associated with increased plasma levels of pro-inflammatory Ils. Cathelicidins appear to have both pro- and anti-inflammatory properties.

The peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) regulates the expression of genes implicated in fatty acid oxidation and oxidative phosphorylation. Its role in liver steatosis is well established, since mice with liver-specific deletion of PGC-1α exhibit lipid accumulation and high-fat diet reduces hepatic PGC-1α expression in mice. In this study, we investigated the role of PGC-1α in the inflammatory changes observed in steatohepatitis induced by high-fat diet.C57black/6 mice were fed a high-fat diet containing 30% fat for 10 weeks. After euthanasia, liver morphology was examined by HE staining and inflammation was determined by IL-6, TNF-α, and IL-1β quantification. Liver gene expression of PGC-1 isoforms was evaluated by real-time PCR and p65 NFκB nuclear translocation by Western blotting. HepG2 cells were treated with linoleic acid overload for 72 h to create an in vitro model of steatohepatitis. RNA interference (RNAi) was used to evaluate the involvement of PGC-1α on inflammatory mediators' production by hepatocytes.The high-fat diet led to a state of nonalcoholic steatohepatitis, associated with increased deposits of intra-abdominal fat, hyperglycemia and hyperlipidemia. Mice liver also exhibited increased proinflammatory cytokines' levels, decreased PGC-1α expression, and marked increase in p65 NFκB nuclear translocation. Linoleic acid treated cells also presented increased expression of proinflammatory cytokines and decreased PGC-1α expression. The knockdown of PGC-1α content caused an increase in IL-6 expression and release via enhanced IκBα phosphorylation and subsequent increase of p65 NFκB nuclear translocation.High-fat diet induces liver inflammation by inhibiting PGC-1α expression and its suppressive effect in NFκB pathway.

Made available in DSpace on 2019-10-06T16:50:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-07-24. Added 1 bitstream(s) on 2019-10-09T18:35:39Z : No. of bitstreams: 1 S2523-31062019000100222.pdf: 3173113 bytes, checksum: 0b6f55fa648748e9f3293c96d1a42acb (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) BACKGROUND: Adaptive immune cells, including CD4+CD69+ and CD4+CD25+FoxP3+ regulatory T (Treg) cells, are important for maintaining immunological tolerance. In human systemic lupus erythematosus (SLE), CD4+CD25+FoxP3+ Treg cells are reduced, whereas CD69 expression is increased, resulting in a homeostatic immune imbalance that may intensify autoreactive T cell activity. To analyze the mechanisms implicated in autotolerance failure, we evaluated CD4+CD69+ and CD4+CD25+FoxP3+ T cells and interleukin profiles in a pristane-induced SLE experimental model. METHODS: For lupus induction, 26 female Balb/c mice received a single intraperitoneal 0.5 ml dose of pristane, and 16 mice received the same dose of saline. Blood and spleen samples were collected from euthanized mice 90 and 120 days after pristane or saline inoculation. Mononuclear cells from peripheral blood (PBMC), peritoneal lavage (PL) and splenocytes were obtained by erythrocyte lysis and cryopreserved for further evaluation by flow cytometry using the GuavaEasyCyte TM HT. After thawing, cells were washed and stained with monoclonal antibodies against CD3, CD4, CD8, CD25, CD28, CD69, FoxP3, CD14 and Ly6C (BD Pharmingen TM). Interleukins were quantified using Multiplex® MAP. The Mann-Whitney test and the Pearson coefficient were used for statistical analysis, and p

Poly(ADP-ribose) polymerase (PARP) is involved in the pathogenesis of cell dysfunction, inflammation and organ failure during septic shock. The goal of the current study was to investigate the efficacy and safety of the clinically approved PARP inhibitor olaparib in experimental models of oxidative stress in vitro and in sepsis in vivo. In mice subjected to cecal ligation and puncture (CLP) organ injury markers, circulating and splenic immune cell distributions, circulating mediators, DNA integrity and survival was measured. In U937 cells subjected to oxidative stress, cellular bioenergetics, viability and DNA integrity were measured. Olaparib was used to inhibit PARP. The results show that in adult male mice subjected to CLP, olaparib (1–10 mg/kg i.p.) improved multiorgan dysfunction. Olaparib treatment reduced the degree of bacterial CFUs. Olaparib attenuated the increases in the levels of several circulating mediators in the plasma. In the spleen, the number of CD4+ and CD8+ lymphocytes were reduced in response to CLP; this reduction was inhibited by olaparib treatment. Treg but not Th17 lymphocytes increased in response to CLP; these cell populations were reduced in sepsis when the animals received olaparib. The Th17/Treg ratio was lower in CLP-olaparib group than in the CLP control group. Analysis of miRNA expression identified a multitude of changes in spleen and circulating white blood cell miRNA levels after CLP; olaparib treatment selectively modulated these responses. Olaparib extended the survival rate of mice subjected to CLP. In contrast to males, in female mice olaparib did not have significant protective effects in CLP. In aged mice olaparib exerted beneficial effects that were less pronounced than the effects obtained in young adult males. In in vitro experiments in U937 cells subjected to oxidative stress, olaparib (1–100 μM) inhibited PARP activity, protected against the loss of cell viability, preserved NAD+ levels and improved cellular bioenergetics. In none of the in vivo or in vitro experiments did we observe any adverse effects of olaparib on nuclear or mitochondrial DNA integrity. In conclusion, olaparib improves organ function and extends survival in septic shock. Repurposing and eventual clinical introduction of this clinically approved PARP inhibitor may be warranted for the experimental therapy of septic shock.

Antimicrobial peptides are an ancient family of molecules that emerged millions of years ago and have been strongly conserved during the evolutionary process of living organisms. Recently, our group described that the human antimicrobial peptide LL-37 migrates to the nucleus, raising the possibility that LL-37 could directly modulate transcription under certain conditions. Here, we showed evidence that LL-37 binds to gene promoter regions, and LL-37 gene silencing changed the transcriptional program of melanoma A375 cells genes associated with histone, metabolism, cellular stress, ubiquitination and mitochondria.

The mechanisms by which exercise affects atherosclerotic plaque stability remain incompletely understood. We evaluated the effects of two training protocols on both atherosclerotic plaque structure and the signaling pathways involved in plaque rupture. Methods Male low-density lipoprotein (LDL) receptor knockout mice were fed a high-fat, high-cholesterol diet (HFD). One group was subjected to moderate exercise using a treadmill for 14 weeks (preventive protocol). The other group started an exercise regimen after 16 weeks of the HFD (therapeutic group). Atherosclerotic plaques within the aorta were evaluated for lipid and collagen contents, as well as for inflammatory markers. Plasma cholesterol and cytokine levels were also determined. Results The mice receiving a HFD developed hypercholesterolemia and atherosclerotic plaques within the aorta. The aortas from the animals in the preventive protocol exhibited smaller lipid cores and higher collagen content. These animals also exhibited lower CD40 expression within the plaques. The aortas of the mice in the therapeutic group exhibited higher collagen content, but no differences in either lipid core size or plaque size were noted. No differences in blood pressure, plasma cholesterol, cytokine levels, plaque size or metalloproteinase 9 expression were observed in the trained animals compared with the sedentary animals. Conclusion Moderate aerobic exercise modified atherosclerotic plaque characteristics and converted the plaques into a more stable phenotype, increasing the collagen content in response to both exercise programs. Furthermore, moderate aerobic exercise reduced the animals' fat content and decreased the activity of the CD40–CD40L signaling pathway in the preventive group.

Syzygium cumini is used worldwide for the treatment of metabolic syndrome-associated outcomes. Previously, we described the antihypertriglyceridemic effect of the hydroethanolic extract of S. cumini leaf (HESc) in monosodium L-glutamate- (MSG-) induced obese rats. This study sought to investigate the molecular mechanisms underlying the antihypertriglyceridemic effect of HESc in MSG-obese rats. Newborn male Wistar rats were injected subcutaneously with MSG (4.0 g/kg/day, obese group) or saline 1.25% (1.0 mL/kg/day, lean group), from 2nd through 10th postnatal day. At 8 weeks old, obese rats started to be orally treated with HESc (0.5 or 1.0 g/kg/day, n=7) or saline 0.9% (1 mL/kg/day, n=7). Lean rats received saline solution (1 mL/kg/day, n=7). Upon 8-week treatment, animals were euthanized for blood and tissue collection. Another set of adult nonobese Wistar rats was used for the assessment of HESc acute effects on Triton WR1339-induced hypertriglyceridemia. HESc reduced weight gain, as well as adipose tissue fat pads, without altering food intake of obese rats. HESc restored fasting serum glucose, triglycerides, total cholesterol, and free fatty acids, as well as insulin sensitivity, to levels similar to lean rats. Additionally, HESc halved the triglyceride content into very low-density lipoprotein particles, as well as healed liver steatosis, in obese rats. Hepatic protein expression of the endoplasmic reticulum chaperone GRP94 was decreased by HESc, which also downregulated the hepatic triglyceride secretion pathway by reducing the splicing of X-box binding protein 1 (XBP-1s), as well as protein disulfide isomerase (PDI) and microsomal triglyceride transfer protein (MTP) translational levels. This action was further corroborated by the acute inhibitory effect of HESc on triglyceride accumulation on Triton WR1339-treated rats. Our data support the downregulation of the XBP-1s/PDI/MTP axis in the liver of MSG-obese rats as a novel feasible mechanism for the antihypertriglyceridemic effect promoted by the polyphenolic phytocomplex present in S. cumini leaf.

Objectifs Le lymphœdeme est une complication courante du traitement du cancer. Les vaisseaux lymphatiques ou les ganglions endommages menent a la stase, induisant l’inflammation, la fibrose et l’accumulation de tissu adipeux. Les methodes de traitement restent palliatives, et les resultats varient. Comme la pathogenie du lymphœdeme n’est toujours pas completement comprise, nous avons adapte un modele experimental deja etabli de stase lymphatique et nous l’utilisons pour etudier le role de l’inflammation dans le developpement du lymphœdeme. Materiel et methodes Le modele experimental est obtenu par ligature des veines et des vaisseaux lymphatiques localises dans l’aspect lateral de la queue des souris. Vingt souris noires mâles C57BL6 ont subi cette procedure, tandis que vingt autres animaux ont ete soumis a la ligature isolee des vaisseaux lymphatiques, preservant les veines. Resultats Les deux modeles experimentaux ont induit le lymphœdeme sur la partie distale des queues des souris. Apres 3 semaines il y avait l’expression des genes impliques dans la differenciation des adipocytes (C/EBP) et de la reponse inflammatoire (TNF-α, IL-6, MCP-1, F4-80). Apres 6 semaines, il y avait un des depots d’adipocytes et les marqueurs de maturite des adipocytes etaient presents (Lipins 1 et 2) ; les mediateurs inflammatoires ont maintenu une expression elevee. Ensuite, un autre groupe a ete traite avec le lipopolysaccharide pour augmenter le processus inflammatoire, ou avec la dexamethasone pour le reduire. Dans cette etude, NOS2 knock-out souris ont ete aussi utilises. Les traitements anti-inflammatoires ont diminue la differenciation des adipocytes et n’interferent pas avec la lymphangiogenese. Conclusion Nous avons developpe une methode adaptee pour generer un lymphœdeme chez la queue de souris qui est plus rapide a effectuer et plus reproductible. En utilisant ce nouveau modele, nous avons pu montrer que l’inflammation precede la differenciation des adipocytes et les depots de graisse dans le lymphœdeme et son blocage diminue la deposition des adipocytes observee chez les animaux atteints.

Roux-en-Y gastric bypass (RYGB) is an effective method to achieve sustained weight loss, but the mechanisms responsible for RYGB effects have not yet been fully characterized. In this study, we profiled the concentrations of 143 lipid metabolites in dry blood spots (DBS) of RYGB patients. DBS from obese patients (BMI range 35&ndash, 44 kg/m2) were collected 7 days before, 15 and 90 days after the surgery. LC-MS/MS was used to quantify acylcarnitines, phosphatidylcholines, sphingomyelins and bile acids. RYGB caused a rapid increase in acylcarnitine levels that proved to be only transient, contrasting with the sustained decrease in phosphatidylcholines and increase of sphingomyelins and bile acids. A PLS-DA analysis revealed a 3-component model (R2 = 0.9, Q2 = 0.74) with key metabolites responsible for the overall metabolite differences. These included the BCAA-derived acylcarnitines and sphingomyelins with 16 and 18 carbons. We found important correlations between the levels of BCAA-derived acylcarnitines and specific sphingomyelins with plasma cholesterol and triacylglycerol concentrations. Along with the marked weight loss and clinical improvements, RYGB induced specific alterations in plasma acylcarnitines, bile acid and phospholipid levels. This calls for more studies on RYGB effects aiming to elucidate the metabolic adaptations that follow this procedure.

We investigated the effects of physical detraining on lipogenesis/lipolysis and cellularity (apoptosis/adipogenesis) in rat subcutaneous (inguinal; SC) and visceral (retroperitoneal; RP) white adipose depots.Three groups of male Wistar rats (6-wk old) were studied: (1) (T) trained for 12 weeks; (2) (D) trained for 8 weeks and detrained for 4 weeks; and (3) (S) age-matched sedentary. Training consisted of treadmill running sessions (1 h/day, 5 days/week, 50-60% maximal race capacity).Physical detraining increased glucose oxidation, lipogenesis, and adipocyte size in the SC and RP depots. The number of apoptotic SC adipocytes was reduced by 53% in the T (p 0.0001) and by 43% in the D (p 0.001) as compared with S. RP adipocyte apoptosis in the T and D was 9.48% and 10.9% greater compared to the S, respectively (p 0.05). In the SC stromal vascular fraction (SVF) of D rats, adiponectin, sterol regulatory element binding protein (SREBP)-1c, Peroxisome proliferator-activated receptor gamma (PPARγ), and Perilipin A mRNA expressions were more pronounced than S group, suggesting a more intense adipogenesis. This putative adipogenic effect was not observed in the RP depot. The physical detraining promoted rapid increase in the SC and RP depots however not through the same mechanisms.Physical detraining induced fat cell hypertrophy (increase of lipogenesis) in both SC and RP whereas hyperplasia (increase of adipogenesis and reduction of apoptosis) was found in SC only. These results indicate the mechanism associated with obesogenic effects of detraining varies with the fat depot.

Sepsis is a dynamic disease, displaying an inflammatory profile that varies over time and for each organ. Controlling the inflammatory response based in targeting a single molecule has been proved useless. We hypothesized that treatment with bone marrow-derived mononuclear cells (BMDMCs) may be more efficient to modulate the systemic inflammatory response to infection. Adult male Balb/c mice were subjected to cecal ligation and puncture (CLP) or endotoxemia model of experimental sepsis. BMDMCs were separated under Ficoll gradient and injected intravenously 1 h after the procedures. Cytokines concentration was quantified in plasma, lungs, heart, and gut. Spleens, lymph nodes, and thymus were used for lymphocytes isolation and cell death assessment. All measurements were performed 2 h after BMDMCs injection. RAW264.7 macrophages and BMDMCs were cocultivated in vitro to investigate the mechanisms involved. Our data showed that an early single intravenous injection of BMDMCs in animals submitted to the murine model of endotoxemia led to the improvement of survival rate; BMDMCs persistency in lung, liver, and spleen after 24 h; decreased necrosis and apoptosis of mononuclear cells; lower TNF-α, but increased IL-10 concentration in plasma; and tissue-specific cytokine profile. In vitro experiments demonstrated that IL-6, IL-10, and nitric oxide production depends on direct contact of BMDMCs to macrophages and that TNF-α production is negatively regulated by PGE2. BMDMCs are efficient in protecting animals from endotoxemia and sepsis, reducing systemic inflammation as well as specifically modulating tissue inflammation, producing the necessary immune regulation to re-equilibrate the inflammatory response.

Sepsis survivors suffer from additional morbidities, including higher risk of readmissions, nervous system disturbances and cognitive dysfunction, and increased mortality, even several years after the initial episode of sepsis. In many ways, the phenotype of sepsis survivors resembles the phenotype associated with accelerated aging. Since telomere shortening is a hallmark of aging, we investigated whether sepsis also leads to telomere shortening. Male balb/c mice were divided into two groups: the control group received 100 µl of normal saline intraperitoneally (i.p.) and the sepsis group received 15 mg/kg of bacterial lipopolysaccharide i.p. After 48 h, animals were euthanized to collect blood, spleen and kidney. The human component of our study utilized blood samples obtained from patients in the trauma department and samples collected 7 d later in those patients who developed sepsis. Telomere length was measured by quantitative polymerase chain reaction. Since oxidative stress is a known inducer of telomere shortening, thiobarbituric acid-reactive substances and superoxide dismutase activity were analyzed to evaluate oxidative stress burden. Induction of endotoxemia in mice resulted in significant telomere shortening in spleen and kidney. Blood cells from patients who progressed to sepsis also exhibited a statistically significant reduction of telomere length. Endotoxemia in mice also induced an early-onset increase in oxidative stress markers but was not associated with a downregulation of telomerase protein expression. We conclude that endotoxemia and sepsis induce telomere shortening in various tissues and hypothesize that this may contribute to the pathogenesis of the delayed pathophysiological events in sepsis survivors.

The rise in the prevalence of obesity and metabolic syndrome turned NAFLD as the most common cause of chronic liver diseases worldwide. Although the role of toll like receptors, especially TLR4, as activators of inflammatory pathways in liver diseases is well established, our goal was to investigate if TLR4 activation could modulate metabolic lipid pathways and alter the onset of NAFLD. We used LDL receptor-deficient mice (LDLrKO) fed with an atherogenic diet as a model. The role of TLR4 activation was evaluated by crossing LDLrKO mice with the TLR4 knockout mice. Animals were fed for 12weeks with high-fat high-cholesterol diet (HFD) containing 18% saturated fat and 1.25% cholesterol. TLR4/LDLr KO mice presented lower triacylglyceride (TAG) plasma levels when compared to LDLrKO, despite the type of diet ingested. HFD induced TAG and cholesterol accumulation in the liver of all mice genotypes studied, but TLR4/LDLr KO presented lower TAG accumulation than LDLrKO mice. Gene expression of TAG synthesis enzymes (ApoB100, MTTP, GPAT1 and GPAT4) was not differentially altered in TLR4/LDLr KO and LDLrKO mice. On the other hand, TLR4 deficiency enhanced the expression of several enzymes involved in the oxidation of fatty acids, as follows: ACOX, CPT-1, MTPa, MTBb, PBE and 3-ketoacyl-CoA thiolase. Acyl-carnitine plasma profile showed an increase in C0 and C2 concentration in TLR4/LDLr KO group, corroborating the hypothesis of increased fat oxidation. Our results indicate that TLR4 may have an important role in the onset of steatosis, once its depletion enhances fatty acid oxidation in the liver of mice, preventing triglyceride accumulation.

Acute respiratory distress syndrome (ARDS) is the most severe lung inflammatory manifestation and has no effective therapy nowadays. Sepsis is one of the main illnesses among ARDS causes. The use of fluid resuscitation is an important treatment for sepsis, but positive fluid balance may induce pulmonary injury. As an alternative, fluid resuscitation with hypertonic saline ((HS) NaCl 7.5%) has been described as a promising therapeutical agent in sepsis-induced ARDS by the diminished amount of fluid necessary. Thus, we evaluated the effect of hypertonic saline in the treatment of LPS-induced ARDS. We found that hypertonic saline (NaCl 7.5%) treatment in rat model of LPS-induced ARDS avoided pulmonary function worsening and inhibited type I collagen deposition. In addition, hypertonic saline prevented pulmonary injury by decreasing metalloproteinase 9 (MMP-9) activity in tissue. Focal adhesion kinase (FAK) activation was reduced in HS group as well as neutrophil infiltration, NOS2 expression and NO content. Our study shows that fluid resuscitation with hypertonic saline decreases the progression of LPS-induced ARDS due to inhibition of pulmonary remodeling that is observed when regular saline is used.

Endotoxin tolerance is defined as a reduced capacity of the host to respond to LPS activation following a first exposure to this stimulus. It affects all leukocytes and regarding macrophages, most studies focus on the reduced ability of these cells to secrete pro-inflammatory cytokines. Therefore, we evaluated other macrophages functions (fungicidal capacity, reactive oxygen species production and antigen presentation) in cells from tolerant mice. We have performed a tolerance model in our laboratory that does not stimulate directly the place from where the cells will be removed (peritoneal cavity). Mouse received subcutaneous injections of LPS in the scruff for 5 days and we analyze the capacity of peritoneal macrophages to phagocyte using three different receptors: Fc, C3b and mannose receptors. We found a reduction in the phagocytosis of erythrocytes and Candida albicans related to the Fc and mannose receptors. These differences can be due to a macrophage reprogramming, as demonstrated by altered expression of cytokines and chemokines. Despite this reduction in phagocytosis capacity, macrophages from tolerant animals exhibited enhanced hydrogen peroxide production and expression of antigen presentation molecules, suggesting that their ability to combat an infection is improved. In summary, our data indicates that LPS tolerance drives macrophages from a predominant release of proinflammatory mediators that amplify inflammation and host damage toward a better killing and antigen presentation state.

A known consequence of the large weight loss after bariatric surgery is the appearance of large skinfolds, particularly in the abdomen region of the patients. The balance between the synthesis of extracellular matrix (ECM) components and their proteolysis, mainly by fibrinolytic systems and matrix metalloproteases (MMPs), may be disturbed in these patients. The causes underlying the deregulation of ECM remodeling that occurs in these patients are not, however, clear. We investigated molecular mechanisms responsible for this dysfunction of ECM remodeling process, comparing it to normal skin. Collagen types, MMP2 and MMP9 expression and activity, interleukins 1β (IL1β) and 6 (IL6), and transcription coactivator PGC-1β expression were analyzed in 16 patients. Ex-obese patients presented increased expression of collagen types III and IV mRNA, increased expression of MMP2, decreased expression and activity of MMP9, and increased expression of PGC-1β in the skin. Inflammation markers IL1β and IL6 mRNA were not different. We have demonstrated that obese patients with extensive weight loss after bariatric surgery have increased expression of PGC-1β in the skin, which can result in a decreased expression and activity of MMP9 and increased collagen types III and IV deposition. These molecular changes may contribute for the formation of saggy skinfolds observed in these patients and impair wound healing.

Long-chain fatty acids are capable of inducing alterations in the homoeostasis of glucose-stimulated insulin secretion (GSIS), but the effect of medium-chain fatty acids (MCFA) is poorly elucidated. In the present study, we fed a normoenergetic MCFA diet to male rats from the age of 1 month to the age of 4 months in order to analyse the effect of MCFA on body growth, insulin sensitivity and GSIS. The 45 % MCFA substitution of whole fatty acids in the normoenergetic diet impaired whole body growth and resulted in increased body adiposity and hyperinsulinaemia, and reduced insulin-mediated glucose uptake in skeletal muscle. In addition, the isolated pancreatic islets from the MCFA-fed rats showed impaired GSIS and reduced protein kinase Bα (AKT1) protein expression and extracellular signal-related kinase isoforms 1 and 2 (ERK1/2) phosphorylation, which were accompanied by increased cellular death. Furthermore, there was a mildly increased cholinergic sensitivity to GSIS. We discuss these findings in further detail, and advocate that they might have a role in the mechanistic pathway leading to the compensatory hyperinsulinaemic status found in this animal model.

Sepsis induces a systemic inflammatory response leading to tissue damage and cell death. LPS tolerance affects inflammatory response. To comprehend potential new mechanisms of immune regulation in endotoxemia, we examined macrophage mRNA expression by macroarray affected by LPS tolerance. LPS tolerance was induced with subcutaneous administration of 1 mg/kg/day of LPS over 5 days. Macrophages were isolated from the spleen and the expression of 1200 genes was quantitatively analyzed by the macroarray technique. The tolerant group displayed relevant changes in the expression of 84 mRNA when compared to naïve mice. A functional group of genes related to cell death regulation was identified. PARP-1, caspase 3, FASL and TRAIL genes were confirmed by RT-PCR to present lower expression in tolerant mice. In addition, reduced expression of the pro-inflammatory genes TNF-α and IFN-γ in the tolerant group was demonstrated. Following this, animals were challenged with polymicrobial sepsis. Flow cytometry analysis showed reduced necrosis and apoptosis in macrophages from the tolerant group compared to the naïve group. Finally, a survival study showed a significant reduction in mortality in the tolerant group. Thus, in the current study we provide evidence for the selective reprogramming of the gene expression of cell death pathways during LPS tolerance and link these changes to protection from cell death and enhanced survival rates.

Many macrophage functions are modulated by fatty acids (FAs), including cytokine release, such as tumor necrosis factor-α (TNF-α). TNF-α is of great interest due to its role in the inflammation process observed in several diseases such as rheumatoid arthritis, atherosclerosis, and obesity. However, the mechanisms by which FA effects occur have not been completely elucidated yet. In this study, we used a mouse monocyte lineage (J774 cells) to evaluate the effect of 50 and 100 μM of saturated (palmitic and stearic acids), monounsaturated (oleic acid) and polyunsaturated (linoleic acid) FAs on TNF-α production. Alterations in gene expression, poly(A) tail length and activation of transcription factors were evaluated. Oleic and linoleic acids, usually known as neutral or pro-inflammatory FA, inhibited LPS-induced TNF-α secretion by the cells. Saturated FAs were potent inducers of TNF-α expression and secretion under basal and inflammatory conditions (in the presence of LPS). Although the effect of the saturated FA was similar, the mechanism involved in each case seem to be distinct, as palmitic acid increased EGR-1 and CREB binding activity and stearic acid increased mRNA poly(A) tail. These results may contribute to the understanding of the molecular mechanisms by which saturated FAs modulate the inflammatory response and may lead to design of associations of dietary and pharmacological strategies to counteract the pathological effects of TNF-α.

Extensive lymphocyte apoptosis may be an important cause of immune suppression in sepsis. Here we investigated the effect of LPS tolerance on lymphocyte apoptosis in an experimental model of polymicrobial infection. Tolerance was induced by the injection of lipopolysaccharide (1.0mg/kg/subcutaneously) once a day for 5 days. Macroarray analysis of mRNA isolated from T-(CD4) lymphocytes was used to identify genes that are differentially expressed during LPS tolerance. In addition, assessment of the expression of apoptosis-associated lymphocyte gene products and apoptotic events was performed on the 8th day; 6h after the terminal challenge with polymicrobial infection or high-dose LPS administration. Survival studies with polymicrobial infection were also conducted. LPS tolerance induced a broad reprogramming of cell death pathways, including a suppression of receptor-mediated and mitochondrial apoptotic pathways, inflammatory caspases, alternate apoptotic pathways, as well as reduced expression of genes involved in necrosis. These alterations led to a marked resistance of lymphocytes against cell death during the subsequent period of sepsis. In addition, LPS tolerance produced an increased differentiation of T-lymphocytes to T(H)1 and T(H)2, with a T(H)1 differentiation predominance. Thus, in the current study we provide an evidence for a marked reprogramming of gene expression of multiple cell death pathways during LPS tolerance. These alterations may play a significant role in the observed protection of the animals from a subsequent lethal polymicrobial sepsis challenge.

Fish oil supplementation has been shown to improve the cachectic state of tumor-bearing animals and humans. Our previous study showed that fish oil supplementation (1 g per kg body weight per day) for 2 generations had anticancer and anticachetic effects in Walker 256 tumor-bearing rats as demonstrated by reduced tumor growth and body weight loss and increased food intake and survival. In this study, the effect of fish oil supplementation for 2 generations on membrane integrity, proliferation capacity, and CD4/CD8 ratio of lymphocytes isolated from mesenteric lymph nodes, spleen, and thymus of Walker 256 tumor-bearing animals was investigated. We also determined fish oil effect on plasma concentration and ex vivo production of cytokines [tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-4 (IL-4), IL-6, and IL-10]. Lymphocytes from thymus of tumor-bearing rats presented lower viability, but this change was abolished by fish oil supplementation. Tumor growth increased proliferation of lymphocytes from all lymphoid organs, and fish oil supplementation abolished this effect. Ex vivo production of TNF-alpha and IL-6 was reduced in supplemented animals, but IL-4 and IL-10 secretion was stimulated in both nontumor and tumor-bearing rats. IL-10 and IFN-gamma plasma levels was also decreased in supplemented animals. These results suggest that the anticachetic effects of fish oil supplementation for a long period of time (2 generations) in Walker 256 tumor-bearing rats may be associated to a decrease in lymphocyte function as demonstrated by reduced viability, proliferation capacity, and cytokine production.

Aims Changes in the gene expression are one of the molecular events involved in the Systemic of Inflammatory Response Syndrome during sepsis. The preconditioning with low doses of lipopolysaccharide (LPS) reduces the expression of pro-inflammatory genes leading to less tissue damage and better outcome. This hyporesponsive state called tolerance is associated to alterations in chromatin structure and nitric oxide (NO) production. In the current study, we demonstrated that tolerance induced by LPS was found to be NO-dependent and related to epigenetic changes. Main methods THP-1 cells were cultivated in RPMI medium (Control), submitted to tolerance (500 ng/mL of LPS 24 h before challenge with 1000 ng/mL of LPS during 24 h Tolerant group) and challenge (1000 ng/mL of LPS during 24 h Directly challenged group). The analyses performed were: cytokines production, histone acetyl transferases/histone deacetylases (HAT/HDAC) activity, nitrosylation of HDAC-2 and -3, expression of acetylated histones H3 and H4. HDAC and Nitric Oxide Synthases (NOS) activities were inhibited with 30 mM trichostatin (TSA) and 100 μM LNAME, respectively. Key findings Administration of low doses of LPS repressed the production of IL-6 and IL-10, however this effect was abolished with the inhibition of NOS activity and by TSA in the case of IL-10. Tolerance modulates the activity of HAT and, consequently, the acetylation of histones H3 and H4. Inhibition of NO decreases acetylation of Histones. The HDACs 2 and 3 were nitrosylated after the tolerance induction. Significance The tolerance to LPS regulates the cytokine production by modulating chromatin structure and this event is NO dependent.

Aim: Sepsis is a critical condition that leads to high mortality and is the most common cause of death in intensive care units. Despite exhaustive efforts by the scientific community, a reliable biomarker for diagnosis, evolution and prognosis of sepsis is still lacking. Results & methodology: Here, using high-throughput proteomics, we describe N-acetylmuramoyl-l-alanine amidase as a novel candidate for differentiating infectious and noninfectious inflammatory syndromes. Discussion & conclusion: This is the first description of N-acetylmuramoyl-l-alanine amidase as a biomarker that can be used alone or in conjunction with other biomarkers to facilitate the diagnosis of sepsis in the critically ill.

Docosahexaenoic (DHA; C22:6 n-3), eicosapentaenoic (EPA; C20:5 n-3), palmitic (PA; C16:0), and stearic (SA; C18:0) acids decrease lymphocyte proliferation in concentrations of >50 muM, as observed in our previous study. However, oleic acid (OA; C18:1 n-9) and linoleic acid (LA; C18:2 n-6) increase lymphocyte proliferation at 25 muM. In this study, the effect of these FAs on the interleukin-2 (IL-2) signaling pathway in human lymphocytes was investigated. Cells were isolated from heparinized venous blood of healthy human donors by density-gradient sedimentation. Cells were stimulated with 5 mug/ml concanavalin A and treated with FAs in the absence or presence of IL-2 for 1 hour. CD25-alpha externalization was analyzed by flow cytometry, and Janus kinase 1 (JAK1), JAK3, signal transducer and activator of transcription (STAT) 5, extracellular signal-regulated kinases (ERKs) 1 and 2, Akt, and protein kinase C (PKC)-zeta phosphorylation were analyzed by Western blotting. The expression of CD25-alpha at the cell surface was increased by DHA, SA, and PA but was unaffected by EPA, OA, and LA. PA, SA, DHA, and EPA decreased JAK1, JAK3, STAT5, and Akt phosphorylation induced by IL-2, but OA and LA did not cause any effect. OA and LA increased ERK1/2 phosphorylation, whereas the other FAs caused a marked decrease. PKC-zeta phosphorylation was decreased by OA and LA and was not altered by the remaining FAs. In conclusion, the inhibitory effect of PA, SA, DHA, and EPA on lymphocyte proliferation observed in our previous study was attributable to a decrease in JAK/STAT, ERK, and Akt pathways activated by IL-2. Probably, OA and LA stimulated lymphocyte proliferation by increasing ERK1/2 phosphorylation through PKC-zeta activation. The inhibition of JAK1, JAK3, STAT5, ERK1/2, and Akt phosphorylation caused by DHA, SA, and PA is associated with an alteration of CD25 expression at the cell surface.

Fatty acids (FAs) have been shown to alter leucocyte function and thus to modulate inflammatory and immune responses. In this review, the effects of FAs on several aspects of lymphocyte, neutrophil and macrophage function are discussed. The mechanisms by which FAs modulate the production of lipid mediators, activity of intracellular signalling pathways, activity of lipid-raft-associated proteins, binding to TLRs (Toll-like receptors), control of gene expression, activation of transcription factors, induction of cell death and production of reactive oxygen and nitrogen species are described in this review. The rationale for the use of specific FAs to treat patients with impaired immune function is explained. Substantial improvement in the therapeutic usage of FAs or FA derivatives may be possible based on an improvement in the understanding of the precise molecular mechanisms of action with respect to the different leucocyte types and outcome with respect to the inflammatory responses.

Background/Aims: Acute restraint stress may induce impaired macrophage phagocytic function. Moderate physical training is associated with beneficial effects on immunological functions. We investigated the effects of moderate physical training on phagocytic function of alveolar macrophages in rats submitted to acute restraint stress. Methods: Thirty male Wistar rats weighing 210–226 g were randomly divided into 4 groups: nontrained rats (n = 7), nontrained rats submitted to stress (n = 8), trained rats (n = 7) and trained rats submitted to stress (n = 8). Trained rats were submitted to a program of moderate running training over a period of 8 weeks. Rats subjected to restraint stress were kept immobilized in glass cylinders (8 cm in diameter and 24 cm long) during 60 min. Phagocytosis capacity of macrophages was evaluated by either Escherichia coli orzymosan stimuli. Results: Restraint stress induced a decrease in phagocytosis of E. coli and zymosan particle stimulation by macrophages. Neither of these alterations was observed in trained animals submitted to acute restraint stress. Conclusions: Our data confirm that acute restraint stress is associated with impaired function of macrophages. Moreover, moderate physical training attenuates the effects of acute stress by a mechanism that involves an increase in tolerance of macrophages.

In the present study, the effect of increasing concentrations of palmitic (PA, C16:0), stearic (SA, C18:0), oleic (OA, C18:1, n-9), linoleic (LA, C18:2n-6), docosahexaenoic (DHA, C22:6 n-3) and eicosapentaenoic (EPA, C20:5 n-3) acids on lymphocyte proliferation was investigated. The maximal non-toxic concentrations of these fatty acids for human lymphocytes in vitro were determined. It was also evaluated whether these fatty acids at non-toxic concentrations affect IL-2 induced lymphocyte proliferation and cell cycle progression. OA and LA at 25 microM increased lymphocyte proliferation and at higher concentrations (75 microM and 100 microM) inhibited it. Both fatty acids promoted cell death at 200 microM concentration. PA and SA decreased lymphocyte proliferation at 50 microM and promoted cell death at concentrations of 100 microM and above. EPA and DHA decreased lymphocyte proliferation at 25 and 50 microM being toxic at 50 and 100 microM, respectively. PA, SA, DHA and EPA decreased the stimulatory effect of IL-2 on lymphocyte proliferation, increasing the percentage of cells in G1 phase and decreasing the proportion of cells in S and G2/M phases. OA and LA caused an even greater pronounced effect. The treatment with all fatty acids increased neutral lipid accumulation in the cells but the effect was more pronounced with PA and DHA. In conclusion, PA, SA, DHA and EPA decreased lymphocyte proliferation, whereas OA and LA stimulated it at non-toxic concentrations.

Acute lung injury (ALI) is a common complication associated with septic shock that directly influences the prognosis of sepsis patients. Currently, one of the main supportive treatment modalities for septic shock is fluid resuscitation. The use of hypertonic saline (HS: 7.5% NaCl) for fluid resuscitation has been described as a promising therapy in experimental models of sepsis-induced ALI, but it has failed to produce similar results in clinical practice. Thus, we compared experimental timing versus clinical timing effectiveness (i.e., early vs. late fluid resuscitation) after the inflammatory scenario was established in a rat model of bacterial lipopolysaccharide-induced ALI. We found that late fluid resuscitation with hypertonic saline (NaCl 7.5%) did not reduce the mortality rates of animals compared with the mortality late associated with early treatment. Late fluid resuscitation with both hypertonic and normal saline increased pulmonary inflammation, decreased pulmonary function, and induced pulmonary injury by elevating metalloproteinase-2 and metalloproteinase-9 activity and collagen deposition in the animals, unlike early treatment. The animals with lipopolysaccharide-induced ALI that received late resuscitation with any kind of fluids demonstrated aggravated pulmonary injury and respiratory function. Moreover, we showed that the therapeutic window for a beneficial effect of fluid resuscitation with hypertonic saline is very narrow.

Circulating dipeptidyl peptidase IV (DPPIV) activity correlates with cardiac dysfunction in humans and experimental heart failure (HF) models. Similarly, inflammatory markers are associated with poorer outcomes in HF patients. However, the contributions of DPPIV to inflammation in HF remain elusive. Therefore, this study aimed to investigate whether the cardioprotective effects of DPPIV inhibition after myocardial injury are accompanied by reduced cardiac inflammation, whether circulating DPPIV activity correlates with the levels of systemic inflammatory markers in HF patients, and whether leukocytes and/or splenocytes may be one of the sources of circulating DPPIV in HF. Experimental HF was induced in male Wistar rats by left ventricular myocardial injury after radiofrequency catheter ablation. The rats were divided into three groups: sham, HF, and HF + DPPIV inhibitor (sitagliptin). Six weeks after surgery, cardiac function, perfusion and inflammatory status were evaluated. Sitagliptin treatment improved cardiac function and perfusion, reduced macrophage infiltration, and diminished the levels of inflammatory biomarkers including TNF-α, IL-1β, and CCL2. In HF patients, serum DPPIV activity correlated with CCL2, suggesting that leukocytes may be the source of circulating DPPIV in HF. Unexpectedly, DPPIV release was higher in splenocytes from HF rats and similar in HF circulating mononuclear cells compared with those from sham, suggesting an organ-specific modulation of DPPIV in HF. Collectively, our data provide new evidence that the cardioprotective effects of DPPIV inhibition in HF may be due to suppression of inflammatory cytokines. Moreover, they suggest that a vicious circle between DPPIV and inflammation may contribute to HF development and progression.

Breast cancer is a prevalent neoplastic disease among women worldwide which treatments still present several side effects and resistance. Considering that cancer cells present derangements in their energetic homeostasis, and that peroxisome proliferator-activated receptor- gamma coactivator 1 (PGC-1) is crucial for cellular metabolism and redox signaling, the main objective of this study was to investigate whether there is a relationship between PGC-1 expression, the proliferation of breast cancer cells and the mechanisms involved. We initially assessed PGC-1β expression in complementary DNA (cDNA) from breast tumor of patients bearing luminal A, luminal B, and HER2-overexpressed and triple negative tumors. Our data showed that PGC-1β expression is increased in patients bearing HER2-overexpressing tumors as compared to others subtypes. Using quantitative PCR and immunoblotting, we showed that breast cancer cells with HER2-amplification (SKBR-3) have greater expression of PGC-1β as compared to a non-tumorous breast cell (MCF-10A) and higher proliferation rate. PGC-1β expression was knocked down with short interfering RNA in HER2-overexpressing cells, and cells decreased proliferation. In these PGC-1β-inhibited cells, we found increased citrate synthase activity and no marked changes in mitochondrial respiration. Glycolytic pathway was decreased, characterized by lower intracellular lactate levels. In addition, after PGC-1β knockdown, SKBR-3 cells showed increased reactive oxygen species production, no changes in antioxidant activity, and decreased expression of ERRα, a modulator of metabolism. In conclusion, we show an association of HER2-overexpression and PGC-1β. PGC-1β knockdown impairs HER2-overexpressing cells proliferation acting on ERRα signaling, metabolism, and redox balance.

DPPIV inhibitors are antidiabetic drugs that confer cardioprotection beyond glucose lowering. We have previously demonstrated that circulating DPPIV associates with poorer cardiovascular outcomes in human and experimental heart failure (HF) and that long-term DPPIV inhibition mitigates the development and/or progression of HF in rats. Since inflammation plays a role on HF pathophysiology, we hypothesized that the cardioprotective effects of DPPIV may be accompanied by reduction of cardiac inflammation in experimental HF. HF was induced in Wistar rats by left ventricular (LV) radiofrequency ablation. Rats were divided into three groups: Sham, HF and HF+IDPPIV inhibitor (sitagliptin 80mg/kg/day). Six-week treatment with sitagliptin significantly improved LV ejection fraction, LV end diastolic volume, cardiac perfusion and mitigated cardiac hypertrophy. Additionally, cardiac infiltrate of macrophages increased in HF rats and treatment with sitaglipitin remarkably attenuated it. Moreover, HF rats not only sho...

Acute lung injury is a condition characterized by exacerbate inflammatory reaction in distal airways and lung dysfunction. Here we investigate the treatment of acute lung injury (ALI) by low level laser therapy (LLLT), an effective therapy used for the treatment of patients with inflammatory disorders or traumatic injuries, due to its ability to reduce inflammation and promote tissue regeneration. However, studies in internal viscera remains unclear. C57BL/6 mice were treated with intratracheal lipopolysaccharide (LPS) (5 mg/kg) or phosphate buffer saline (PBS). Six hours after instillation, two groups were irradiated with laser at 660 nm and radiant exposure of 10 J/cm2 . Intratracheal LPS inoculation induced a marked increase in the number of inflammatory cells in perivascular and alveolar spaces. There was also an increase in the expression and secretion of cytokines (TNF-α, IL-1β, IL-6,) and chemokine (MCP-1). The LLLT application induced a significant decrease in both inflammatory cells influx and inflammatory mediators secretion. These effects did not affect lung mechanical properties, since no change was observed in tissue resistance or elastance. In conclusion LLLT is able to reduce inflammatory reaction in lungs exposed to LPS without affecting the pulmonary function and recovery.

Summary Background & Aims Previous study from our laboratory showed the toxicity of oleic (OA) and linoleic acids (LA) on Jurkat and Raji cells and human lymphocytes in vitro. The mechanisms involved in the toxicity induced by OA and LA on Jurkat cells were determined in vitro. Methods Jurkat cells were treated in the presence of OA and LA (25, 50, 100 and 200 μM). The parameters investigated were: triglycerides and cholesterol ester concentrations determined by enzymatic assay, activation of peroxisome proliferator activated receptor (PPAR) by electrophoretic mobility shift assay, caspase 3, 6 and 8 activities by spectrofluorometric assay, tumor necrosis factor- α (TNF- α ) and interferon- γ production by enzyme linked absorbent assay (ELISA), expression of pro- (Bax) and anti- (Bcl-2) apoptotic genes by real time polymerase chain reaction and expression of pleiotropic genes by macroarray technique Results Evidence is presented herein that the increase in triglycerides concentrations induced by OA is more pronounced than that caused by LA in Jurkat cells. Importantly, triglycerides accumulation may be a mechanism to protect lymphocytes against the toxicity induced by fatty acids. Both fatty acids raised PPAR activation, caspase 3 and 6 activities and TNF-α production. LA in toxic concentrations modulated the expression of genes related to cell cycle, apoptosis, proliferation, oxidative stress, and cytokine receptors. Conclusion The findings reported herein support the cell death induced by OA and LA involved triglycerides accumulation, PPAR activation, caspase 3 and 6 activities and TNF- α production.