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The SARS-CoV-2 pandemic have been affecting millions of people worldwide, since the beginning of 2020. COVID-19 can cause a wide range of clinical symptoms, which varies from asymptomatic presentation to severe respiratory insufficiency, exacerbation of immune response, disseminated microthrombosis and multiple organ failure, which may lead to dead. Due to the rapid spread of SARS-CoV-2, the development of vaccines to minimize COVID-19 severity in the world population is imperious. One of the employed techniques to produce vaccines against emerging viruses is the synthesis of recombinant proteins, which can be used as immunizing agents. Based on the exposed, the aim of the present study was to verify the systemic and immunological effects of IM administration of recombinant Nucleocapsid protein (NP), derived from SARS-CoV-2 and produced by this research group, in 2 different strains of rats (Rattus norvegicus); Wistar and Lewis. For this purpose, experimental animals received 4 injections of NP, once a week, and were submitted to biochemical and histological analysis. Our results showed that NP inoculations were safe for the animals, which presented no clinical symptoms of worrying side effects, nor laboratorial alterations in the main biochemical and histological parameters, suggesting the absence of toxicity induced by NP. Moreover, NP injections successfully triggered the production of specific anti-SARS-CoV-2 IgG antibodies by both Wistar and Lewis rats, showing the sensitization to have been well sufficient for the immunization of these strains of rats. Additionally, we observed the local lung activation of the Bronchus-Associated Lymphoid Tissue (BALT) of rats in the NP groups, suggesting that NP elicits specific lung immune response. Although pre-clinical and clinical studies are still required, our data support the recombinant NP produced by this research group as a potential immunizing agent for massive vaccination, and may represent advantages upon other recombinant proteins, since it seems to induce specific pulmonary protection.

Background: A low-sodium (LS) diet reduces blood pressure, contributing to the prevention of cardiovascular diseases. However, intense dietary sodium restriction impairs insulin sensitivity and worsens lipid profile. Considering the benefits of aerobic exercise training (AET), the effect of LS diet and AET in hepatic lipid content and gene expression was investigated in LDL receptor knockout (LDLr-KO) mice. Methods: Twelve-week-old male LDLr-KO mice fed a normal sodium (NS) or LS diet were kept sedentary (S) or trained (T) for 90 days. Body mass, plasma lipids, insulin tolerance testing, hepatic triglyceride (TG) content, gene expression, and citrate synthase (CS) activity were determined. Results were compared by 2-way ANOVA and Tukey’s post-test. Results: Compared to NS, LS increased body mass and plasma TG, and impaired insulin sensitivity, which was prevented by AET. The LS-S group, but not the LS-T group, presented greater hepatic TG than the NS-S group. The LS diet increased the expression of genes related to insulin resistance (ApocIII, G6pc, Pck1) and reduced those involved in oxidative capacity (Prkaa1, Prkaa2, Ppara, Lipe) and lipoprotein assembly (Mttp). Conclusion: AET prevented the LS-diet-induced TG accumulation in the liver by improving insulin sensitivity and the expression of insulin-regulated genes and oxidative capacity.

Atherosclerosis is a chronic inflammatory disease characterized by the formation of atheroma plaque in the arterial wall, process that causes long-term obstruction of the arteries. Postmenopausal women tend to have greater body adiposity, factor that corroborate to increased risk of cardiovascular events and development of atherosclerosis. Therefore, the aim of this study was to evaluate the association of experimental model of atherosclerosis with ovarian deprivation and consumption of high-fat diet in metabolic, hemodynamic, and autonomic outcomes. 21 female ApoE-Ko were divided into 3 groups (n = 7 in each): control treated with normolipidic diet (C); ovariectomized treated with normolipidic diet (Ovx); ovariectomized treated with high-fat diet (OvxHF). Hemodynamic parameters, baroreflex sensitivity and cardiovascular autonomic modulation were evaluated. Values (mean ± standard error of mean) were analyzed by One-way ANOVA, followed by Tukey's post hoc (p

Advanced glycation end products (AGEs) play an important role in oxidative stress and inflammation, processes implicated in the development and progression of kidney dysfunction. In the present study, we investigated the participation of the pro-oxidant protein thioredoxin-interacting protein (TXNIP) and of epigenetic mechanisms on kidney tissue (in vivo, in non-diabetic rats) and on terminally differentiated glomerular podocytes (in vitro) chronically exposed to AGEs. AGEs induced total kidney and glomerular TXNIP expression and decreased H3K27me3 content. Concomitant treatment with the antioxidant N-acetyl-cysteine (NAC) reversed only the increased TXNIP expression. TXNIP expression positively correlated with proteinuria and negatively correlated with H3K27me3 content. In vitro studies in podocytes showed that 72 h exposure to AGEs decreased nephrin expression and increased Txnip, Nox4, Col4a1, and epithelial-to-mesenchymal transition (EMT) markers (Acta2, Snail1, and Tgfb1). Podocytes treatment with NAC reversed Nox4, Col4a1, Acta2, and Tgfb1 increased expression but did not abrogate the reduced expression of nephrin. MiR-29a expression was downregulated by AGEs in vivo, but not in vitro. In conclusion, treatment of non-diabetic rats with AGEs induced TXNIP expression and decreased the contents of the repressive epigenetic mark H3K27me3 and of miR-29a, potentially driving injury to glomerular filtration barrier and podocytes dysfunction.

Telomere length (TL) is a biomarker of cell aging, and its shortening has been linked to several age-related diseases. In Alzheimer's disease (AD), telomere shortening has been associated with neuroinflammation and oxidative stress. The majority of studies on TL in AD were based on leucocyte DNA, with little information about its status in the central nervous system. In addition to other neuroprotective effects, lithium has been implicated in the maintenance of TL. The present study aims to determine the effect of chronic lithium treatment on TL in different regions of the mouse brain, using a triple-transgenic mouse model (3xTg-AD). Eighteen transgenic and 22 wild-type (Wt) male mice were treated for eight months with chow containing 1.0 g (Li1) or 2.0 g (Li2) of lithium carbonate/kg, or standard chow (Li0). DNA was extracted from parietal cortex, hippocampus and olfactory epithelium and TL was quantified by real-time PCR. Chronic lithium treatment was associated with longer telomeres in the hippocampus (Li2, p = 0.0159) and in the parietal cortex (Li1, p = 0.0375) of 3xTg-AD compared to Wt. Our findings suggest that chronic lithium treatment does affect telomere maintenance, but the magnitude and nature of this effect depend on the working concentrations of lithium and characteristics of the tissue. This effect was observed when comparing 3xTg-AD with Wt mice, suggesting that the presence of AD pathology was required for the lithium modulation of TL.

Because of the paucity of information regarding metabolic effects of advanced glycation end products (AGEs) on liver, we evaluated effects of AGEs chronic administration in (1) insulin sensitivity; (2) hepatic expression of genes involved in AGEs, glucose and fat metabolism, oxidative stress and inflammation and; (3) hepatic morphology and glycogen content. Rats received intraperitoneally albumin modified (AlbAGE) or not by advanced glycation for 12 weeks. AlbAGE induced whole-body insulin resistance concomitantly with increased hepatic insulin sensitivity, evidenced by activation of AKT, inactivation of GSK3, increased hepatic glycogen content, and decreased expression of gluconeogenesis genes. Additionally there was reduction in hepatic fat content, in expression of lipogenic, pro-inflamatory and pro-oxidative genes and increase in reactive oxygen species and in nuclear expression of NRF2, a transcription factor essential to cytoprotective response. Although considered toxic, AGEs become protective when administered chronically, stimulating AKT signaling, which is involved in cellular defense and insulin sensitivity.

Monogenic familial hypercholesterolemia is characterized by impaired cellular uptake of apolipoprotein B containing lipoproteins. However, its consequences on whole-body cholesterol metabolism are unclear. We investigated cholesterol metabolism in wild-type mice (control) and in knockout (KO) mice for the low-density lipoprotein receptor (LDLR-KO) and for apolipoprotein E (apoE-KO) containing the genetic basis of the C57BL/6J mice, under a cholesterol-free diet. Cholesterol and "non-cholesterol" sterols (cholestanol, desmosterol, and lathosterol) were measured in plasma, tissues, as well as in feces as cholesterol and its bacterial modified products (neutral sterols) using gas chromatography/mass spectrometry, and bile acids were measured by an enzymatic method. Compared to controls, LDLR-KO mice have elevated plasma and whole-body cholesterol concentrations, but total fecal sterols are not modified, characterizing unaltered body cholesterol synthesis together with impaired body cholesterol excretion. ApoE-KO mice presented the highest concentrations of plasma cholesterol, whole-body cholesterol, cholestanol, total fecal sterols, and cholestanol, compatible with high cholesterol synthesis rate; the latter seems attributed to elevated body desmosterol (Bloch cholesterol synthesis pathway). Nonetheless, whole-body lathosterol (Kandutsch-Russel cholesterol synthesis pathway) decreased in both KO models, likely explaining the diminished fecal bile acids. We have demonstrated for the first time quantitative changes of cholesterol metabolism in experimental mouse models that explain differences between LDLR-KO and apoE-KO mice. These findings contribute to elucidate the metabolism of cholesterol in human hypercholesterolemia of genetic origin.

Background:The autoimmunity and inflammation phenomena observed in systemic sclerosis (SSc) lead to the dysregulation of type V collagen (Col V) and extracellular matrix (ECM) deposition of Col V, collagen I (Col I) and collagen III (Col III) fibers, characteristic of pulmonary fibroproliferative disease. T cells are the predominant inflammatory infiltrate in the lungs and are believed to produce cytokines that drive the synthesis of matrix proteins by fibroblasts, resulting in excessive fibrosis. Although most studies to date have focused on CD4+ T and CD20+ B immune cells in some SSc subsets, cytotoxic CD8+ T cells are also involved in the pathogenesis of SSc. We previously reported that reactivity to the ColV-self antigen predicts late SSc lung fibrosis in mice immunized with Col V after 120 days; however, the profile of underlying immune matrices in early SSc lung fibrosis remains unclear.Objectives:To quantify T and B-cells and collagen fibers in the days following immunization with Col V in a mouse model mimicking human lung involvement in SSc.Methods:Female C57BL/6 mice (n=18) were immunized subcutaneously with human Col V (150 μg) in complete Freund´s adjuvant, followed by two intramuscular boosters (IM-COLV). The control group (n=18) did not receive Col V. Three groups of animals (n=6 each) were euthanized on days 15, 30 and 45 after immunization. The lung sections were fixed in 10% buffered formalin, embedded in paraffin, and sectioned at 4 µm. Immunohistochemistry, immunofluorescence and histomorphometry were performed to phenotype and quantify CD4 and CD8 T-cells, CD20 B-cells, and Col I, III and V, respectively.Results:We found that T-cells (CD4+), cytotoxic T cells (CD8+), and B-cells (CD20+) were present in high densities in the lungs of IM-COLV mice, with variation in the absolute abundance of each of these immune cell lineages at 15, 30 and 45 days after immunization with Col V. In fact, a significantly higher density of CD4+, CD8+, and CD20+ was detected early along the connective matrix of alveolar septa at 15 days after Col V immunization. In addition, higher density of Col I, III and V fibers were also observed early (Table 1). Although early SSc-lung fibrosis is generally considered to be linked to inflammatory cytokines, we also infer that cytotoxic T-cells may induce alveolar cells apoptosis by triggering kinases on macrophages and then accelerate fibrotic scar.Conclusion:Our study quantitatively establishes that the densities of CD4+ CD8+ and CD20+, as well as Col I, Col III and Col V can be characterized as markers of early lung fibrosis in SSc. These findings suggest that cytotoxic T cells may induce apoptosis and secrete profibrotic cytokines to induce the deposition of collagen fibers in the lungs, inferring that early B cell depletion may improve early lung fibrosis in SSc.Post-Col V Immunization (Days)153045ControlIM-COLVControlIM-COLVControlIM-COLVImmune CellsCD4+ cells/mm2 (%)13.85 ± 0.4634.14 ± 1.55*12.63 ± 0.5623.38 ± 0.8314.32 ± 0.5223.62 ± 0.40CD8+ cells/mm2 (%)13.84 ± 0.6039.40 ± 0.99**12.42 ± 0.5222.87 ± 0.8512.93 ± 0.5723.07±1.00CD20+ cells/mm2 (%)12.12 ± 1.0133.84±1.43#11.70 ± 1.1415.23 ± 6.7012.46 ± 0.5028.24 ± 0.67Collagen FibersCol V (area fraction)0.05 ± 0.0030.12±0.010.08 ± 0.0030.14±0.01††0.07 ± 0.0030.11±0,003Col I (area fraction)0.16 ± 0,010.38±0.05##0.14 ± 0.010.25±0.030.15 ± 0.020.24±0.01Col III (area fraction)0,16 ± 0,0030.28±0.01†0.14 ± 0.0040.25±0.050.13± 0,0070.16±0.02Total Col µg/mg2.7 ± 0.293.99 ± 0.583.15 ± 0.193.77 ± 0.272.87 ± 0.304.99 ± 0.60Data are shown as mean ± SD. Comparisons were done using ANOVA/Dunn´s Multiple comparison testP#15 vs 30 and 45 days; ## 15 vs 30 and 45 days; † 15 vs 45 days; †† 30 vs 45 days. Col I, collagen type I; Col III, collagen type III; Col V, collagen type V.Disclosure of Interests:Vitoria Elias: None declared, Zelita Queiroz: None declared, Sergio Catanozi: None declared, Antonio Santos Filho: None declared, Sandra Fernezlian: None declared, Ana Paula Velosa: None declared, Percival D. Sampaio-Barros Speakers bureau: Actelion, Boehringer Ingelheim, Abbvie, Lilly, Novartis, Consultant of: Abbvie, Bayer, Boehringer Ingelheim, Lilly, Novartis, Pfizer, Vera Capelozzi: None declared, Walcy Teodoro: None declared

Aims Advanced glycated albumin (AGE-albumin) adversely impairs macrophage lipid homeostasis in vitro, which may be prevented by angiotensin receptor blockers. In vivo studies are inconclusive whether AGE-albumin itself plays important role in early-stage atherogenesis. We aimed at investigating how AGE-albumin by itself drives atherosclerosis development in dyslipidemic non-diabetic mice and if its effects are due to the activation of renin–angiotensin system in the arterial wall and the expression of genes and proteins involved in lipid flux. Methods and results Murine albumin glycation was induced by incubation with 10mM glycolaldehyde and C-albumin with PBS alone. Twelve-week-old-male apoE knockout mice were submitted to a daily IP injection of control (C) or AGE-albumin (2mg/mL) during 30days with or without losartan (LOS: 100mg/L; C+LOS and AGE+LOS). Aortic arch was removed, and gene expression was determined by RT-PCR and protein content by immunofluorescence. Plasma lipid and glucose levels were similar among groups. Systolic blood pressure was similarly reduced in both groups treated with LOS. In comparison to C-albumin, aortic lipid infiltration was 5.3 times increased by AGE-albumin, which was avoided by LOS. LOS prevented the enhancement induced by AGE-albumin in Ager, Tnf and Cybb mRNA levels but did not reduce Olr1 . Nfkb and Agt mRNA levels were unchanged by AGE-albumin. LOS similarly reduced Agtr1a mRNA level in both C and AGE-albumin groups. In AGE-albumin-treated mice, immunofluorescence for carboxymethyl-lysine, 4-hydroxynonenal and RAGE was respectively, 4.8, 2.6 and 1.7 times enhanced in comparison to C-albumin. These increases were all avoided by LOS. Conclusions AGE-albumin evokes a pre-stage of atherogenesis in dyslipidemic mice independently of the presence of diabetes mellitus or modulation in the RAS in part by the induction of lipid peroxidation and inflammation.

Interesterified fats are currently being used to replace trans fatty acids. However, their impact on biological pathways involved in the atherosclerosis development was not investigated. Weaning male LDLr-KO mice were fed for 16 weeks on a high-fat diet (40% energy as fat) containing polyunsaturated (PUFA), TRANS, palmitic (PALM), palmitic interesterified (PALM INTER), stearic (STEAR) or stearic interesterified (STEAR INTER). Plasma lipids, lipoprotein profile, arterial lesion area, macrophage infiltration, collagen content and inflammatory response modulation were determined. Macrophage cholesterol efflux and the arterial expression of cholesterol uptake and efflux receptors were also performed. The interesterification process did not alter plasma lipid concentrations. Although PALM INTER did not increase plasma cholesterol concentration as much as TRANS, the cholesterol enrichment in the LDL particle was similar in both groups. Moreover, PALM INTER induced the highest IL-1β, MCP-1 and IL-6 secretion from peritoneal macrophages as compared to others. This inflammatory response elicited by PALM INTER was confirmed in arterial wall, as compared to PALM. These deleterious effects of PALM INTER culminate in higher atherosclerotic lesion, macrophage infiltration and collagen content than PALM, STEAR, STEAR INTER and PUFA. These events can partially be attributed to a macrophage cholesterol accumulation, promoted by apoAI and HDL 2 -mediated cholesterol efflux impairment and increased Olr-1 and decreased Abca1 and Nr1h3 expressions in the arterial wall. Interesterified fats containing palmitic acid induce atherosclerosis development by promoting cholesterol accumulation in LDL particles and macrophagic cells, activating the inflammatory process in LDLr-KO mice.

A low-sodium (LS) diet has been shown to reduce blood pressure (BP) and the incidence of cardiovascular diseases. However, severe dietary sodium restriction promotes insulin resistance (IR) and dyslipidemia in animal models and humans. Thus, further clarification of the long-term consequences of LS is needed. Here, we investigated the effects of chronic LS on gastrocnemius gene and protein expression and lipidomics and its association with IR and plasma lipids in LDL receptor knockout mice. Three-month-old male mice were fed a normal sodium diet (NS; 0.5% Na; n = 12–19) or LS (0.06% Na; n = 14–20) over 90 days. Body mass (BM), BP, plasma total cholesterol, triacylglycerol (TG), glucose, hematocrit, and IR were evaluated. LS increased BM (9%), plasma TG (51%), blood glucose (19%), and IR (46%) when compared with the NS. RT-qPCR analysis revealed that genes involved in lipid uptake and oxidation were increased by the LS: Fabp3 (106%), Prkaa1 (46%), and Cpt1 (74%). Genes and proteins (assessed by Western blotting) involved in insulin signaling were not changed by the LS. Similarly, lipid species classically involved in muscle IR, such as diacylglycerols and ceramides detected by ultra-high-performance liquid chromatography coupled to mass spectrometry, were also unchanged by LS. Species of phosphatidylcholines (68%), phosphatidylinositol (90%), and free fatty acids (59%) increased while cardiolipins (41%) and acylcarnitines (9%) decreased in gastrocnemius in response to LS and were associated with glucose disposal rate. Together these results suggest that chronic LS alters glycerophospholipid and fatty acids species in gastrocnemius that may contribute to glucose and lipid homeostasis derangements in mice.

Interesterified fats are being widely used by the food industry in an attempt to replace trans fatty acids. The effect of interesterified fats containing palmitic or stearic acids on lipid metabolism and inflammatory signaling pathways in adipose and hepatic tissues was evaluated. Male LDLr-KO mice were fed a high-fat diet containing polyunsaturated (PUFA), palmitic (PALM), palmitic interesterified (PALM INTER), stearic (STEAR), or stearic interesterified (STEAR INTER) fats for 16 weeks. The expression of genes and protein levels involved in lipid metabolism and inflammatory processes in liver and white adipose tissue was determined by quantitative RT-PCR and by Western blot, respectively. The infiltration of inflammatory cells in hepatic and adipose tissues was determined by eosin and hematoxylin, while liver collagen content was determined by Sirius Red staining. Both interesterified fats increased liver collagen content and JNK phosphorylation. Additionally, the STEAR INTER group developed nonalcoholic steatohepatitis (NASH) associated with higher neutrophil infiltration. PALM INTER induced adipose tissue expansion and enlargement of adipocytes. Furthermore, PALM INTER triggered increased IKK phosphorylation and TNF&alpha, protein content, conditions associated with the upstream activation of the NFkB signaling pathway. STEAR INTER induced NASH, while PALM INTER triggered hepatic fibrosis and adipocyte hypertrophy with inflammatory response in LDLr-KO mice.

Phytosterol metabolism is unknown in the hypercholesterolemia of genetic origin. We investigated the metabolism of phytosterols in a cholesterol-free, phytosterol-containing standard diet in hypercholesterolemic mice knockouts for low density lipoprotein receptor (LDLR) and apolipoprotein E (apoE) mice compared to wild-type mice (controls). Phytosterols were measured in mice tissues by GCMS. ApoE-KO mice absorbed less phytosterols than LDLR-KO and the latter absorbed less phytosterols than control mice, because the intestinal campesterol content was low in both KO mice, and sitosterol was low in the intestine in apoE-KO mice as compared to LDLR-KO mice. Although the diet contained nine times more sitosterol than campesterol, the concentration of sitosterol was lower than that of campesterol in plasma in LDLR-KO, and in the liver in controls and in LDLR-KO, but only in apoE-KO. On the other hand, in the intestine sitosterol was higher than campesterol in controls, and in LDLR-KO but with a tendency only in apoE-KO. Because of the high dietary supply of sitosterol, sitosterol was better taken up by the intestine than campesterol, but the amount of sitosterol was lower than that of campesterol in the liver, while in the whole body the amounts of these phytosterols do not differ from each other. Therefore, via intestinal lymph less sitosterol than campesterol was transferred to the body. However, as compared to controls, in apoE-KO mice, but not in LDLR-KO mice, the increase in campesterol and sitosterol in plasma and in the whole body indicating that apoE-KO mice have a marked defect in the elimination of both phytosterols from the body.

Adaptive immune cells, including CD4For 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 0.05 considered significant.Compared with the controls, SLE-induced animals presented increased numbers of CD4Increased numbers of CD4

Made available in DSpace on 2018-11-26T22:38:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-05-25 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Little is known about advanced glycation end products (AGEs) participation in glucose homeostasis, a process in which skeletal muscle glucose transporter GLUT4 (Scl2 alpha 4 gene) plays a key role. This study investigated (1) the in vivo and in vitro effects of AGEs on Slc2 alpha 4/GLUT4 expression in skeletal muscle of healthy rats, and (2) the potential involvement of endoplasmic reticulum and inflammatory stress in the observed regulations. For in vivo analysis, rats were treated with advanced glycated rat albumin (AGE-albumin) for 12 weeks; for in vitro analysis, soleus muscles from normal rats were incubated with bovine AGE-albumin for 2.5 to 7.5 hours. In vivo, AGE-albumin induced whole-body insulin resistance; decreased (similar to 30%) Slc2 alpha 4 mRNA and GLUT4 protein content; and increased (similar to 30%) the nuclear content of nuclear factor NF-kappa-B p50 subunit (NFKB1), and cellular content of 78 kDa glucose-regulated protein (GRP78). In vitro, incubation with AGE-albumin decreased (similar to 50%) the Slc2 alpha 4/GLUT4 content; and increased cellular content of GRP78/94, phosphorylated-IKK-alpha/beta, nuclear content of NFKB1 and RELA, and the nuclear protein binding into Slc2 alpha 4 promoter NFKB-binding site. The data reveal that AGEs impair glucose homeostasis in non-diabetic states of increased AGEs concentration; an effect that involves activation of endoplasmic reticulum-and inflammatory-stress and repression of Slc2 alpha 4/GLUT4 expression. Univ Sao Paulo, Inst Biomed Sci, Dept Physiol & Biophys, Sao Paulo, Brazil Univ Sao Paulo, Fac Med, Hosp Clin HCFMUSP, Lab Carboidrato & Radioimunoensaio LIM 18, Sao Paulo, Brazil Univ Sao Paulo, Fac Med, Hosp Clin HCFMUSP, Lab Lipides LIM 10, Sao Paulo, SP, Brazil Univ Estadual Paulista, Fac Sci & Technol, Dept Physiotherapy, Sao Paulo, Brazil Univ Nove Julho UNINOVE, Programa Posgrad Med, Sao Paulo, Brazil Univ Estadual Paulista, Fac Sci & Technol, Dept Physiotherapy, Sao Paulo, Brazil FAPESP: 2012/20432-0 FAPESP: 2016/25155-5 FAPESP: 2012/18724-2 FAPESP: 2013/00713-7 FAPESP: 2014/17251-9 CNPq: 2016/15603-0

Apolipoprotein E knockout (apoE-KO) mice present synaptic loss, cognitive dysfunction, and high plasma lipid levels that may affect brain function simulating Alzheimer disease. Plasma and brain sterols were measured in apoE-KO and in wild type control mice on a cholesterol-free, phytosterol-containing diet by gas chromatography coupled to a mass spectrometer. Plasma cholesterol and phytosterols (campesterol and sitosterol) were higher in apoE-KO compared to control mice. Cholesterol precursors (desmosterol and lathosterol) were not detected in plasma of control mice but were present in apoE-KO mice. In the brain amounts of cholesterol, desmosterol, campesterol and 24-hydroxycholesterol were significantly lower in apoE-KO than in controls. There is a tendency in apoE-KO for lower values of 7α-hydroxycholesterol and 7β-hydroxycholesterol. Cholesterol content, synthesis rates (desmosterol) and export of 24-hydroxycholesterol are reduced in the brain of the severe hypercholesterolemic apoE-KO mice.

Because collagen type V (Col V) can be exposed in tissue injury, we hypothesized that oral administration of this collagen species modulates the inflammation and remodeling of experimental synovitis, avoiding joint destruction, and that the modulation may differ according to the temporal administration. Arthritis (IA, n = 20) was induced in Lewis rats by intraarticular (ia) injection of 500 μg of methylated bovine serum albumin (mBSA) emulsified in complete Freund's adjuvant (CFA) (10 μl) followed by an intraarticular booster of mBSA (50 μg) in saline (50 μl) administered at 7 and 14 days. The control group received saline (50 μl, ia). After the first intraarticular injection, ten IA animals were supplemented via gavage with Col V (500 μg/300 μl) daily for 30 days (IA/Suppl). The control group received saline (50 μL) and Col V supplement in the same way (Suppl). Col V oral administration in IA/Suppl led to 1) inhibited edema and severe inflammatory cell infiltration, 2) decreased collagen fiber content, 3) decreased collagen type I, 4) inhibited lymphocyte subpopulations and macrophages, 5) inhibited IL-1β, IL-10, IL-17 and TNF-α production and 6) increased expression of caspase-9 in the synovial tissue. In conclusion, Col V supplementation decreased synovial inflammation and the fibrotic response, possibly by increased the apoptosis of inflammatory cells.

Scope There have been conflicting reports on the usefulness of phytosterols (PS) in preventing atherosclerosis. We evaluated the effects of dietary PS supplementation in LDLr-KO male mice on the plasma and aorta sterol concentrations and on atherosclerotic lesion development. Methods and results Mice were fed a high fat diet (40% of energy) supplemented with or without PS (2% w/w, n = 10). Plasma and arterial wall cholesterol and PS concentrations, lesion area, macrophage infiltration, and mRNA expression from LOX-1, CD36, ABCA1 and ABCG1 in peritoneal macrophages were measured. After 16 weeks, the plasma cholesterol concentration in PS mice was lower than that in the controls ( p = 0.02) and in the arterial wall ( p = 0.03). Plasma PS concentrations were higher in PS-fed animals than in controls ( p n = 5) was smaller than that in controls ( p = 0.0062) and the macrophage area ( p = 0.0007). PS correlates negatively with arterial lipid content and macrophage ( r = −0.76; p p Conclusions Despite inducing an increase in PS plasma concentration, PS supplementation is not associated with its accumulation in the arterial wall and prevents atherosclerotic lesion development.

This study investigated the influence of sodium restriction and antihypertensive drugs on atherogenesis utilizing hypertensive (H) low-density lipoprotein-receptor knockout mice treated or not with losartan (Los) or hydralazine (Hyd) and fed low-sodium (LS) or normal-sodium (NS) chow. Despite reducing the blood pressure (BP) of H-LS mice, the LS diet caused arterial lipid infiltration due to increased plasma total cholesterol (TC) and triglycerides (TG). Los and Hyd reduced the BP of H-LS mice, and Los effectively prevented arterial injury, likely by reducing plasma TG and nonesterified fatty acids. Aortic lipid infiltration was lower in Los-treated H-LS mice (H-LS+Los) than in normotensive (N)-LS and H-LS mice. Aortic angiotensin II type 1 (AT1) receptor content was greater in H-NS than H-LS mice and in H-LS+Hyd than H-LS+Los mice. Carboxymethyl-lysine (CML) and receptor for advanced glycation end products (RAGE) immunostaining was greater in H-LS than H-NS mice. CML and RAGE levels were lower in LS animals treated with antihypertensive drugs, and Hyd enhanced the AT1 receptor level. Hyd also increased the gene expression of F4/80 but not tumor necrosis factor-α, interleukin (IL)-1β, IL-6, IL-10, intercellular adhesion molecule-1 or cluster of differentiation 66. The novelty of the current study is that in a murine model of simultaneous hypertension and hyperlipidemia, the pleiotropic effect of chronic, severe sodium restriction elicited aortic damage even with reduced BP. These negative effects on the arterial wall were reduced by AT1 receptor antagonism, demonstrating the influence of angiotensin II in atherogenesis induced by a severely LS diet.

Background Persistent systemic inflammation has been widely recognized in patients with chronic kidney disease (CKD), and is associated with increased risk of morbidity and mortality. Intervention therapies aiming for the blockade of inflammatory cytokines are considered attractive approaches for CKD patients with signs of chronic inflammation. In this context, thalidomide, due to its potent anti-inflammatory and immunomodulatory properties, may represent an alternative strategy of treatment. In the present study, we developed an experimental model of CKD with uraemia in mice, induced by a diet rich in adenine, which causes progressive renal dysfunction, resembling the human uraemic features. Inflammatory parameters were analysed in this model of CKD and the potential beneficial effects of thalidomide as an anti-inflammatory drug was also investigated. Methods C57/BL-6 mice were fed with an adenine-containing diet during a period of 6 weeks. Thirty mice were divided into three groups: Control group (animals receiving normal diet), ADE group (mice receiving adenine-containing diet) and ADE + TLD group (CKD mice receiving thalidomide, 30 mg/kg/day, by gavage). Besides biochemical and histopathological changes, local and systemic inflammatory parameters were also analysed, including expression of cytokines interleukin (IL)-1β, tumour necrosis factor-α, IL-6, IL-4 and IL-10 in kidney samples by real-time RT-PCR and quantification of serum levels of cytokines. Finally, the electrophoretic mobility shift assay (EMSA) for NF-κB was also examined. Results Adenine-fed mice developed advanced CKD characterized by a marked increase in serum urea, creatinine, phosphorus and intact parathyroid hormone (iPTH) levels. In addition, histological changes of tubulointerstitial injury, characterized by deposition of crystals in the kidney, accompanied by tubular dilatation, degeneration of proximal tubular epithelium with loss of the brush border, inflammatory cellular infiltration, foreign-body granuloma formation and interstitial fibrosis were also evident. By immunohistochemistry, Mac-2- and α-SMA-positive cells were identified in the tubulointerstitial compartment. Treatment with thalidomide significantly reduced serum urea, creatinine, phosphorus and iPTH levels and protected against tubulointerstitial injury. Local and systemic inflammation in the mice model of adenine-induced CKD was confirmed by the findings of significantly high expression of cytokine mRNA levels and NF-κB activation in the kidney tissue as well as marked increased serum levels of inflammatory cytokines. Thalidomide treatment significantly reduced gene expression of these cytokines and the activation of the NF-κB in the renal tissue and the circulating levels of cytokines. Conclusions Dietary adenine caused advanced CKD with uraemia in mice providing a useful experimental model to study molecular and morphological changes associated with this disease. The negative impact of inflammation in this CKD model was overcome by the marked anti-inflammatory effects of thalidomide, promoting renal protection.

Aim: To assess the renal effects of chronic exposure to advanced glycation end-products (AGEs) in the absence of diabetes and the potential impact of concomitant treatment with the antioxidant N-acetyl cysteine (NAC). Methods: Wistar rats received intraperitoneally 20 mg/kg/day of albumin modified (AlbAGE) or not (AlbC) by advanced glycation for 12 weeks and oral NAC (600mg/L; AlbAGE+NAC and AlbC+NAC, respectively). Biochemical, urinary and renal morphological analyses; carboxymethyl-lysine (CML, an AGE), CD68 (macrophage infiltration), and 4-hydroxynonenal (4-HNE, marker of oxidative stress) immunostaining; intrarenal mRNA expression of genes belonging to pathways related to AGEs (Ager, Ddost, Nfkb1), renin-angiotensin system (Agt, Ren, Ace), fibrosis (Tgfb1, Col4a1), oxidative stress (Nox4, Txnip), and apoptosis (Bax, Bcl2); and reactive oxidative species (ROS) content were performed. Results: AlbAGE significantly increased urine protein-to-creatinine ratio; glomerular area; renal CML content and macrophage infiltration; expression of Ager, Nfkb1, Agt, Ren, Tgfb1, Col4a1, Txnip, Bax/Bcl2 ratio; and 4-HNE and ROS contents. Some of these effects were attenuated by NAC concomitant treatment. Conclusion: Because AGEs are highly consumed in modern diets and implicated in the progression of different kidney diseases, NAC could be a therapeutic intervention to decrease renal damage, considering that long-term restriction of dietary AGEs is difficult to achieve in practice.

Pyramidal neuron loss in the hippocampal CM region is a very early hallmark of Alzheimer disease (AD). Lithium might be a therapeutic strategy for AD due to its neuroprotective and neurotrophic properties. This study used modern stereological techniques to investigate possible CM pyramidal neuron loss in 11-month-old triple transgenic AD (3xTg-AD) mice, and also the effects of therapeutic and subtherapeutic lithium doses on the number and density of CAl pyramidal neurons and volume of CAl pyramidal layer in 3xTg-AD and wild-type mice treated from 3 toil months of age. 3xTg-AD mice displayed CM pyramidal layer atrophy that is likely due to reduced neuronal volume because of the absence of neuronal loss. Both lithium treatments of 3xTg-AD mice, which already expressed AD-like pathology, had no effect on CM atrophy. However, lithium treatment of wild-type mice, at low (subtherapeutic) doses, induced a significant increase in total CM pyramidal neuron number that led to a significant increase in total CAl pyramidal layer volume. The lithium-induced increase in CM neuron number is highly consistent with previous evidence that adult neurogenesis can be exogenously induced in the CM pyramidal layer with impact on total CAl neuron number, thus raising the possibility of the chronic use of low-dose lithium as a strategy to help compensate for neuronal loss in CM and perhaps other typically non-neurogenic brain regions in various neurological diseases. With regard to AD, low-dose lithium intervention must be initiated as early as possible in the course of neuropathology for beneficial effects to occur. (C) 2016 Elsevier GmbH. All rights reserved.

Objective: We investigated the effect of advanced glycated albumin (AGE-albumin) on macrophage sensitivity to inflammation elicited by S100B calgranulin and lipopolysaccharide (LPS) and the mechanism by which HDL modulates this response. We also measured the influence of the culture medium, isolated from macrophages treated with AGE-albumin, on reverse cholesterol transport (RCT). Methods and results: Macrophages were incubated with control (C) or AGE-albumin in the presence or absence of HDL, followed by incubations with S100B or LPS. Also, culture medium obtained from cells treated with C- or AGE-albumin, following S100B or LPS stimulation was utilized to treat naive macrophages in order to evaluate cholesterol efflux and the expression of HDL receptors. In comparison with C-albumin, AGE-albumin, promoted a greater secretion of cytokines after stimulation with S100B or LPS. A greater amount of cytokines was also produced by macrophages treated with AGE-albumin even in the presence of HDL. Cytokine-enriched medium, drawn from incubations with AGE-albumin and S100B or LPS impaired the cholesterol efflux mediated by apoA-I (23% and 37%, respectively), HDL2 (43% and 47%, respectively) and HDL3 (20% and 8.5%, respectively) and reduced ABCA-1 protein level (16% and 26%, respectively). Conclusions: AGE-albumin primes macrophages for an inflammatory response impairing the RCT. Moreover, AGE-albumin abrogates the anti-inflammatory role of HDL, which may aggravate the development of atherosclerosis in DM.

Galvao FHF, Soler W, Pompeu E, Waisberg DR, Mello ES, Costa ACL, Teodoro W, Velosa AP, Capelozzi VL, Antonangelo L, Catanozi S, Martins A, Malbouisson LMS, Cruz RJ, Figueira ER, Filho JAR, Chaib E, D′Albuquerque LAC. Immunoglobulin G profile in hyperacute rejection after multivisceral xenotransplantation. Xenotransplantation 2012; 19: 298–304. © 2012 John Wiley & Sons A/S. Abstract: Introduction: Xenotransplantation is a potential solution for the high mortality of patients on the waiting list for multivisceral transplantation; nevertheless, hyperacute rejection (HAR) hampers this practice and motivates innovative research. In this report, we describe a model of multivisceral xenotransplantation in which we observed immunoglobulin G (IgG) involvement in HAR. Methods: We recovered en bloc multivisceral grafts (distal esophagus, stomach, small intestine, colon, liver, pancreas, and kidneys) from rabbits (n = 20) and implanted them in the swine (n = 15) or rabbits (n = 5, control). Three hours after graft reperfusion, we collected samples from all graft organs for histological study and to assess IgG fixation by immunofluorescence. Histopathologic findings were graded according to previously described methods. Results: No histopathological features of rejection were seen in the rabbit allografts. In the swine-to-rabbit grafts, features of HAR were moderate in the liver and severe in esophagus, stomach, intestines, spleen, pancreas, and kidney. Xenograft vessels were the central target of HAR. The main lesions included edema, hemorrhage, thrombosis, myosites, fibrinoid degeneration, and necrosis. IgG deposition was intense on cell membranes, mainly in the vascular endothelium. Conclusions: Rabbit-to-swine multivisceral xenotransplants undergo moderate HAR in the liver and severe HAR in the other organs. Moderate HAR in the liver suggests a degree of resistance to the humoral immune response in this organ. Strong IgG fixation in cell membranes, including vascular endothelium, confirms HAR characterized by a primary humoral immune response. This model allows appraisal of HAR in multiple organs and investigation of the liver’s relative resistance to this immune response.

We analyzed the impact of chronic exposure to urban air pollution on the development of atherosclerosis. Hyperlipemic mice (LDLR −/− ) were submitted to a high fat diet and air pollution for four months. We measured the susceptibility of LDL to oxidative modifications (TBARS), the presence of anti-oxLDL and an apoB-derived peptide (apoB-D) in blood and the degree of atherosclerosis in the aortic arch. Air pollution increased the susceptibility of LDL to oxidation as well as anti-oxLDL and anti-apo-B levels. These levels were even higher than in mice submitted to a high fat diet and non-polluted air. The lipid content of the atherosclerotic plaques in the aorta was increased in groups with a high cholesterol diet independently of the air quality. However, the thickness of the arterial wall was greater in mice fed a high lipid diet with polluted air. Thus, we conclude that urban air pollution exacerbates the susceptibility of LDL to oxidation, atherogenesis and vascular remodeling in hyperlipemic mice and that an immune response accompanies this process.

Thyroid hormone receptor β (TRβ also listed as THRB on the MGI Database)-selective agonists activate brown adipose tissue (BAT) thermogenesis, while only minimally affecting cardiac activity or lean body mass. Here, we tested the hypothesis that daily administration of the TRβ agonist GC-24 prevents the metabolic alterations associated with a hypercaloric diet. Rats were placed on a high-fat diet and after a month exhibited increased body weight (BW) and adiposity, fasting hyperglycemia and glucose intolerance, increased plasma levels of triglycerides, cholesterol, nonesterified fatty acids and interleukin-6. While GC-24 administration to these animals did not affect food ingestion or modified the progression of BW gain, it did increase energy expenditure, eliminating the increase in adiposity without causing cardiac hypertrophy. Fasting hyperglycemia remained unchanged, but treatment with GC-24 improved glucose tolerance by increasing insulin sensitivity, and also normalized plasma triglyceride levels. Plasma cholesterol levels were only partially normalized and liver cholesterol content remained high in the GC-24-treated animals. Gene expression in liver, skeletal muscle, and white adipose tissue was only minimally affected by treatment with GC-24, with the main target being BAT. In conclusion, during high-fat feeding treatment with the TRβ-selective agonist, GC-24 only partially improves metabolic control probably as a result of accelerating the resting metabolic rate.

Background Dietary salt restriction has been reported to adversely modify the plasma lipoprotein profile in hypertensive and in normotensive subjects. We investigated the effects of the low sodium intake (LSI) on the plasma lipoprotein profile and on inflammation and thrombosis biomarkers during the fasting and postprandial periods. Methods Non-obese, non-treated hypertensive adults (n = 41) were fed strictly controlled diets. An initial week on a control diet (CD, Na = 160 mmol/day) was followed by 3 weeks on LSI (Na = 60 mmol/day). At admission and on the last day of each period, the 24-h ambulatory blood pressure was monitored and blood was drawn after an overnight fasting period and after a fat-rich test meal. Results The dietary adherence was confirmed by 24-h urinary sodium excretion. Fasting triglyceride (TG), chylomicron-cholesterol, hsC-reactive protein (CRP), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) concentrations, renin activity, aldosterone, insulin, and homeostasis model assessment insulin resistance (HOMA-IR) values were higher, but non-esterified fatty acids (NEFA) were lower on LSI than on CD. For LSI, areas under the curve (AUC) of TG, chylomicron-cholesterol, apoB and the cholesterol/apoB ratio were increased, whereas AUC-NEFA was lowered. LSI did not modify body weight, hematocrit, fasting plasma cholesterol, glucose, adiponectin, leptin, fibrinogen and factor VII (FVII), and AUC of lipoprotein lipase and of lipoprotein remnants. Conclusion LSI induced alterations in the plasma lipoproteins and in inflammatory markers that are common features of the metabolic syndrome.

Background The pig is an essential model for liver transplantation research and training. However, it develops hemodynamic instability during the anhepatic phase, requiring a short anhepatic phase or an extracorporeal circulation not appropriate for training purposes because it increases the risk of intraoperative complications. In this article we describe an economical and reproductive experimental model for training surgeon fellows in liver transplantation, without veno-venous bypass, using a supraceliac aortic cross-clamping maneuver. Material and methods After liver liberation, we cross-clamped the supraceliac aorta and cross-clamped and divided the infrahepatic inferior vena cava (IVC), bile duct (BD), hepatic artery (HA), portal vein (PV), and suprahepatic IVC. We rapidly removed and flushed the liver ex situ, repositioned it orthotopically, and performed anastomosis in suprahepatic IVC, infrahepatic IVC and PV, reperfusing the liver. Lastly, we anastomosed the HA and BD. We also performed pulmonary artery catheter exams and recovery blood samples serially before and after graft reperfusion (beginning of anesthesia = basal; 5 min after reperfusion and 120 min after reperfusion = end-point) for hemodynamic and metabolic assessment. Results Transplantation fellows were able to perform the operations assisted by a senior surgeon. The median procedure time was 211 min (188-233 min). One pig died due to hemorrhage and 5 remained alive for up to 2 h after liver reperfusion, achieving at this time normal hemodynamic and metabolic parameters. Conclusions This model is suitable for training and experimentation, avoids venovenous bypass, is low cost, avoids immunological reaction, and prevents hemodynamic and metabolic complications.