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Mutations in the mitochondrial genome (mtDNA) are ubiquitous in humans and can lead to a broad spectrum of disorders. However, due to the presence of multiple mtDNA molecules in the cell, co-existence of mutant and wild-type mtDNAs (termed heteroplasmy) can mask disease phenotype unless a threshold of mutant molecules is reached. Importantly, the mutant mtDNA level can change across lifespan as mtDNA segregates in an allele- and cell-specific fashion, potentially leading to disease. Segregation of mtDNA is mainly evident in hepatic cells, resulting in an age-dependent increase of mtDNA variants, including non-synonymous potentially deleterious mutations. Here we modeled mtDNA segregation using a well-established heteroplasmic mouse line with mtDNA of NZB/BINJ and C57BL/6N origin on a C57BL/6N nuclear background. This mouse line showed a pronounced age-dependent NZB mtDNA accumulation in the liver, thus leading to enhanced respiration capacity per mtDNA molecule. Remarkably, liver-specific atg7 (autophagy related 7) knockout abolished NZB mtDNA accumulat ion, resulting in close-to-neutral mtDNA segregation through development into adulthood. prkn (parkin RBR E3 ubiquitin protein ligase) knockout also partially prevented NZB mtDNA accumulation in the liver, but to a lesser extent. Hence, we propose that age-related liver mtDNA segregation is a consequence of macroautophagic clearance of the less-fit mtDNA. Considering that NZB/BINJ and C57BL/6N mtDNAs have a level of divergence comparable to that between human Eurasian and African mtDNAs, these findings have potential implications for humans, including the safe use of mitochondrial replacement therapy.Abbreviations: Apob: apolipoprotein B; Atg1: autophagy-related 1; Atg7: autophagy related 7; Atp5a1: ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1; BL6: C57BL/6N mouse strain; BNIP3: BCL2/adenovirus E1B interacting protein 3; FCCP: carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; MAP1LC3A: microtubule-associated protein 1 light chain 3 alpha; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; mt-Atp8: mitochondrially encoded ATP synthase 8; MT-CO1: mitochondrially encoded cytochrome c oxidase I; MT-CO2: mitochondrially encoded cytochrome c oxidase II; mt-Co3: mitochondrially encoded cytochrome c oxidase III; mt-Cytb: mitochondrially encoded cytochrome b; mtDNA: mitochondrial DNA; MUL1: mitochondrial ubiquitin ligase activator of NFKB 1; nDNA: nuclear DNA; Ndufa9: NADH:ubiquinone oxireductase subunit A9; NDUFB8: NADH:ubiquinone oxireductase subunit B8; Nnt: nicotinamide nucleotide transhydrogenase; NZB: NZB/BINJ mouse strain; OXPHOS: oxidative phosphorylation; PINK1: PTEN induced putative kinase 1; Polg2: polymerase (DNA directed), gamma 2, accessory subunit; Ppara: peroxisome proliferator activated receptor alpha; Ppia: peptidylprolyl isomerase A; Prkn: parkin RBR E3 ubiquitin protein ligase; P10: post-natal day 10; P21: post-natal day 21; P100: post-natal day 100; qPCR: quantitative polymerase chain reaction; Rpl19: ribosomal protein L19; Rps18: ribosomal protein S18; SD: standard deviation; SEM: standard error of the mean; SDHB: succinate dehydrogenase complex, subunit B, iron sulfur (Ip); SQSTM1: sequestosome 1; Ssbp1: single-stranded DNA binding protein 1; TFAM: transcription factor A, mitochondrial; Tfb1m: transcription factor B1, mitochondrial; Tfb2m: transcription factor B2, mitochondrial; TOMM20: translocase of outer mitochondrial membrane 20; UQCRC2: ubiquinol cytochrome c reductase core protein 2; WT: wild-type.

Transient global ischemia is a leading cause of learning and memory dysfunction and induces a pattern of delayed neuronal death in the CA1 subfield of the hippocampus by down-regulating GluR2 mRNA AMPA receptors in this cerebral area. This study sought to investigate the neuroprotective effect of coumestrol against spatial memory impairment induced by global ischemia that leads to neural death by reducing the GluR2 receptors content in the hippocampal CA1 area. Our studies demonstrated that coumestrol administration prevented spatial memory deficits in mice. These findings suggest a cognitive enhancement role of coumestrol against cognitive impairment in ischemic events.

Background The activation of the hypothalamic–pituitary–adrenal (HPA) axis is essential for metabolic adaptation in response to fasting. However, the neurocircuitry connecting changes in the peripheral energy stores to the activity of hypothalamic paraventricular corticotrophin-releasing factor (CRFPVN) neurons, the master controller of the HPA axis activity, is not completely understood. Our main goal was to determine if hypothalamic arcuate nucleus (ARC) POMC and AgRP neurons can communicate fasting-induced changes in peripheral energy stores, associated to a fall in plasma leptin levels, to CRFPVN neurons to modulate the HPA axis activity in mice. Results We observed increased plasma corticosterone levels associate with increased CRFPVN mRNA expression and increased CRFPVN neuronal activity in 36 h fasted mice. These responses were associated with a fall in plasma leptin levels and changes in the mRNA expression of Agrp and Pomc in the ARC. Fasting-induced decrease in plasma leptin partially modulated these responses through a change in the activity of ARC neurons. The chemogenetic activation of POMCARC by DREADDs did not affect fasting-induced activation of the HPA axis. DREADDs inhibition of AgRPARC neurons reduced the content of CRFPVN and increased its accumulation in the median eminence but had no effect on corticosterone secretion induced by fasting. Conclusion Our data indicate that AgRPARC neurons are part of the neurocircuitry involved in the coupling of PVNCRF activity to changes in peripheral energy stores induced by prolonged fasting.

Obesity is mainly caused by excess energy intake and physical inactivity, and the number of overweight/obese individuals has been steadily increasing for decades. Previous studies showed that rodents fed westernized diets exhibit endocrine pancreas deterioration and a range of metabolic disorders. This study evaluated the effects of moderated aerobic treadmill exercise training on pancreatic islet cell viability and function in mice consuming a high-fat and sucrose diet. In the present study, 60-day-old male C57BL/6J mice were divided into four groups: control (C), fed a standard diet AIN-93M (3.83 kcal/g; 70% carbohydrate (cornstarch and dextrinized starch were chosen as the major source of carbohydrate for the AIN-93 diet. In addition, a small amount of sucrose), 20% protein (casein), and 10% fat (soybean) with no training (i.e., sedentary); C + training (CTR, fed the standard diet with eight weeks of exercise; high-fat diet + sucrose (HFDS), fed a high fat and sucrose diet (5.2 kcal/g; 20% carbohydrate (cornstarch and dextrinized starch were chosen as the major source of carbohydrate), 20% protein (casein), 60% fat (Lard was chosen as the major source of fat and a small amount of soybean) + 20% sucrose diluted in drinking water with no training; and HFDS + training (HFDSTR). After eight weeks, the HFDS mice displayed increased body weight (P

The present study aims to compare the responses between male and female C57BL/6 mice to multiple metabolic challenges to understand the importance of sex in the control of energy homeostasis.Male and female C57BL/6 mice were subjected to nutritional and hormonal challenges, such as food restriction and refeeding, diet-induced obesity, feeding response to ghrelin and leptin, ghrelin-induced growth hormone secretion, and central responsiveness to ghrelin and leptin. The hypothalamic expression of transcripts that control energy homeostasis was also evaluated.Male mice lost more weight and lean body mass in response to food restriction, compared to females. During refeeding, males accumulated more body fat and exhibited lower energy expenditure and glycemia, as compared to females. Additionally, female mice exhibited a higher protection against diet-induced obesity and related metabolic imbalances in comparison to males. Low dose ghrelin injection elicited higher food intake and growth hormone secretion in male mice, whereas the acute anorexigenic effect of leptin was more robust in females. However, the sex differences in the feeding responses to ghrelin and leptin were not explained by variations in the central responsiveness to these hormones nor by differences in the fiber density from arcuate nucleus neurons. Female, but not male, mice exhibited compensatory increases in hypothalamic Pomc mRNA levels in response to diet-induced obesity.Our findings revealed several sexually differentiated responses to metabolic challenges in C57BL/6 mice, highlighting the importance of taking into account sex differences in metabolic studies.

Leishmaniasis remains an important neglected tropical infection caused by the protozoan Leishmania and affects 12 million people in 98 countries. The treatment is limited with severe adverse effects. In the search for new therapies, the drug repositioning and combination therapy have been successfully applied to neglected diseases. The aim of the present study was to evaluate the in vitro and in vivo anti-Leishmania (Leishmania) amazonensis potential of triclosan, an approved topical antimicrobial agent used for surgical procedures. in vitro phenotypic studies of drug-treated parasites were performed to evaluate the lethal action of triclosan, accompanied by an isobolographic ex-vivo analysis with the association of triclosan and miltefosine. The results showed that triclosan has activity against L. (L.) amazonensis intracellular amastigotes, with a 50% inhibitory concentration of 16 μM. By using fluorescent probes and transmission electron microscopy, a pore-forming activity of triclosan toward the parasite plasma membrane was demonstrated, leading to depolarization of the mitochondrial membrane potential and reduction of the reactive oxygen species levels in the extracellular promastigotes. The in vitro interaction between triclosan and miltefosine in the combination therapy assay was classified as additive against intracellular amastigotes. Leishmania-infected mice were treated with topical triclosan (1% base cream for 14 consecutive days), and showed 89% reduction in the parasite burden. The obtained results contribute to the investigation of new alternatives for the treatment of cutaneous leishmaniasis and suggest that the coadministration of triclosan and miltefosine should be investigated in animal models.

This study investigated whether regulation of the renin-angiotensin system (RAS) by enalapril and/or aerobic exercise training (AET) causes browning of the subcutaneous white adipose tissue (sWAT). C57BL/6 mice were fed either a standard chow or a high-fat (HF) diet for 16 weeks. At Week 8, HF-fed animals were divided into sedentary (HF), enalapril (HF-E), AET (HF-T), and enalapril plus AET (HF-ET) groups. Subsequently, sWAT was extracted for morphometry, determination of RAS expression, and biomarkers of WAT browning. The HF group displayed adipocyte hypertrophy and induction of the classical RAS axis. Conversely, all interventions reduced adiposity and induced the counterregulatory RAS axis. However, only AET raised plasma irisin, increased peroxisome proliferator-activated receptor-γ coactivator-1α, and uncoupling protein-1 levels, and the expression of PR-domain containing 16 in sWAT. Therefore, we concluded that AET-induced sWAT browning was independent of the counterregulatory axis shifting of RAS in HF diet-induced obesity.

β2 -adrenoceptor agonists improve autophagy and re-establish proteostasis in cardiac cells; therefore, suggesting autophagy as a downstream effector of β2 -adrenoceptor signaling pathway. Here, we used the pharmacological and genetic tools to determine the autophagy effect of sustained β2 -adrenoceptor activation in rodents with neurogenic myopathy, which display impaired skeletal muscle autophagic flux. Sustained β2 -adrenoceptor activation using Formoterol (10 μg kg-1 day-1 ), starting at the onset of neurogenic myopathy, prevents disruption of autophagic flux in skeletal muscle 14 days after sciatic nerve constriction. These changes are followed by reduction of the cytotoxic protein levels and increased skeletal muscle cross-sectional area and contractility properties. Of interest, sustained administration of Formoterol at lower concentration (1 μg kg-1 day-1 ) induces similar improvements in skeletal muscle autophagic flux and contractility properties in neurogenic myopathy, without affecting the cross-sectional area. Sustained pharmacological inhibition of autophagy using Chloroquine (50 mg kg-1 day-1 ) abolishes the beneficial effects of β2 -adrenoceptor activation on the skeletal muscle proteostasis and contractility properties in neurogenic myopathy. Further supporting an autophagy mechanism for β2 -adrenoceptor activation, skeletal muscle-specific deletion of ATG7 blunts the beneficial effects of β2 -adrenoceptor on skeletal muscle proteostasis and contractility properties in neurogenic myopathy in mice. These findings suggest autophagy as a critical downstream effector of β2 -adrenoceptor signaling pathway in skeletal muscle.

What is the central question of this study? What is the effect of an obesogenic diet on the expression of microRNAs (miRNAs) involved in cardiac hypertrophy in female mice? What is the main finding and its importance? Female mice fed an obesogenic diet exhibited cardiac hypertrophy associated with increased levels of miRNA-143-3p, decreased mRNA levels of Sox6 and increased mRNA levels of Myh7. Inhibition of miRNA-143-3p increased Sox6 mRNA levels and reduced Myh7 expression in cardiomyocytes, and prevented angiotensin II-induced cardiomyocyte hypertrophy. The results indicate that the miRNA-143-3p-Sox6-Myh7 pathway may play a key role in obesity-induced cardiac hypertrophy.Obesity induces cardiometabolic disorders associated with a high risk of mortality. We have previously shown that the microRNA (miRNA) expression profile is changed in obesity-induced cardiac hypertrophy in male mice. Here, we investigated the effect of an obesogenic diet on the expression of miRNAs involved in cardiac hypertrophy in female mice. Female mice fed an obesogenic diet displayed an increased body weight gain, glucose intolerance, insulin resistance and dyslipidaemia. In addition, obese female mice exhibited cardiac hypertrophy associated with increased levels of several miRNAs, including miR-143-3p. Bioinformatic analysis identified Sox6, regulator of Myh7 gene transcription, as a predicted target of miR-143-3p. Female mice fed an obesogenic diet exhibited decreased mRNA levels of Sox6 and increased expression of Myh7 in the heart. Loss-of-function studies in cardiomyocytes revealed that inhibition of miR-143-3p increased Sox6 mRNA levels and reduced Myh7 expression. Collectively, our results indicate that obesity-associated cardiac hypertrophy in female mice is accompanied by alterations in diverse miRNAs, and suggest that the miR-143-3p-Sox6-Myh7 pathway may play a key role in obesity-induced cardiac hypertrophy.

Diabetic neuropathy, often associated with diabetes mellitus, is a painful condition with no known effective treatment except glycemic control. Studies with neuropathic pain models report alterations in cannabinoid and opioid receptor expression levels; receptors whose activation induces analgesia. We examined whether interactions between CB(1)R and opioid receptors could be targeted for the treatment of diabetic neuropathy. For this, we generated antibodies that selectively recognize native CB(1)R-MOR and CB(1)R-DOR heteromers using a subtractive immunization strategy. We assessed the levels of CB(1)R, MOR, DOR, and interacting complexes using a model of streptozotocin-induced diabetic neuropathy and detected increased levels of CB(1)R, MOR, DOR, and CB(1)R-MOR complexes compared with those in controls. An examination of G-protein signaling revealed that activity induced by the MOR, but not the DOR agonist, was potentiated by low nanomolar doses of CB(1)R ligands, including antagonists, suggesting an allosteric modulation of MOR signaling by CB(1)R ligands within CB(1)R-MOR complexes. Because the peptide endocannabinoid, hemopressin, caused a significant potentiation of MOR activity, we examined its effect on mechanical allodynia and found that it blocked allodynia in wild-type mice and mice with diabetic neuropathy lacking DOR (but have CB(1)R-MOR complexes). However, hemopressin does not alter the levels of CB(1)R-MOR complexes in diabetic mice lacking DOR but increases the levels of CB(1)R-DOR complexes in diabetic mice lacking MOR. Together, these results suggest the involvement of CB(1)R-MOR and CB(1)R-DOR complexes in diabetic neuropathy and that hemopressin could be developed as a potential therapeutic for the treatment of this painful condition.

PurposeSome first-line cytotoxic chemotherapics, e.g. doxorubicin, paclitaxel and oxaliplatin, induce activation of the immune system through immunogenic cell death (ICD). Tumor cells undergoing ICD function as a vaccine, releasing damage-associated molecular patterns (DAMPs), which act as adjuvants, and neoantigens of the tumor are recognized as antigens. ICD induction is rare, however it yields better and long-lasting antitumor responses to chemotherapy. Advanced metastatic melanoma (AMM) is incurable for more than half of patients. The discovery of ICD inducers against AMM is an interesting drug discovery strategy with high translational potential. Here we evaluated ICD induction of four highly cytotoxic chromomycins A (CA5-8).MethodsICD features and DAMPs were evaluated using several in vitro techniques with metastatic melanoma cell line (B16-F10) exposed to chromomcins A5-8 such as flow cytometry, western blot, RT-PCR and luminescence. Additionally in vivo vaccination assays with CA5-treated cells in a syngeneic murine model (C57Bl/6) were performed to confirm ICD evaluating the immune cells activation and their antitumor activity.ResultsB16-F10 treated with CA5-8 and doxorubicin exhibited ICD features such as autophagy and apoptosis, externalization of calreticulin, and releasing of HMGB1. However, CA5-treated cells had the best profile, also inducing ATP release, ERp57 externalization, phosphorylation of eIF2α and altering expression of transcription of genes related to autophagy, endoplasmic reticulum stress, and apoptosis. Bona fide ICD induction by CA5 was confirmed by vaccination of C57BL/6 mice with CA5-treated cells which activated antigen-presenting cells and T lymphocytes and stimulated antitumor activity.ConclusionCA5 induces bona fide immunogenic cell death on melanoma.

In nature, confrontations between conspecifics are recurrent and related, in general, due to the lack of resources such as food and territory. Adequate defence against a conspecific aggressor is essential for the individual's survival and the group integrity. However, repeated social defeat is a significant stressor promoting several behavioural changes, including social defence per se . What would be the neural basis of these behavioural changes? To build new hypotheses about this, we here investigate the effects of repeated social stress on the neural circuitry underlying motivated social defence behaviour in male mice. We observed that animals re-exposed to the aggressor three times spent more time in passive defence during the last exposure than in the first one. These animals also show less activation of the amygdalar and hypothalamic nuclei related to the processing of conspecific cues. In turn, we found no changes in the activation of the hypothalamic dorsal pre-mammillary nucleus (PMD) that is essential for passive defence. Therefore, our data suggest that the balance between the activity of circuits related to conspecific processing and the PMD determines the pattern of social defence behaviour. Changes in this balance may be the basis of the adaptations in social defence after repeated social defeat.

Made available in DSpace on 2022-04-28T19:50:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-02-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) We compared the parameters related to glucose homeostasis, and liver and muscle proteomes in fluorosis-susceptible (A/J; S) and fluorosis-resistant (129P3/J; R) mice in response to fluoride (F) exposure and exercise. Ninety male mice (45 R-mice and 45 S-mice) were randomized into three groups: (SI; RI) No-F, No-Exercise, (SII; RII) 50 ppm F, No-Exercise, (SIII; RIII) 50 ppm F, Exercise. Overall, mean F concentrations in the plasma and femur were significantly higher in R-mice compared with S-mice. In R-mice, exercise resulted in an increase in F accumulation in the femur. In S-mice, the mean plasma glucose level was significantly higher in Group II compared with Groups I and III. There was an increase in liver proteins involved in energy flux and antioxidant enzymes in non-exercise groups (I, II) of S-mice in comparison with the corresponding groups of R-mice. The results also showed a decrease in muscle protein expression in Group I S-mice compared with their R-mice counterparts. In conclusion, the findings suggest an increased state of oxidative stress in fluorosis-susceptible mice that might be exacerbated by the treatment with F. In addition, fluorosis-susceptible mice have plasma glucose levels higher than fluorosis-resistant mice on exposure to F, and this is not affected by exercise. Bauru School of Dentistry University of São Paulo, SP Department of Physical Education School of Sciences São Paulo State University, SP School of Human and Health Sciences University of Huddersfield Centre for Oral Health Research School of Dental Sciences Newcastle University School of Health and Life Sciences Teesside University Department of Physical Education School of Sciences São Paulo State University, SP CAPES: 001 FAPESP: 2014/50798-1 FAPESP: 2015/05278-2 FAPESP: 2015/12109-2 FAPESP: 2015/50406-9 FAPESP: 2016/20020-4

Obesity is a major concern for global healthcare systems. Systemic low-grade inflammation in obesity is a major risk factor for insulin resistance. Leptin is an adipokine secreted by the adipose tissue that functions by controlling food intake, leading to satiety. Leptin levels are increased in obesity. Here, we show that leptin enhances the effects of LPS in macrophages, intensifying the production of cytokines, glycolytic rates and morphological and functional changes in the mitochondria through an mTORC2-dependent, mTORC1-independent mechanism. Leptin also boosts the effects of IL-4 in macrophages, leading to increased oxygen consumption, expression of macrophage markers associated with a tissue repair phenotype, and wound healing. In vivo, hyperleptinemia caused by diet-induced obesity increases the inflammatory response by macrophages. Deletion of leptin receptor and subsequently of leptin signaling in myeloid cells (ObR-/-) is sufficient to improve insulin resistance in obese mice and decrease systemic inflammation. Our results indicate that leptin acts as a systemic nutritional checkpoint to regulate macrophage fitness and contributes to obesity-induced inflammation and insulin resistance. Thus, specific interventions aimed at downstream modulators of leptin signaling may represent new therapeutic targets to treat obesity-induced systemic inflammation.

Can exposure time to equilibration solutions during oocyte vitrification affect the lipid profile of oocytes and embryonic development? Could vitrification media supplemented with oleic, linoleic acids and L-carnitine effectively minimize damage induced by vitrification on embryo development and oocyte membrane lipid profile?Experimental study including 936 oocytes from C57BL/6J mice, randomly divided into fresh IVF (control) and equilibration solution groups. Oocytes were exposed to equilibration solution from Irvine Scientific, Tvitri-4 or Tvitri-4 supplemented with L-carnitine and fatty acids for 7 or 10 min, vitrified-warmed, and submitted to IVF. The lipid profile of oocytes immediately after equilibration solution exposure was also asessed using the same equilibration times and solution compositions.Longer equilibration time resulted in lower oocyte survival and blastocyst rates, and reduced relative abundance of structural lipids, i.e. phosphatidylcholines and sphingomyelins, varying according to equilibration solution composition. It also induced membrane disruptions resembling bubbles in the oocyte surface predominantly in equilibration solution from Irvine Scientific, rarely in Tvitri-4 and absent in Tvitri-4 supplemented with L-carnitine and fatty acids. To reveal the metabolic pathways associated with the equilibration phase of vitrification, lipid pathway analysis was conducted; both P-values and pathway impact values showed that the linoleic acid metabolism (P = 0.00223; impact =1) and alpha-Linolenic acid metabolism (P = 0.00084; impact = 0.33) were the most pathway perturbed, followed by glycerophospholipid metabolism (P = 0.0167; impact = 0.25) CONCLUSION: A longer equilibration phase pre-vitrification can influence embryo development and induce changes in oocyte lipid composition related to membrane integrity. The results suggest internalization of oleic and linoleic acids added to equilibration solution by the oocyte, which, to some extent, contributed to membrane phospholipids preservation, regardless of the equilibration times assessed.

The alveolar bone repair process may be influenced by multiple local and systemic factors, which include immune system cells and mediators. Macrophages allegedly play important roles in the repair process, and the transition of an initial inflammatory M1 profile into a pro-reparative M2 profile theoretically contributes to a favorable repair outcome. In this context, considering immunoregulatory molecules as potential targets for improving bone repair, this study evaluated the role of the immunoregulatory molecule FTY720, previously described to favor the development of the M2 phenotype, in the process of alveolar bone healing in C57Bl/6 (WT) mice. Experimental groups submitted to tooth extraction and maintained under control conditions or treated with FTY720 were evaluated by microtomographic (μCT), histomorphometric, immunohistochemical and molecular analysis to characterize healing and host response features at 0, 1, 3, 7 and 14 days. Our results demonstrated that the FTY720 group presented higher bone tissue density, higher bone tissue volume, greater tissue volume fraction, greater number and thickness of trabeculae and a higher number of osteoblasts and osteoclasts than the control group. Accordingly, the bone markers BMP2, BMP7, ALPL, SOST and RANK mRNA expressions increased in the FTY720 treated group. Furthermore, the levels of FIZZ, ARG2 and IL-10 mRNA increased in the FTY720 group together with the presence of CD206

Adolescents are more sensitive than adults to the neural and behavioral effects of psychostimulants, and exhibit greater vulnerability to drug abuse, dependence or relapse into these conditions. We have reported that cocaine pretreatment during adolescence promotes the expression of behavioral sensitization to a greater extent than when the pretreatment occurs at adulthood. Behavioral sensitization has been associated to the transition from drug use to addiction and is postulated to indicate heightened sensitivity to the appetitive motivational effects of drugs. The relationship between behavioral sensitization and conventional measures of drug reward, such as conditioned place preference (CPP), has yet to be thoroughly investigated, and little is known about age-related differences in this phenomenon. The present study tested cocaine-induced CPP in adolescent and adult mice exposed to cocaine (or vehicle) pretreatment, either in an intermittent or "binge" (i.e., heavy cocaine use on a single occasion, which increases the likelihood of experiencing cocaine-related problems) fashion. Cocaine administration induced behavioral sensitization to a greater extent in adolescent than in adult mice. Cocaine-induced CPP was fairly similar in vehicle pretreated adolescent and adult mice, yet greater in adolescent vs. adults after cocaine-induced sensitization. The results confirmed the higher sensitivity of adolescent mice to cocaine-induced behavioral sensitization and suggest its association with greater sensitivity to cocaine's rewarding effects.

Doxorubicin causes cardiotoxicity and exercise intolerance. Pre-conditioning exercise training seems to prevent doxorubicin-induced cardiac damage. However, the effectiveness of the cardioprotective effects of exercise training concomitantly with doxorubicin treatment remains largely unknown. To determine whether low-to-moderate intensity aerobic exercise training during doxorubicin treatment would prevent cardiotoxicity and exercise intolerance, we performed exercise training concomitantly with chronic doxorubicin treatment in mice. Ventricular structure and function were accessed by echocardiography, exercise tolerance by maximal exercise test, and cardiac biology by histological and molecular techniques. Doxorubicin-induced cardiotoxicity, evidenced by impaired ventricular function, cardiac atrophy, and fibrosis. Exercise training did not preserve left ventricular ejection fraction or reduced fibrosis. However, exercise training preserved myocardial circumferential strain alleviated cardiac atrophy and restored cardiomyocyte cross-sectional area. On the other hand, exercise training exacerbated doxorubicin-induced body wasting without affecting survival. Finally, exercise training blunted doxorubicin-induced exercise intolerance. Exercise training performed during doxorubicin-based chemotherapy can be a valuable approach to attenuate cardiotoxicity.

Ethnopharmacological relevance Sphagneticola trilobata (L.) Pruski is a plant species belonging to the Asteraceae family. Kaurenoid acid (KA) is a diterpene metabolite and one of the active ingredients of Sphagneticola trilobata (L.) Pruski. Extracts containing KA are used in traditional medicine to treat pain, inflammation, and infection. Aim The goal of the present study was to investigate the in vivo effects of KA (1–10 mg/kg, per oral gavage) upon LPS inoculation in mice by intraperitoneal (i.p.) or intraplantar (i.pl.; subcutaneous plantar injection) routes at the dose of 200 ng (200 μL or 25 μL, respectively). Methods In LPS paw inflammation, mechanical and thermal hyperalgesia MPO activity and oxidative imbalance (TBARS, GSH, ABTS and FRAP assays) were evaluated. In LPS peritonitis we evaluated leukocyte migration, cytokine production, oxidative stress, and NF-κB activation. Results KA inhibited LPS-induced mechanical and thermal hyperalgesia, MPO activity and modulated redox status in the mice paw. Pre- and post-treatment with KA inhibited migration of neutrophils and monocytes in LPS peritonitis. KA inhibited the pro-inflammatory/hyperalgesic cytokine (e.g., TNF-α, IL-1β and IL-33) production while enhanced anti-inflammatory/analgesic cytokine IL-10 in peritoneal cavity. In agreement with the effect of KA over pro-inflammatory cytokines it inhibited oxidative stress (total ROS, superoxide production and superoxide positive cells) and NF-κB activation during peritonitis. Conclusion KA efficiently dampens LPS-induced peritonitis and hyperalgesia in vivo, suggesting it as a suitable candidate to control excessive inflammation and pain during gram-negative bacterial infections and bringing mechanistic explanation to the ethnopharmacological application of Sphagneticola trilobata (L.) Pruski in inflammation and infection.

Background Sepsis is an emergency medical condition that can lead to death and it is defined as a life-threatening organ dysfunction caused by immune dysregulation in response to an infection. It is considered the main killer in intensive care units. Sepsis associated-encephalopathy (SAE) is mostly caused by a sepsis-induced systemic inflammatory response. Studies report SAE in 14–63% of septic patients. Main SAE symptoms are not specific and usually include acute impairment of consciousness, delirium and/or coma, along with electroencephalogram (EEG) changes. For those who recover from sepsis and SAE, impaired cognitive function, mobility and quality of life are often observed months to years after hospital discharge, and there is no treatment available today to prevent that. Inflammation and oxidative stress are key players for the SAE pathophysiology. Gold nanoparticles have been demonstrated to own important anti-inflammatory properties. It was also reported 20 nm citrate-covered gold nanoparticles (cit-AuNP) reduce oxidative stress. In this context, we tested whether 20 nm cit-AuNP could alleviate the acute changes caused by sepsis in brain of mice, with focus on inflammation. Sepsis was induced in female C57BL/6 mice by cecal ligation and puncture (CLP), 20 nm cit-AuNP or saline were intravenously (IV) injected 2 h after induction of sepsis and experiments performed 6 h after induction. Intravital microscopy was used for leukocyte and platelet adhesion study in brain, blood brain barrier (BBB) permeability carried out by Evans blue assay, cytokines measured by ELISA and real time PCR, cell adhesion molecules (CAMs) by flow cytometry and immunohistochemistry, and transcription factors, by western blotting. Results 20 nm cit-AuNP treatment reduced leukocyte and platelet adhesion to cerebral blood vessels, prevented BBB failure, reduced TNF- concentration in brain, and ICAM-1 expression both in circulating polymorphonuclear (PMN) leukocytes and cerebral blood vessels of mice with sepsis. Furthermore, 20 nm cit-AuNP did not interfere with the antibiotic effect on the survival rate of mice with sepsis. Conclusions Cit-AuNP showed important anti-inflammatory properties in the brain of mice with sepsis, being a potential candidate to be used as adjuvant drug along with antibiotics in the treatment of sepsis to avoid SAE

The hypothalamus mediates important exercise-induced metabolic adaptations, possibly via hormonal signals. Hypothalamic leptin receptor (LepR)- and steroidogenic factor 1 (SF1)-expressing neurons are directly responsive to growth hormone (GH) and deletion of GH receptor (GHR) in these cells impairs neuroendocrine responses during situations of metabolic stress. In the present study, we determined whether GHR ablation in LepR- or SF1-expressing cells modifies acute and chronic metabolic adaptations to exercise. Male mice carrying deletion of GHR in LepR- or SF1-expressing cells were submitted to 8 weeks of treadmill running training. Changes in aerobic performance and exercise-induced metabolic adaptations were determined. Mice carrying GHR deletion in LepR cells showed increased aerobic performance after 8 weeks of treadmill training, whereas GHR ablation in SF1 cells prevented improvement in running capacity. Trained mice carrying GHR ablation in SF1 cells exhibited increased fat mass and reduced cross-sectional area of the gastrocnemius muscle. In contrast, deletion of GHR in LepR cells reduced fat mass and increased gastrocnemius muscle hypertrophy, energy expenditure and voluntary locomotor activity in trained mice. Although glucose tolerance was not significantly affected by targeted deletions, glycemia before and immediately after maximum running tests was altered by GHR ablation. In conclusion, GHR signaling in hypothalamic neurons regulates the adaptation capacity to aerobic exercise in a cell-specific manner. These findings suggest that GH may represent a hormonal cue that informs specific hypothalamic neurons to produce exercise-induced acute and chronic metabolic adaptations.

Chikungunya virus (CHIKV) is responsible for a self-limited illness that can evolve into long-lasting painful joint inflammation. In this study, we report a novel experimental CHIKV vaccine formulation of lipid nanoparticles loaded with a recombinant protein derived from the E2 structural protein. This antigen fragment, designated ∆E2.1, maintained the antigenicity of the native viral protein and was specifically recognized by antibodies induced in CHIKV-infected patients. The antigen has been formulated into nanoparticles consisting of nano-multilamellar vesicles (NMVs) combined with the adjuvant monophosphoryl lipid A (MPLA). The vaccine formulation demonstrated a depot effect, leading to controlled antigen release, and induced strong antibody responses significantly higher than in mice immunized with the purified protein combined with the adjuvant. More relevantly, E2-specific antibodies raised in mice immunized with ∆E2.1-loaded NMV-MPLA neutralized CHIKV under in vitro conditions. Taken together, the results demonstrated that the new nanoparticle-based vaccine formulation represents a promising approach for the development of effective anti-CHIKV vaccines.

Background: Evidence has revealed an association between familial hypercholesterolemia and cognitive impairment. In this regard, a connection between cognitive deficits and hippocampal blood-brain barrier (BBB) breakdown was found in low-density lipoprotein receptor knockout mice (LDLr–/–), a mouse model of familial hypercholesterolemia. Objective: Herein we investigated the impact of a hypercholesterolemic diet on cognition and BBB function in C57BL/6 wild-type and LDLr–/–mice. Methods: Animals were fed with normal or high cholesterol diets for 30 days. Thus, wild-type and LDLr–/–mice were submitted to memory paradigms. Additionally, BBB integrity was evaluated in the mice’s prefrontal cortices and hippocampi. Results: A tenfold elevation in plasma cholesterol levels of LDLr–/–mice was observed after a hypercholesterolemic diet, while in wild-type mice, the hypercholesterolemic diet exposure increased plasma cholesterol levels only moderately and did not induce cognitive impairment. LDLr–/–mice presented memory impairment regardless of the diet. We observed BBB disruption as an increased permeability to sodium fluorescein in the prefrontal cortices and hippocampi and a decrease on hippocampal claudin-5 and occludin mRNA levels in both wild-type and LDLr–/–mice treated with a hypercholesterolemic diet. The LDLr–/–mice fed with a regular diet already presented BBB dysfunction. The BBB-increased leakage in the hippocampi of LDLr–/–mice was related to high microvessel content and intense astrogliosis, which did not occur in the control mice. Conclusion: Therefore, LDLr–/–mice seem to be more susceptible to cognitive impairments and BBB damage induced by exposure to a high cholesterol diet. Finally, BBB disruption appears to be a relevant event in hypercholesterolemia-induced brain alterations.

Este trabalho teve o objetivo de observar a resposta imune de camundongos BALB/c desencadeada por cepas de T. cruzi de graus variados virulência. A maioria dos estudos relacionados com a imunossupressão, durante a infecção experimental de camundongos, desencadeada pelo T. cruzi são restritos a cepas de alta virulência não levando em consideração sua diversidade na natureza. Durante a realização deste trabalho, procurou-se associar a supressão da resposta imune durante a infecção experimental pelo I cruzi em camundongos BALB/c com a virulência determinadas pelas cepas Y (de alta virulência), PF (avirulenta ) e SC38 (de média virulência). Este trabalho enfoca aspectos relacionados à imunidade humoral, através da dosagem de anticorpos dirigidos contra antígenos heterólogos, dosagem de anticorpos gamaglobulina total anti-Z cruzi, anticorpos isotípicos IgGl, IgG2a e IgE específicos para T. cruzi, bem como imunidade celular, através da dosagem de citocinas EL-4, IL-10, IFN-yeIL-12. Os resultados obtidos sugerem que a atividade imunossupressiva promovida pelo T. cruzi é variável de acordo com a virulência da cepa e que o grau de imunossupressão podem estar relacionado com as características biológicas particulares de cada cepa. As dosagens dos anticorpos isotípicos mostraram que a cepa SC38 induz produção de anticorpos IgG2a, em maior quantidade comparadas com a PF e Y, indicando um possível controle da infecção. A cepa PF mostrou que induz a produção de IgGl embora significativamente menor que aquelas encontradas nos grupos Y e SC38, indicando uma tendência a ativar linfócitos do tipo Th2. Os grupos Y e SC38 produziram IgE, embora sem diferença significativa entre os dois grupos. Durante a análise da produção de citocinas dos sobrenadantes de cultura de esplenócitos, oriundos de camundongos BALB/c mfectados com as diferentes cepas de T. cruzi, foi observada uma maior produção de IL-10 no grupo SC38, indicando uma forte tendência a ativar linfócitos do tipo Th2. Estes resultados refletem-se com a IL481 que apresentou maior expressividade para a SC38, embora a produção desta citocina tenha sido baixa em todos os grupos Embora a produção do IFN-y tenha sido igual nos sobrenadantes de culturas dos esplenócitos oriundos dos camundongos inoculados com as cepas de T. cruzi, no decorrer dos 90 dias, foi observado que a produção desta citocina é precoce, aparecendo do sétimo ao décimo quinto dia com as cepas Y e PF nas culturas que apresentavam apenas o meio. Com a cepa SC38, o aparecimento de IFN-y foi observado a partir do trigésimo dia sendo que a concentração desta citocina sobe gradualmente até o nonagésimo dia. Os resultados da produção de IFN-y refletem-se na produção de óxido nítrico (NO), onde nos esplenócitos estimulados com os antígenos solúveis da cepa Y o NO é detectado até o décimo quinto dia, enquanto que nos estimulados com antígenos da cepa SC38 a produção de NO é observada a partir do trigésimo dia subindo gradualmente até o nonagésimo. Isso demonstra um controle na produção de NO no grupo SC38. Baseado nos dados obtidos durante este trabalho, podemos afirmar que a atividade intrínseca imunossupressiva do T. cruzi está intimamente relacionada com a produção do NO, podendo esta produção ser determinada pelas características biológicas individuais de cada cepa. A produção de IL-10 e de IFN-y também são determinados pelas características individuais de cada cepa onde a IL-10 parece favorecer a sobrevivência dos camundongos, diminuindo os efeitos intrínsecos imunossupressivos induzidos pelo parasita. Além dessas citocinas, a produção expressiva, de anticorpos IgG2a e IgGl parece ser importante no controle da infecção pelo do T cruzi. The main puipose of this investigation was to observe the imtnune response of Balb/c mouse strain as a result of infection with T. cruzi straíns which present various leveis o f virulence. The majority of the immunosuppression studies involved in the I trypanosomiasis emphasized the infection with high virulent parasite strain. In the present study it was employed parasites which present high virulence (Y strain), median virulence (SC38) and low virulence (PF). It was determined the leveis o f heterologous antíbodies against sheep red bíood cells and specific antíbodies against T. cruzi from isotypes IgGl, IgG2a and IgE in the serum samples. It was also studied the secretíon of IL-4, IL-10, IFN-y and IL-12 cytolanes by spleen cells . The results showed that the immunosuppressive activity is variable according to the degree o f virulence o f the parasites which may be related with the particular biological features o f each strain. The infection by the median virulent strain, SC38, elicited the higher production of IgG2a antíbodies when compared to the other strains. These data may indicate one possibíe mechanism to control the infection. The low virulent strain, PF, generates production o f IgGl, even though in lower levei when compared with other strains. The IgE production was detected in serum samples from animais infected with the Y and SC38 strains, but without signifícant difference. The higher leveis of IL-10 and IL-4 were observed in the supematants from spleen cells from animais infected with SC38 strain. It was observed that the early production (days 7 and 15) of IFN-y by unstimulated spleen cells was present in the animais infected with the Y and PF strains o f T. cruzi, The production o f this cytokine in the SC38 strain infected group was delayed since it was detected only by day 30 and the leveis o f secretíon of IFN-y were increasing homogeneously untíí day 90. The nitric oxide production by spleen cells basically followed the same pattem as the one found for IFN-y production.84 Taken together, the resuits obtained in the present study showed that the intrinsic immunosuppressive activity that follows the T. cruzi infection is closely related to the levei ofnitric oxide production which may determine the biological feature of each strain. Also, the IL-10 and IFN-y may act antagonistically in this immunosuppressive process. On the other hand, the production of IgG2a and IgGl may play an important role controlling I cruzi infection. Dissertação (Mestrado)

Natural products are ecofriendly agents that can be used against parasitic diseases. Eimeria species cause eimeriosis in many birds and mammals and resistance to available medications used in the treatment of eimeriosis is emerging. We investigated the in vitro and in vivo activity of Morus nigra leaf extracts (MNLE) against sporulation of oocysts and infection of mice with Eimeria papillata. Phytochemical analysis of MNLE showed the presence of seven compounds and the in vitro effects of MNLE, amprolium, DettolTM, formalin, ethanol, and phenol were studied after incubation with oocysts before sporulation. Furthermore, infection of mice with E. papillata induced an oocyst output of approximately 12 × 105 oocysts/g of feces. MNLE significantly decreased oocyst output to approximately 86% and the total number of parasitic stages in the jejunum by approximately 87%. In addition, the reduction in the number of goblet cells in the jejuna of mice was increased after treatment. These findings suggest that mulberry exhibited powerful anticoccidial activity. Resumo Os produtos naturais são agentes ecologicamente corretos que podem ser usados contra doenças parasitárias. As espécies de Eimeria causam eimeriose em muitas aves e mamíferos e a resistência aos medicamentos disponíveis usados no tratamento da eimeriose está emergindo. Foram investigadas as atividades in vitro e in vivo dos extratos de folhas de Morus nigra (MNLE) contra esporulação de oocistos e infecção de camundongos com Eimeria papillata. A análise fitoquímica do MNLE mostrou a presença de sete compostos e os efeitos in vitro do MNLE, amprolium, DettolTM, formalina, etanol e fenol foram estudados após incubação com oocistos antes da esporulação. Além disso, a infecção de camundongos com E. papillata induziu uma produção de oocistos de aproximadamente 12 × 105 oocistos / g de fezes. O MNLE reduziu significativamente a produção de oocistos para aproximadamente 86%, e o número total de estágios parasitários no jejuno em aproximadamente 87%. Além disso, a redução no número de células caliciformes no jejuno de camundongos aumentou após o tratamento. Esses achados sugerem que a amoreira exibia uma poderosa atividade anticoccidiana.

Palmitoleic acid (POA, 16:1n-7) is a lipokine that has potential nutraceutical use to treat non-alcoholic fatty liver disease. We tested the effects of POA supplementation (daily oral gavage, 300 mg/Kg, 15 days) on murine liver inflammation induced by a high fat diet (HFD, 59% fat, 12 weeks). In HFD-fed mice, POA supplementation reduced serum insulin and improved insulin tolerance compared with oleic acid (OA, 300 mg/Kg). The livers of POA-treated mice exhibited less steatosis and inflammation than those of OA-treated mice with lower inflammatory cytokine levels and reduced toll-like receptor 4 protein content. The anti-inflammatory effects of POA in the liver were accompanied by a reduction in liver macrophages (LM, CD11c+; F4/80+; CD86+), an effect that could be triggered by peroxisome proliferator activated receptor (PPAR)-γ, a lipogenic transcription factor upregulated in livers of POA-treated mice. We also used HFD-fed mice with selective deletion of PPAR-γ in myeloid cells (PPAR-γ KOLyzCre+) to test whether the beneficial anti-inflammatory effects of POA are dependent on macrophages PPAR-γ. POA-mediated improvement of insulin tolerance was tightly dependent on myeloid PPAR-γ, while POA anti-inflammatory actions including the reduction in liver inflammatory cytokines were preserved in mice bearing myeloid cells deficient in PPAR-γ. This overlapped with increased CD206+ (M2a) cells and downregulation of CD86+ and CD11c+ liver macrophages. Moreover, POA supplementation increased hepatic AMPK activity and decreased expression of the fatty acid binding scavenger receptor, CD36. We conclude that POA controls liver inflammation triggered by fat accumulation through induction of M2a macrophages independently of myeloid cell PPAR-γ., Graphical abstract Unlabelled Image, Highlights • Palmitoleic acid (POA) supplementation reduced serum insulin and improved insulin tolerance; • Livers of POA-treated mice exhibited less steatosis and inflammation; • POA lowered the liver M1 macrophages population and the expression of inflammation-related immune-cell markers; • POA increased PPAR-γ, a transcription factor that regulates anti-inflammatory effects in macrophages; • However, POA reduced liver inflammation even in mice that lack PPAR-γ expression in myeloid cells; • POA controls liver inflammation through induction of M2a macrophages independently of PPAR-γ in myeloid cells.

Rocky Mountain spotted fever (RMSF) is a life-threatening tick-borne disease caused by Rickettsia rickettsii, which is widely distributed throughout the Americas. Over 4000 cases of RMSF are recorded annually in the United States, while only around 100 cases are reported in Brazil. Conversely, while case fatality rates in the United States oscillate around 5%, in Brazil they can surpass 70%, suggesting that differences in tick vectoring capacity, population sensitivity, and/or variability in virulence of the rickettsial strains may exist. In this study, we compared the susceptibility of C3H/HeN mice to two highly virulent strains of R. rickettsii, one from the United States (Sheila Smith) and the other from Brazil (Taia&ccedil, u). Animals inoculated with the Taia&ccedil, u strain succumbed to infection earlier and exhibited severe histological lesions in both liver and spleen sooner than mice infected with the Sheila Smith strain. These differences in survival and signs of the disease are not related to a greater proliferation of the Taia&ccedil, u strain, as there were no significant differences in the rickettsial load in mice tissues inoculated with either strain. The present study is the first step to experimentally assess differences in fatality rates of RMSF in two different regions of the American continent.

The application of a bronchoconstrictor, usually Methacholine (MCh), in respiratory mechanics studies is usually accompanied by the assessment of respiratory mechanics in a dose–response curve. The MCh used in the dose–response curve can be inhaled (i.h.) and intravenous (i.v.) and there are studies comparing i.v. bolus and i.h. MCh in both mice and rats. However, MCh i.v. can be injected at short time interval (bolus) or in continuous infusion. This comparison is relevant since the way MCh is applied influences the mathematical model. We chose an aging process scenario to compare both protocols. This study aims to compare respiratory mechanics of 3-, 6-, and 10-month SAMR1 mice and how both administration methods (continuous infusion and bolus) impact respiratory mechanics evaluation. Both protocols were capable of assessing the difference among ages and doses in: peak or plateau; and area under the curve analysis. The respiratory mechanics parameters were Rn, G, and H (two-way analysis of variance: groups and doses with a P Impact statement Respiratory mechanics studies are associated with fundamental research and translational studies; the present work thus investigates this particular matter. Our current research describes differences and similarities between two different ways of administrating a very prevalent bronchoconstrictor (methacholine) in an aging process scenario. The core issue of our work is related with troubles we find with the bolus protocol and the application of the mathematical model used to assess the respiratory mechanics. Our findings reveal the continuous infusion as an alternative to these problems and we hope to provide the proper foundations to a more reliable assessment in the respiratory field.

Influenza viruses are respiratory pathogens of public health concern worldwide with up to 650,000 deaths occurring each year. Seasonal influenza virus vaccines are employed to prevent disease, but with limited effectiveness. Development of a universal influenza virus vaccine with the potential to elicit long-lasting, broadly cross-reactive immune responses is necessary for reducing influenza virus prevalence. In this study, we have utilized lipid nanoparticle-encapsulated, nucleoside-modified mRNA vaccines to intradermally deliver a combination of conserved influenza virus antigens (hemagglutinin stalk, neuraminidase, matrix-2 ion channel, and nucleoprotein) and induce strong immune responses with substantial breadth and potency in a murine model. The immunity conferred by nucleoside-modified mRNA-lipid nanoparticle vaccines provided protection from challenge with pandemic H1N1 virus at 500 times the median lethal dose after administration of a single immunization, and the combination vaccine protected from morbidity at a dose of 50 ng per antigen. The broad protective potential of a single dose of combination vaccine was confirmed by challenge with a panel of group 1 influenza A viruses. These findings support the advancement of nucleoside-modified mRNA-lipid nanoparticle vaccines expressing multiple conserved antigens as universal influenza virus vaccine candidates.

Made available in DSpace on 2022-04-29T08:46:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-01-01 Background Fluoride has become widely used in dentistry because of its effectiveness in caries control. However, evidence indicates that excessive intake interferes with the metabolic processes of different tissues. Thus, this study aimed to investigate the effects of long-term exposure to F on the parotid salivary gland of mice, from the analysis of oxidative, proteomic and genotoxic parameters. Materials and methods The animals received deionized water containing 0, 10 or 50 mg/L of F, as sodium fluoride, for 60 days. After, parotid glands were collected for analysis of oxidative biochemistry, global proteomic profile, genotoxicity assessment and histopathological analyses. Results The results revealed that exposure to fluoride interfered in the biochemical homeostasis of the parotid gland, with increased levels of thiobarbituric acid reactive species and reduced glutathione in the exposed groups; as well as promoted alteration of the glandular proteomic profile in these groups, especially in structural proteins and proteins related to oxidative stress. However, genotoxic assessment demonstrated that exposure to fluoride did not interfere with DNA integrity in these concentrations and durations of exposure. Also, it was not observed histopathological alterations in parotid gland. Conclusions Thus, our results suggest that long-term exposure to fluoride promoted modulation of the proteomic and biochemical profile in the parotid glands, without inducing damage to the genetic component. These findings reinforce the importance of rationalizing the use of fluorides to maximize their preventative effects while minimizing the environmental risks. Laboratory of Functional and Structural Biology Institute of Biological Sciences Federal University of Para, PA Laboratory of Clinical Immunology and Oxidative Stress Pharmacy Faculty Institute of Health Science Federal University of Para, PA Evandro Chagas Institute, PA Department of Biological Sciences Bauru Dental School University of São Paulo, SP Department of Chemistry University of Nebraska-Lincoln Department of Preventive and Restorative Dentistry School of Dentistry São Paulo State University (UNESP), SP Department of Preventive and Restorative Dentistry School of Dentistry São Paulo State University (UNESP), SP

To endure prolonged fasting, animals undergo important acute physiological adjustments. However, whether severe fasting also leads to long-term metabolic adaptations is largely unknown. Forty-eight-hour fasting caused a pronounced weight loss in adult C57BL/6 male mice. Seven days of refeeding increased body adiposity to levels above baseline, whereas fasting-induced reductions in lean body mass and energy expenditure were not fully recovered. Respiratory exchange ratio and locomotor activity also remained altered. A fasting/refeeding cycle led to persistent suppression of Pomc mRNA levels and significant changes in the expression of histone deacetylases and DNA methyltransferases in the hypothalamus. Additionally, histone acetylation in the ventromedial nucleus of the hypothalamus was reduced by prolonged fasting and remained suppressed after refeeding. Mice subjected to 48-h fasting 30 days earlier exhibited higher body weight and fat mass compared to aged-matched animals that were never food-deprived. Furthermore, a previous fasting experience altered the changes in body weight, lean mass, energy expenditure and locomotor activity induced by a second cycle of fasting and refeeding. Notably, when acutely exposed to high-palatable/high-fat diet, mice that went through cumulative fasting episodes presented higher calorie intake and reduced energy expenditure and fat oxidation, compared to mice that had never been subjected to fasting. When chronically exposed to high-fat diet, mice that experienced cumulative fasting episodes showed higher gain of body and fat mass and reduced energy expenditure and calorie intake. In summary, cumulative episodes of prolonged fasting lead to hypothalamic epigenetic changes and long-lasting metabolic adaptations in mice.

Inflammatory bowel diseases are debilitating illnesses characterized by severe inflammation of the gastrointestinal tract. Treatments currently available are expensive and ineffective. We here investigated the role of red-light emitting diode (LED) on dextran sodium sulfate (DSS)-induced colitis. DSS was added to the drinking water of male mice at days 0, 2, 4 and withdrawn at day 6. LED irradiation was performed daily for 90s from day 6 to 9 on the right and left sides of the ventral surface and beside the external anal region. LED treatment decreased the amount of crypt dysplasia/edema, inflammatory infiltrates and ulcers, attenuated apoptosis and increased proliferation of crypt cells. Also, LED treatment induced expression of annexin A1 in the damaged epithelium, preserved the organization of claudin-1 and skewed cytokine profiling towards a more anti-inflammatory status. Thus, LED treatment promotes structural protection and modulates the inflammatory response, constituting a potential non-invasive and low-cost combined therapy to help patients achieve disease remission.

Multiple neuroendocrine, autonomic and behavioral responses are regulated by the paraventricular nucleus of the hypothalamus (PVH). Previous studies have shown that PVH neurons express the growth hormone (GH) receptor (GHR), although the role of GH signaling on PVH neurons is still unknown. Given the great heterogeneity of cell types located in the PVH, we performed a detailed analysis of the neurochemical identity of GH-responsive cells to understand the possible physiological importance of GH action on PVH neurons. GH-responsive cells were detected via the phosphorylated form of the signal transducer and activator of transcription-5 (pSTAT5) in adult male mice that received an intraperitoneal GH injection. Approximately 51% of GH-responsive cells in the PVH co-localized with the vesicular glutamate transporter 2. Rare co-localizations between pSTAT5 and vesicular GABA transporter or vasopressin were observed, whereas approximately 20% and 38% of oxytocin and tyrosine hydroxylase (TH) cells, respectively, were responsive to GH in the PVH. Approximately 55%, 35% and 63% of somatostatin, thyrotropin-releasing hormone (TRH) and corticotropin-releasing hormone (CRH) neurons expressed GH-induced pSTAT5, respectively. Additionally, 8%, 49% and 75% of neuroendocrine TH, TRH and CRH neurons, and 67%, 32% and 74% of nonneuroendocrine TH, TRH and CRH neurons were responsive to GH in the PVH of Fluoro-Gold-injected mice. Our findings suggest that GH action on PVH neurons is involved in the regulation of the thyroid, somatotropic and adrenal endocrine axes, possibly influencing homeostatic and stress responses.

Recommendations of therapeutic exercise in Duchenne muscular dystrophy are still controversial. The hypothesis that a low-intensity training (LIT) protocol leads to muscle adaptations on mdx mice model was tested. Dystrophic male mice with 8 weeks old were separated in exercised (mdxE, n= 8) and sedentary (mdxC, n= 8) groups. Wild-type mice were used as control (WT, n= 8) group. Exercised group underwent a LIT protocol (9 m/min, 30 min, 3 days/wk, 60 days) on a horizontal treadmill. At day 60 all animals were analyzed regarding parameters of markers of muscle lesion and extracellular matrix turnover of muscle tissue by collagens fibers on tibial anterior muscle. Histomorphometry attested that centrally located nuclei fibers and the coefficient of variance of minimal Feret’s diameter was similar in mdxE and mdxC groups (P= 1.000) and both groups presented higher mean values than WT group (P< 0.001). Fraction area of collagen fibers of mdxE group was lower than mdxC group (P= 0,027) and similar to WT group (P= 0,751). Intramuscular area of Col3 of the mdxE group was higher than mdxC and WT groups (P

Bisphenol A (BPA) is an endocrine disrupting chemical able to promote hormone-responsive tumors. The major route of BPA contamination being oral, the aim of the present study was to investigate BPA effects on oral cells. Here, we evaluated the impact of sub-chronic in vivo exposure to BPA and its in vitro effects on neoplastic and non-neoplastic oral cells. We evaluated the oral mucosa of mice chronically exposed to BPA (200 mg/L). The response of keratinocytes (NOK–SI) and Head and Neck (HN) Squamous Cell Carcinoma (SCC), HN12 and HN13 cell lines to BPA was examined. In vivo, BPA accumulated in oral tissues and caused an increase in epithelial proliferative activity. BPA disrupted the function of keratinocytes by altering pro-survival and proliferative pathways and the secretion of cytokines and growth factors. In tumor cells, BPA induced proliferative, invasive, pro-angiogenic, and epigenetic paths. Our data highlight the harmful effects of BPA on oral mucosa and, tumorigenic and non-tumorigenic cells. Additionally, BPA may be a modifier of oral cancer cell behavior by prompting a functional shift to a more aggressive phenotype.

A inflamação e a angiogênese atuam concomitantimente em diversas doenças crônicas. A maior parte destes processos são mediados através da interação entre a matriz extracelular e os receptores de integrina. A Alternagina-C (ALT-C) é uma proteína tipo desintegrina, derivada do veneno da serpente Bothrops alternatus. Estudos com a ALT-C mostraram efeitos na adesão celular mediada por colágeno tipo I e a capacidade de modular via integrina α2β1 diversos fatores de crescimento. Nosso objetivo foi investigar os efeitos da ALT-C em componentes chaves da angiogênese inflamatória induzida pelo modelo de implantes sintéticos de esponja em camundongos. O tratamento com ALT-C inibiu os principais componentes do tecido fibrovascular (inflamação e angiogênese) induzido pelos implantes sintéticos subcutâneos, e aumentou a fibrogênese. Efeitos anti-inflamatórios foram observados através da inibição de mastócitos e de NAG (atividade de macrófagos), e também pela inibição da citocina TNF-α e das quimiocinas MCP-1 e CXCL-1. A redução do conteúdo de hemoglobina, número de vasos e das concentrações das citocinas VEGF e FGF mostraram que a ALT-C é também anti-angiogênica. Todos essse fatores revelam que a ALT-C é uma forte candidata no desenvolvimento de terapias anti-inflamatórias e antiangiogênicas em processos inflamatórios crônicos. Inflammation and angiogenesis act concomitantly in several chronic diseases. Most of steps of these processes are mediated by integration between extracellular matrix and integrin receptors. Alternagin-C (ALT-C), is a disintegrin-like derived from the venom of Bothrops alternatus. Studies with ALT-C have showed effects in cell adhesion mediated by collagen type I and ability of modulate growth factors associated integrin α2β1. Our aim was to investigate the effects of ALT-C on key components of inflammatory angiogenesis in the murine sponge model. Polyester-polyurethane sponges, used as framework for fibrovascular tissue growth, were implanted in Balb/c mice. Treatment with ALT-C inhibited the main components keys fibrovascular tissue (inflammation and angiogenesis) induced by synthetic subcutaneous implants and also changing the fibrogenic component. Anti-inflammatory effects were observed by inhibition of mast cell and NAG (macrophage activity), and also by the inhibition of cytokines TNF-α and MCP-1 and chemokine CXCL-1. The reduction in hemoglobin content, number of vessels and the concentrations of VEGF and FGF cytokines, have shown that ALT-C is also anti-angiogenic. All these factors show that Alt-C is a strong candidate for the development of anti-inflammatory and anti-angiogenic therapies Dissertação (Mestrado)

Nonalcoholic fatty liver disease (NAFLD) prevails in obesity and is linked to several health complications including dyslipidemia and atherosclerosis. How exactly NAFLD induces atherogenic dyslipidemia to promote cardiovascular diseases is still elusive. Here, we identify Tsukushi (TSK) as a hepatokine induced in response to NAFLD. We show that both endoplasmic reticulum stress and inflammation promote the expression and release of TSK in mice. In humans, hepatic TSK expression is also associated with steatosis, and its circulating levels are markedly increased in patients suffering from acetaminophen-induced acute liver failure (ALF), a condition linked to severe hepatic inflammation. In these patients, elevated blood TSK levels were associated with decreased transplant-free survival at hospital discharge, suggesting that TSK could have a prognostic significance. Gain- and loss-of-function studies in mice revealed that TSK impacts systemic cholesterol homeostasis. TSK reduces circulating HDL cholesterol, lowers cholesterol efflux capacity, and decreases cholesterol-to-bile acid conversion in the liver. Our data identify the hepatokine TSK as a blood biomarker of liver stress that could link NAFLD to the development of atherogenic dyslipidemia and atherosclerosis.

Introduction: Nuocytes play an important role in Type 2 immunity. However, the contribution of ILC2s to allergic rhinitis remains to be clearly elucidated. Objective: To evaluate the role of nuocytes from mesenteric lymph node on allergic responses in mice. Methods: After intraperitoneal administration of interleukin IL-25 and IL-33 to wild-type and Il17br-/-Il1rl1-/- double-deficient mice, nuocytes were purified from the the nasal-associated lymphoid tissue and mesenteric lymph nodes. Then, we assessed productions of IL-5 and IL-13 in nuocytes’ cultures. Finally, we adoptively transferred the mesenteric lymph node-derived nuocytes from wild-type and Il17br-/-Il1rl1-/- mice to the murine model of allergic rhinitis to evaluate their roles in nasal allergic responses. Results: We showed that nuocytes in the mesenteric lymph nodes of wild-type mice were upregulated after application of IL-25 and IL-33, and were induced to produce IL-5 and IL-13. Numbers of sneezing and nasal rubbing as well as eosinophils were all enhanced after the adoptive transfer of wild-type nuocytes. Concentrations of IL-5, IL-13, IL-25 and IL-33 in nasal lavage fluid of allergic mice were also increased. However, nuocytes fromIl17br-/-Il1rl1-/- mice did not increase sneezing and nasal rubbing and eosinophilia, and upregulate the above cytokines in the nasal lavage fluid. Conclusion: The findings demonstrate that nuocytes from the mesenteric lymph nodes of wildtype mice promote allergic responses in a mouse model. Resumo Introdução: Os nuócitos desempenham um papel importante na imunidade do tipo 2. No entanto, a contribuição das interleucinas ILC2s na rinite alérgica ainda precisa ser elucidada. Objetivo: Avaliar o papel dos nuócitos de linfonodos mesentéricos nas respostas alérgicas em camundongos. Método: Após a administração intraperitoneal de interleucina (IL)-25 e IL-33 em camundongos do tipo selvagem e camundongos Il17br-/-Il1rl1-/- com deficiência dupla, os nuócitos foram purificados do tecido linfoide associado a mucosa nasal e linfonodos mesentéricos. Em seguida, avaliamos as produções de IL-5 e IL-13 em culturas de nuócitos. Finalmente, transferimos adotivamente os nuócitos derivados de linfonodos mesentéricos de camundongos do tipo selvagem e camundongos Il17br-/-Il1rl1-/- para o modelo murino de rinite alérgica para avaliar seu papel nas respostas alérgicas nasais. Resultados: Mostramos que os nuócitos nos linfonodos mesentéricos de camundongos do tipo selvagem estavam up-regulados após a aplicação de IL-25 e IL-33 e foram induzidos a produzir IL-5 e IL-13. Os espirros e friçcão nasal, bem como os eosinófilos, aumentaram após a transferência adotiva de nuócitos do tipo selvagem. As concentrações de IL-5, IL-13, IL-25 e IL-33 no líquido da lavagem nasal de camundongos alérgicos também estavam aumentadas. Entretanto, os nuócitos de camundongos Il17br-/-Il1rl1-/- não aumentaram os espirros e a friçcão nasal ou eosinofilia e up-regularam as citocinas acima no líquido de lavagem nasal. Conclusão: Os achados demonstram que os nuócitos dos linfonodos mesentéricos de camundongos selvagens promovem respostas alérgicas em um modelo de camundongo.

Objective To evaluate the effects of oxidative stress on insulin signaling in cardiac tissue of obese mice. Methods Thirty Swiss mice were equally divided (n=10) into three groups: Control Group, Obese Group, and Obese Group Treated with N-acetylcysteine. After obesity and insulin resistance were established, the obese mice were treated with N-acetylcysteine at a dose of 50mg/kg daily for 15 days via oral gavage. Results Higher blood glucose levels and nitrite and carbonyl contents, and lower protein levels of glutathione peroxidase and phosphorylated protein kinase B were observed in the obese group when compared with their respective control. On the other hand, treatment with N-acetylcysteine was effective in reducing blood glucose levels and nitrite and carbonyl contents, and significantly increased protein levels of glutathione peroxidase and phosphorylated protein kinase B compared to the Obese Group. Conclusion Obesity and/or a high-lipid diet may result in oxidative stress and insulin resistance in the heart tissue of obese mice, and the use of N-acetylcysteine as a methodological and therapeutic strategy suggested there is a relation between them. RESUMO Objetivo Avaliar os efeitos do estresse oxidativo sobre a sinalização da insulina em tecido cardíaco de camundongos obesos. Métodos Utilizaram-se 30 camundongos Swiss subdivididos igualmente (n=10) em três grupos: Grupo Controle, Grupo Obeso e Grupo Obeso Tratado com N-acetilcisteína. Após estabelecidas a obesidade e a resistência à insulina, os camundongos obesos foram tratados diariamente, durante 15 dias, via gavagem oral, com N-acetilcisteína na dose de 50mg/kg. Resultados Observaram-se maiores níveis de glicose sanguínea, conteúdos de nitrito e carbonil, e menores níveis proteicos de glutationa peroxidase e proteína quinase B fosforilada no Grupo Obeso quando comparado a seu respectivo controle. Por outro lado, o tratamento com N-acetilcisteína se mostrou eficiente em diminuir os níveis glicêmicos, os conteúdos de nitrito e carbonil, e aumentar significativamente os níveis proteicos de glutationa peroxidase e proteína quinase B fosforilada, quando comparados ao Grupo Obeso. Conclusão Obesidade e/ou dieta hiperlipídica levam a estresse oxidativo e à resistência à insulina no tecido cardíaco de camundongos obesos, e o uso da N-acetilcisteína como estratégia metodológica e terapêutica sugeriu haver relação entre ambos.