Sandra Rodrigues

Absence of melatonin induces night-time hepatic insulin resistance and increased gluconeogenesis due to stimulation of nocturnal unfolded protein response

It is known that the circadian rhythm in hepatic phosphoenolpyruvate carboxykinase expression (a limiting catalytic step of gluconeogenesis) and hepatic glucose production is maintained by both daily oscillation in autonomic inputs to the liver and night feeding behavior. However, increased glycemia and reduced melatonin (Mel) levels have been recently shown to coexist in diabetic patients at the end of the night period. In parallel, pinealectomy (PINX) is known to cause glucose intolerance with increased basal glycemia exclusively at the end of the night. The mechanisms that underlie this metabolic feature are not completely understood. Here, we demonstrate that PINX rats show night-time hepatic insulin resistance characterized by reduced insulin-stimulated RAC-α serine/threonine-protein kinase phosphorylation and increased phosphoenolpyruvate carboxykinase expression. In addition, PINX rats display increased conversion of pyruvate into glucose at the end of the night. The regulatory mechanism suggests the participation of unfolded protein response (UPR), because PINX induces night-time increase in activating transcription factor 6 expression and prompts a circadian fashion of immunoglobulin heavy chain-binding protein, activating transcription factor 4, and CCAAT/enhancer-binding protein-homologous protein expression with Zenith values at the dark period. PINX also caused a night-time increase in Tribble 3 and regulatory-associated protein of mammalian target of rapamycin; both were reduced in liver of PINX rats treated with Mel. Treatment of PINX rats with 4-phenyl butyric acid, an inhibitor of UPR, restored night-time hepatic insulin sensitivity and abrogated gluconeogenesis in PINX rats. Altogether, the present data show that a circadian oscillation of UPR occurs in the liver due to the absence of Mel. The nocturnal UPR activation is related with night-time hepatic insulin resistance and increased gluconeogenesis in PINX rats.

Maternal immune activation in late gestation increases neuroinflammation and aggravates experimental autoimmune encephalomyelitis in the offspring.

Multiple sclerosis (MS) is characterized by an autoimmune response against myelin antigens driven by autoreactive T cells. Several lines of evidence indicate that environmental factors, such as previous infection, can influence and trigger autoimmune responses. However, the importance of the gestational period, particularly under inflammatory conditions, on the modulation of MS and related neuroinflammation by the offspring is unknown. This study aimed to evaluate the impact of prenatal exposure to lipopolysaccharide (LPS) during late gestation on the neuroinflammatory response in primary mixed glial cultures and on the progression of experimental autoimmune encephalomyelitis (EAE, an animal model of MS) in the offspring. LPS (Escherichia coli 0127:B8, 120μg/kg) was administered intraperitoneally to pregnant C57BL/6J mice on gestational day 17, and the offspring were assigned to two experiments: (1) mixed glial cultures generated using the brain of neonates, stimulated in vitro with LPS, and (2) adult offspring immunized with MOG35-55. The EAE clinical symptoms were followed for 30days. Different sets of animals were sacrificed either during the onset (7days post-immunization [p.i.]), when spleen and lymph nodes were collected, or the peak of disease (20days p.i.), when CNS were collected for flow cytometry, cytokine production, and protein/mRNA-expression analysis. The primary CNS cultures from the LPS-treated group produced exaggerated amounts of IL-6, IL-1β and nitrites after in vitro stimulus, while IL-10 production was lowered compared to the data of the control group. Prenatal exposure to LPS worsened EAE disease severity in adult offspring, and this worsening was linked to increased CNS-infiltrating macrophages, Th1 cells and Th17 cells at the peak of EAE severity; additionally, exacerbated gliosis was evidenced in microglia (MHC II) and astrocytes (GFAP protein level and immunoreactivity). The IL-2, IL-6 and IL-17 levels in the spleen and lymph nodes were increased in the offspring of the LPS-exposed dams. Our results indicate that maternal immune activation during late gestation predispose the offspring to increased neuroinflammation and potentiate the autoimmune response and clinical manifestation of EAE.

IL‐18, TNF, and IFN‐γ alleles and genotypes are associated with susceptibility to chronic hepatitis B infection and severity of liver injury

This study evaluated the association of polymorphisms in the IL‐18 (−607C/A and −137C/G), IFNγ (+874 A/T), and TNF (−238 A/G and −308 A/G) genes with susceptibility to HBV infection and severity of liver injury. A total of 259 chronic HBV‐infected patients followed at the University Hospital, Faculty of Medicine of Ribeirão Preto, São Paulo, Brazil, and 202 healthy individuals were studied. Four Single Nucleotide Polymorphisms (SNPs) were amplified by Polymerase Chain Reaction (PCR). Liver biopsy was performed in 212 HBV‐infected patients and classified according to severity of liver fibrosis (scores 0–4) and necroinflammatory activity (HAI scores 0–18). TNF‐308*A allele (P < 0.001; OR = 2.16) and TNF −308 AA genotype (P = 0.026; OR = 5.43) were associated with susceptibility to HBV infection. An association was found between severe liver fibrosis when compared to mild fibrosis and the following polymorphisms: Alleles IL‐18 ‐137*G (P = 0.004; OR = 3.45), TNF −308*A (P < 0.001; OR = 3.39), and IFNγ +874*T (P = 0.029; OR = 1.85) and IL‐18 −137 GG genotype (P = 0.009; OR = 3.70). No significant association was found between IL‐18 (−607 A/C) polymorphism and severity of liver fibrosis. Alleles IL‐18 −137*G (P = 0.028; OR = 2.64) and TNF‐308*A (P = 0.002; OR = 3.06) and IL‐18 −137 GG genotype (P = 0.011; OR = 4.20) were associated with severe necroinflammatory activity (HAI>12) when compared to mild necroinflammatory activity (HAI 1–8). The results suggest that IL‐18 −137C/G, TNF‐308 G/A and IFNγ +874 A/T SNPs were associated to more severe liver injury in chronic HBV infection. TNF −308*A allele and TNF −308 AA genotype could play a role in the susceptibility to HBV infection. J. Med. Virol. 87:1689–1696, 2015.

Long-term disruption of maternal glucose homeostasis induced by prenatal glucocorticoid treatment correlates with miR-29 upregulation.

Excess of glucocorticoids (GCs) during pregnancy is strongly associated with the programming of glucose intolerance in the offspring. However, the impact of high GC levels on maternal metabolism is not clearly documented. This study aimed to test the hypothesis that mothers exposed to elevated levels of GCs might also display long-term disturbances in glucose homeostasis. Dexamethasone (DEX) was administered noninvasively to the mothers via drinking water between the 14th and the 19th days of pregnancy. Mothers were subjected to glucose and insulin tolerance tests at 1, 2, 3, 6, and 12 mo postweaning. Pregnant rats not treated with DEX and age-matched virgin rats were used as controls. Pancreatic islets were isolated at the 20th day of pregnancy and 12 mo postweaning in order to evaluate glucose-stimulated insulin secretion. The expression of the miR-29 family was also studied due to its responsiveness to GCs and its well-documented role in the regulation of pancreatic β-cell function. Rats treated with DEX during pregnancy presented long-term glucose intolerance and impaired insulin secretion. These changes correlated with 1) increased expression of miR-29 and its regulator p53, 2) reduced expression of syntaxin-1a, a direct target of miR-29, and 3) altered expression of genes related to cellular senescence. Our data demonstrate that the use of DEX during pregnancy results in deleterious outcomes to the maternal metabolism, hallmarked by reduced insulin secretion and glucose intolerance. This maternal metabolic programming might be a consequence of time-sustained upregulation of miR-29s in maternal pancreatic islets.

The HLA-G 14-base pair deletion allele and the deletion/deletion genotype are associated with persistent HBe antigenemia in chronic hepatis B infection

BACKGROUND AND AIMS HLA-G has well-recognized immunomodulatory properties, and this molecule is frequently expressed in the livers of hepatitis B virus (HBV)-infected patients. Because the HLA-G 14 bp-insertion/deletion polymorphism (rs371194629) has been associated with the magnitude of HLA-G expression, we evaluated this polymorphism in the recognized evolutionary forms of chronic HBV infection. METHODS We studied 196 chronic HBV-infected patients (118 HBeAg-negative chronic hepatitis, 53 HBeAg-positive chronic hepatitis and 25 inactive carriers exhibiting low levels of serum HBVDNA and persistently normal ALT levels), and 202 healthy individuals. Chronic hepatitis HLA-G typing was performed using PCR-amplified DNA hybridized with specific primers. RESULTS The frequencies of the insertion/deletion alleles and genotypes were very similar in patients and controls. After patient stratification according to the evolutionary form of the chronic HBV infection, the frequencies of the deletion allele (P=0.0460; OR=1.26; 95%CI=1.01-1.45) and of the deletion/deletion genotype (P=0.0356; OR=2.08; 95%CI=1.05-4.09) were overrepresented in HBeAg-positive patients when compared to HBeAg-negative patients. No differences were observed when HBV inactive carriers were compared to HBeAg-negative chronic hepatitis patients. CONCLUSIONS Because the 14-bp deletion allele has been associated with increased HLA-G production and because HLA-G may down regulate the cytotoxic activity of TCD8 and NK cells, patients exhibiting the 14-bp deletion allele at single or double doses are at increased risk for developing chronic forms of HBV associated with persistent viremia and worse prognoses.

TNFa-e Microsatellite‚ HLA-DRB1 and-DQB1 Alleles and Haplotypes in Brazilian Patients Presenting Recently Diagnosed Type 1 Diabetes Mellitus

Abstract:  TNF microsatellite and HLA class II polymorphisms were studied in 28 recently diagnosed Brazilian patients presenting type 1 diabetes mellitus (T1DM) and in 120 healthy controls. TNFa‐e and HLA‐DRB1/DQB1 alleles were identified using sets of sequence‐specific primers. Compared to controls, the DRB1*03 and DQB1*02 allele groups, TNFa1 allele, and the TNFa4‐b5‐c1‐d4‐e3 and TNFa10‐b5‐c1‐d4‐e3 haplotypes were overrepresented in patients. TNF microsatellite together with HLA polymorphisms is associated with type 1 diabetes in Brazilian patients, corroborating the participation of the MHC genes in disease susceptibility.

Repeated Domperidone treatment modulates pulmonary cytokines in LPS-induced acute lung injury in mice

&NA; The dopaminergic antagonist drug Domperidone has immunomodulatory effects. We investigated the effects of repeated Domperidone treatment in a model of Lypopolyssacharide (LPS)‐induced acute lung inflammation. Adult C57BL/6J mice were treated with either Vehicle or Domperidone for 5 days, and challenged intranasally with LPS in the following day. The behavior of mice was analyzed in the open field and elevated plus‐maze test before and 24 h after LPS challenge. The bronchoalveolar lavage fluid, blood and lung tissue were collected 24 h and 48 h after LPS challenge. Domperidone treatment increased LPS‐induced tumor necrosis factor (TNF) and interleukin (IL)‐6 production in the bronchoalveolar lavage fluid, without altering tissue damage and the number of immune cells in the lungs and circulation. Locomotor and anxiety‐like behavior were unchanged after Domperidone and/or LPS treatment. Cytokine data indicate that Domperidone promotes a change in activity of other cell types, likely alveolar epithelial cells, without affecting immune cell migration in the present model. Due to the role of these cytokines in progression of inflammation, Domperidone treatment may exacerbate a subsequent inflammatory injury.

Selective regulation of hepatic lipid metabolism by the AMP-activated protein kinase pathway in late-pregnant rats

The liver plays an essential role in maternal metabolic adaptation during late pregnancy. With regard to lipid metabolism, increased secretion of very low-density lipoprotein (VLDL) is characteristic of late pregnancy. Despite this well-described metabolic plasticity, the molecular changes underlying the hepatic adaptation to pregnancy remain unclear. As AMPK is a key intracellular energy sensor, we investigated whether this protein assumes a causal role in the hepatic adaptation to pregnancy. Pregnant Wistar rats were treated with vehicle or AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) for 5 days starting at gestational day 14. At the end of treatment, the rats were subjected to an intraperitoneal pyruvate tolerance test and in situ liver perfusion with pyruvate. The livers were processed for Western blot analysis, quantitative PCR, thin-layer chromatography, enzymatic activity, and glycogen content measurements. Blood biochemical profiles were also assessed. We found that AMPK and ACC phosphorylation were reduced in the livers of pregnant rats in parallel with a reduced level of hepatic gluconeogenesis of pyruvate. This effect was accompanied by both a reduction in the levels of hepatic triglycerides (TG) and an increase in circulating levels of TG. Treatment with AICAR restored hepatic levels of TG to those observed in nonpregnant rats. Additionally, AMPK activation reduced the upregulation of genes related to VLDL synthesis and secretion observed in the livers of pregnant rats. We conclude that the increased secretion of hepatic TG in late pregnancy is concurrent with a transcriptional profile that favors VLDL production. This transcriptional profile results from the reduction in hepatic AMPK activity.

Prolonged fasting elicits increased hepatic triglyceride accumulation in rats born to dexamethasone-treated mothers

We investigated the effect of dexamethasone during the last week of pregnancy on glucose and lipid metabolism in male offspring. Twelve-week old offspring were evaluated after fasting for 12-hours (physiological) and 60-hours (prolonged). Physiological fasting resulted in glucose intolerance, decreased glucose clearance after pyruvate load and increased PEPCK expression in rats born to dexamethasone-treated mothers (DEX). Prolonged fasting resulted in increased glucose tolerance and increased glucose clearance after pyruvate load in DEX. These modulations were accompanied by accumulation of hepatic triglycerides (TG). Sixty-hour fasted DEX also showed increased citrate synthase (CS) activity, ATP citrate lyase (ACLY) content, and pyruvate kinase 2 (pkm2), glucose transporter 1 (slc2a1) and lactate dehydrogenase-a (ldha) expressions. Hepatic AKT2 was increased in 60-hour fasted DEX, in parallel with reduced miRNAs targeting the AKT2 gene. Altogether, we show that metabolic programming by prenatal dexamethasone is characterized by an unexpected hepatic TG accumulation during prolonged fasting. The underlying mechanism may depend on increased hepatic glycolytic flux due to increased pkm2 expression and consequent conversion of pyruvate to non-esterified fatty acid synthesis due to increased CS activity and ACLY levels. Upregulation of AKT2 due to reduced miRNAs may serve as a permanent mechanism leading to increased pkm2 expression.

Maxadilan-simile expression in Nyssomyia neivai, a sandfly vector in an endemic region of Brazil, and its immunogenicity in patients with American tegumentary leishmaniasis

BACKGROUND Maxadilan (Max) is a salivary component in the sandfly Lutzomyia longipalpis (Lutz & Neiva 1912), a vector of visceral leishmaniasis. Max has a powerful vasodilatory effect and is a candidate vaccine that has been tested in experimental leishmaniasis. Nyssomyia neivai (Pinto 1926) is a vector of the pathogen responsible for American tegumentary leishmaniasis (ATL) in Brazil. OBJECTIVE We searched for Max expression in Ny. neivai and for antibodies against Max in ATL patients. METHODS cDNA and protein were extracted from the cephalic segment, including salivary glands, of Ny. neivai and analysed by polymerase chain reaction, DNA sequencing, and blotting assays. The results were compared with data obtained from Lu. longipalpis samples. We quantified antibodies against Max in serum samples from 41 patients with ATL (31 and 10 with the cutaneous and mucocutaneous forms, respectively) and 63 controls from the endemic northeastern region of São Paulo state, using enzyme-linked immunosorbent assay. FINDINGS Recognition of a Max-simile peptide by specific antibodies confirmed expression of a Max sequence in Ny. neivai (GenBank EF601123.1). Compared to controls, patients with ATL presented higher levels of antibodies against Max (p = 0.004); 24.4% of the patients with ATL and 3.2% of the controls presented anti-Max levels above the cutoff index (p = 0.014). The anti-Max levels were not associated with the specific clinical form of ATL, leishmanin skin test response, absence or presence of amastigotes in histopathologic exam, results of indirect immunofluorescence testing for leishmaniasis, or duration of cutaneous form disease. MAIN CONCLUSION High serum anti-Max levels did not protect patients against ATL, but confirmed previous natural exposure to Ny. neivai bites in this ATL endemic region.