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-1ß 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.

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.

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 ß-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.

Preparation and characterization of collagen-nanohydroxyapatite biocomposite scaffolds by cryogelation method for bone tissue engineering applications.

Recent efforts of bone repair focus on development of porous scaffolds for cell adhesion and proliferation. Collagen-nanohydroxyapatite (HA) scaffolds (70:30; 50:50; and 30:70 mass percentage) were produced by cryogelation technique using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide as crosslinking agents. A pure collagen scaffold was used as control. Morphology analysis revealed that all cryogels had highly porous structure with interconnective porosity and the nanoHA aggregates were randomly dispersed throughout the scaffold structure. Chemical analysis showed the presence of all major peaks related to collagen and HA in the biocomposites and indicated possible interaction between nanoHA aggregates and collagen molecules. Porosity analysis revealed an enhancement in the surface area as the nanoHA percentage increased in the collagen structure. The biocomposites showed improved mechanical properties as the nanoHA content increased in the scaffold. As expected, the swelling capacity decreased with the increase of nanoHA content. In vitro studies with osteoblasts cells showed that they were able to attach and spread in all cryogels surfaces. The presence of collagen-nanoHA biocomposites resulted in higher overall cellular proliferation compared to pure collagen scaffold. A statistically significant difference between collagen and collagen-nanoHA cryogels was observed after 21 day of cell culture. These innovative collagen-nanoHA cryogels could have potentially appealing application as scaffolds for bone regeneration.

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.

Mechanism of acute silver toxicity in the euryhaline copepod Acartia tonsa.

Acute silver effects on whole-body ion regulation and Na(+),K(+)-ATPase activity were evaluated in the euryhaline copepod Acartia tonsa. Experiments were run at 20 degrees C, three different salinities (5, 15 and 30 ppt), in either the absence or the presence of food (diatom Thalassiosira weissflogii; 2 x 10(4)cells/mL). Standard static-renewal procedures were used. Copepods were acutely (48 h) exposed to silver (AgNO(3)) concentrations equivalent to the 48-h EC10 (dissolved Ag=3, 49, and 94 microg/L), 48-h EC30 (dissolved Ag=5, 71, and 125 microg/L) or 48-h EC50 (dissolved Ag=7, 83, and 173 microg/L) values in the absence of food or to the 48-h EC50 (dissolved Ag=35, 90, and 178 microg/L) values in the presence of food. These values were previously determined under the same experimental conditions at salinities 5, 15 and 30 ppt, respectively. Endpoints analyzed were whole-body ion concentrations (Na(+), Cl(-), and Mg(2+)) and Na(+),K(+)-ATPase activity. In starved copepods, lower whole-body Na(+) and Mg(2+) concentrations were observed in salinities 5 and 30 ppt, respectively. Also a higher whole-body Na(+),K(+)-ATPase activity was observed in all salinities tested. Data from fed copepods indicate that all these salinity effects were completely associated with starvation. Silver exposure induced a decrease in the whole-body Mg(2+) concentration in starved copepods in salinities 5 and 30 ppt and a Na(+),K(+)-ATPase inhibition in both starved and fed copepods in all salinities tested. Thus, food addition in the experimental media completely protected against silver effects on Mg(2+) concentration, but not against those on Na(+),K(+)-ATPase activity. In starved copepods, enzyme inhibition was dependent on silver concentration and a relationship between this parameter and mortality was observed in all salinities tested. Therefore, Na(+),K(+)-ATPase molecules seem to be a key site for acute silver toxicity in marine invertebrates, as reported for freshwater fish and crustaceans.

Blood meal identification from mosquitoes collected at a commercial alligator farm.

Outbreaks of West Nile virus on a Florida alligator farm prompted an investigation of which species of mosquitoes were feeding on the animals at the farm. Mosquitoes were collected on 4 separate overnight trips in September and October 2003 by using CO2-baited Centers for Disease Control light traps and wooden resting boxes that were placed inside or near the alligator housing pens. Mosquitoes were identified to species, bloodfed individuals were separated, and their abdomens were removed for DNA extraction. The DNA was tested to determine the vertebrate origin of the blood meal by polymerase chain reaction (PCR) amplification by using 4 primer sets specific to crocodilians, alligators, mammals, and birds. PCR products were sequenced to identify hosts. Of the 37 mosquito blood meals tested, 13 blood meals were positively identified to species, and 7 blood meals of those 13 were from Alligator mississippiensis, the American alligator. Alligator blood was found in Culex erraticus, Mansonia dyari, and Ma. titillans, and to our knowledge, this represents the first report of these mosquito species feeding on American alligators.