Ethanol-induced colitis prevents oral tolerance induction in mice

The gut mucosa is a major site of contact with antigens from food and microbiota. Usually, these daily contacts with natural antigens do not result in inflammatory reactions; instead they result in a state of systemic hyporesponsiveness named oral tolerance. Inflammatory bowel diseases (IBD) are associated with the breakdown of the immunoregulatory mechanisms that maintain oral tolerance. Several animal models of IBD/colitis are available. In mice, these include targeted disruptions of the genes encoding cytokines, T cell subsets or signaling proteins. Colitis can also be induced by intrarectal administration of chemical substances such as 2,4,6-trinitrobenzene sulfonic acid in 50% ethanol. We report here a novel model of colitis induced by intrarectal administration of 50% ethanol alone. Ethanol-treated mice develop an inflammatory reaction in the colon characterized by an intense inflammatory infiltrate in the mucosa and submucosa of the large intestine. They also present up-regulation of both interferon gamma (IFN-gamma) and interleukin-4 (IL-4) production by cecal lymph node and splenic cells. These results suggest a mixed type of inflammation as the substrate of the colitis. Interestingly, cells from mesenteric lymph nodes of ethanol-treated mice present an increase in IFN-gamma production and a decrease in IL-4 production indicating that the cytokine balance is altered throughout the gut mucosa. Moreover, induction of oral tolerance to ovalbumin is abolished in these animals, strongly suggesting that ethanol-induced colitis interferes with immunoregulatory mechanisms in the intestinal mucosa. This novel model of colitis resembles human IBD. It is easy to reproduce and may help us to understand the mechanisms involved in IBD pathogenesis.

Lightweight, liquid-cooled, direct-drive generator for highpower wind turbines: motivation, concept, and performance

Thesis: A liquid-cooled, direct-drive, permanent-magnet, synchronous generator with helical, double-layer, non-overlapping windings formed from a copper conductor with a coaxial internal coolant conduit offers an excellent combination of attributes to reliably provide economic wind power for the coming generation of wind turbines with power ratings between 5 and 20MW. A generator based on the liquid-cooled architecture proposed here will be reliable and cost effective. Its smaller size and mass will reduce build, transport, and installation costs. Summary: Converting wind energy into electricity and transmitting it to an electrical power grid to supply consumers is a relatively new and rapidly developing method of electricity generation. In the most recent decade, the increase in wind energy’s share of overall energy production has been remarkable. Thousands of land-based and offshore wind turbines have been commissioned around the globe, and thousands more are being planned. The technologies have evolved rapidly and are continuing to evolve, and wind turbine sizes and power ratings are continually increasing. Many of the newer wind turbine designs feature drivetrains based on Direct-Drive, Permanent-Magnet, Synchronous Generators (DD-PMSGs). Being low-speed high-torque machines, the diameters of air-cooled DD-PMSGs become very large to generate higher levels of power. The largest direct-drive wind turbine generator in operation today, rated just below 8MW, is 12m in diameter and approximately 220 tonne. To generate higher powers, traditional DD-PMSGs would need to become extraordinarily large. A 15MW air-cooled direct-drive generator would be of colossal size and tremendous mass and no longer economically viable. One alternative to increasing diameter is instead to increase torque density. In a permanent magnet machine, this is best done by increasing the linear current density of the stator windings. However, greater linear current density results in more Joule heating, and the additional heat cannot be removed practically using a traditional air-cooling approach. Direct liquid cooling is more effective, and when applied directly to the stator windings, higher linear current densities can be sustained leading to substantial increases in torque density. The higher torque density, in turn, makes possible significant reductions in DD-PMSG size. Over the past five years, a multidisciplinary team of researchers has applied a holistic approach to explore the application of liquid cooling to permanent-magnet wind turbine generator design. The approach has considered wind energy markets and the economics of wind power, system reliability, electromagnetic behaviors and design, thermal design and performance, mechanical architecture and behaviors, and the performance modeling of installed wind turbines. This dissertation is based on seven publications that chronicle the work. The primary outcomes are the proposal of a novel generator architecture, a multidisciplinary set of analyses to predict the behaviors, and experimentation to demonstrate some of the key principles and validate the analyses. The proposed generator concept is a direct-drive, surface-magnet, synchronous generator with fractional-slot, duplex-helical, double-layer, non-overlapping windings formed from a copper conductor with a coaxial internal coolant conduit to accommodate liquid coolant flow. The novel liquid-cooling architecture is referred to as LC DD-PMSG. The first of the seven publications summarized in this dissertation discusses the technological and economic benefits and limitations of DD-PMSGs as applied to wind energy. The second publication addresses the long-term reliability of the proposed LC DD-PMSG design. Publication 3 examines the machine’s electromagnetic design, and Publication 4 introduces an optimization tool developed to quickly define basic machine parameters. The static and harmonic behaviors of the stator and rotor wheel structures are the subject of Publication 5. And finally, Publications 6 and 7 examine steady-state and transient thermal behaviors. There have been a number of ancillary concrete outcomes associated with the work including the following. X Intellectual Property (IP) for direct liquid cooling of stator windings via an embedded coaxial coolant conduit, IP for a lightweight wheel structure for lowspeed, high-torque electrical machinery, and IP for numerous other details of the LC DD-PMSG design X Analytical demonstrations of the equivalent reliability of the LC DD-PMSG; validated electromagnetic, thermal, structural, and dynamic prediction models; and an analytical demonstration of the superior partial load efficiency and annual energy output of an LC DD-PMSG design X A set of LC DD-PMSG design guidelines and an analytical tool to establish optimal geometries quickly and early on X Proposed 8 MW LC DD-PMSG concepts for both inner and outer rotor configurations Furthermore, three technologies introduced could be relevant across a broader spectrum of applications. 1) The cost optimization methodology developed as part of this work could be further improved to produce a simple tool to establish base geometries for various electromagnetic machine types. 2) The layered sheet-steel element construction technology used for the LC DD-PMSG stator and rotor wheel structures has potential for a wide range of applications. And finally, 3) the direct liquid-cooling technology could be beneficial in higher speed electromotive applications such as vehicular electric drives.

Glutathione peroxidase induction protects Saccharomyces cerevisiae sod1deltasod2delta double mutants against oxidative damage

Saccharomyces cerevisiae mutants deficient in superoxide dismutase genes (sod1delta, sod2delta and the double mutant) were subjected to H2O2 stress in the stationary phase. The highest sensitivity was observed in the sod2delta mutant, while the sod1deltasod2delta double mutant was not sensitive. Sod mutants had lower catalase activity (44%) than wild-type cells, independent of H2O2 stress. Untreated cells of sod1deltasod2delta double mutants showed increased glutathione peroxidase activity (126%), while sod1delta had lower activity (77%) than the wild type. Glutathione levels in sod1delta were increased (200-260%) after exposure to various H2O2 concentrations. In addition, the highest malondialdehyde levels could be observed without H2O2 treatment in sod1delta (167%) and sod2delta (225%) mutants. In contrast, the level of malondialdehyde in the sod1deltasod2delta double mutant was indistinguishable from that of the wild type. These results suggest that resistance to H2O2 by sod1deltasod2delta cells depends on the induction of glutathione peroxidase and is independent of catalase, and that glutathione is a primary antioxidant in the defense against H2O2 in stationary phase sod1delta mutants.

Cytotoxicity of chlorhexidine digluconate to murine macrophages and its effect on hydrogen peroxide and nitric oxide induction

Chlorhexidine, even at low concentrations, is toxic for a variety of eukaryotic cells; however, its effects on host immune cells are not well known. We evaluated in vitro chlorhexidine-induced cytotoxicity and its effects on reactive oxygen/nitrogen intermediate induction by murine peritoneal macrophages. Thioglycollate-induced cells were obtained from Swiss mice by peritoneal lavage with 5 ml of 10 mM phosphate-buffered saline, washed twice and resuspended (10(6) cells/ml) in appropriate medium for each test. Cell preparations contained more than 95% macrophages. The cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay and the presence of hydrogen peroxide (H2O2) and nitric oxide (NO) by the horseradish peroxidase-dependent oxidation of phenol red and Griess reaction, respectively. The midpoint cytotoxicity values for 1- and 24-h exposures were 61.12 ± 2.46 and 21.22 ± 2.44 µg/ml, respectively. Chlorhexidine did not induce synthesis or liberation of reactive oxygen/nitrogen intermediates. When macrophages were treated with various sub-toxic doses for 1 h (1, 5, 10, and 20 µg/ml) and 24 h (0.5, 1, and 5 µg/ml) and stimulated with 200 nM phorbol myristate acetate (PMA) solution, the H2O2 production was not altered; however, the NO production induced by 10 µg/ml lipopolysaccharide (LPS) solution varied from 14.47 ± 1.46 to 22.35 ± 1.94 µmol/l and 13.50 ± 1.42 to 20.44 ± 1.40 µmol/l (N = 5). The results showed that chlorhexidine has no immunostimulating activity and sub-toxic concentrations did not affect the response of macrophages to the soluble stimulus PMA but can interfere with the receptor-dependent stimulus LPS.

Induction of oral tolerance and the effect of interleukin-4 on murine skin allograft rejection

We studied the effect of oral and portal vein administration of alloantigens on mouse skin allograft survival. Graft receptor BALB/c mice received spleen cells (30, 90, 150 or 375 x 10(6)) from donor C57BL/6 mice intragastrically on three successive days, starting seven days before the skin graft. Allograft survival was significantly increased with the feeding of 150 x 10(6) allogeneic spleen cells by one gavage (median survival of 12 vs 14 days, P <= 0.005) or when 300 x 10(6) cells were given in six gavage (12 vs 14 days, P < 0.04). A similar effect was observed when 150 x 10(6) spleen cells were injected into the portal vein (12 vs 14 days, P <= 0.03). Furthermore, prolonged allograft survival was observed with subcutaneous (12 vs 16 days, P <= 0.002) or systemic (12 vs 15 days, P <= 0.016) application of murine interleukin-4 (IL-4), alone or in combination with spleen cell injection into the portal vein (12 vs 18 days, P <= 0.0018). Taken together, these results showed that tolerance induction with spleen cells expressing fully incompatible antigens by oral administration or intraportal injection partially down-modulates skin allograft rejection. Furthermore, these findings demonstrated for the first time the effect of subcutaneous or systemic IL-4 application on allograft skin survival suggesting its use as a beneficial support therapy in combination with a tolerance induction protocol.

Risk factors for excess weight loss and hypernatremia in exclusively breast-fed infants

Data were prospectively obtained from exclusively breast-fed healthy term neonates at birth and from healthy mothers with no obstetric complication to determine risk factors for excess weight loss and hypernatremia in exclusively breast-fed infants. Thirty-four neonates with a weight loss > or = 10% were diagnosed between April 2001 and January 2005. Six of 18 infants who were eligible for the study had hypernatremia. Breast conditions associated with breast-feeding difficulties (P < 0.05), primiparity (P < 0.005), less than four stools (P < 0.001), pink diaper (P < 0.001), delay at initiation of first breast giving (P < 0.01), birth by cesarean section (P < 0.05), extra heater usage (P < 0.005), extra heater usage among mothers who had appropriate conditions associated with breast-feeding (P < 0.001), mean weight loss in neonates with pink diaper (P < 0.05), mean uric acid concentration in neonates with pink diaper (P < 0.0001), fever in hypernatremic neonates (P < 0.02), and the correlation of weight loss with both serum sodium and uric acid concentrations (P < 0.02) were determined. Excessive weight loss occurs in exclusively breast-fed infants and can be complicated by hypernatremia and other morbidities. Prompt initiation of breast-feeding after delivery and prompt intervention if problems occur with breast-feeding, in particular poor breast attachment, breast engorgement, delayed breast milk "coming in", and nipple problems will help promote successful breast-feeding. Careful follow-up of breast-feeding dyads after discharge from hospital, especially regarding infant weight, is important to help detect inadequate breast-feeding. Environmental factors such as heaters may exacerbate infant dehydration.

Primary mechanism of apoptosis induction in a leukemia cell line by fraction FA-2-b-ß prepared from the mushroom Agaricus blazei Murill

Agaricus blazei Murill is a native Brazilian mushroom which functions primarily as an anticancer substance in transplanted mouse tumors. However, the mechanism underlying this function of A. blazei Murill remains obscure. The present study was carried out to investigate the effect of fraction FA-2-b-ß, an RNA-protein complex isolated from A. blazei Murill, on human leukemia HL-60 cells in vitro. Typical apoptotic characteristics were determined by morphological methods using DNA agarose gel electrophoresis and flow cytometry. The growth suppressive effect of fraction FA-2-b-ß on HL-60 cells in vitro occurred in a dose- (5-80 µg/mL) and time-dependent (24-96 h) manner. The proliferation of HL-60 cells (1 x 10(5) cells/mL) treated with 40 µg/mL of fraction FA-2-b-ß for 24-96 h and with 5-80 µg/mL for 96 h resulted in inhibitory rates ranging from 8 to 54.5%, and from 4.9 to 86.3%, respectively. Both telomerase activity determined by TRAP-ELISA and mRNA expression of the caspase-3 gene detected by RT-PCR were increased in HL-60 cells during fraction FA-2-b-ß treatment. The rate of apoptosis correlated negatively with the decrease of telomerase activity (r = 0.926, P < 0.05), but correlated positively with caspase-3 mRNA expression (r = 0.926, P < 0.05). These data show that fraction FA-2-b-ß can induce HL-60 cell apoptosis and that the combined effect of down-regulation of telomerase activity and up-regulation of mRNA expression of the caspase-3 gene could be the primary mechanism of induction of apoptosis. These findings provide strong evidence that fraction FA-2-b-ß could be of interest for the clinical treatment of acute leukemia.

Low expression of APAF-1XL in acute myeloid leukemia may be associated with the failure of remission induction therapy

Apoptotic protease activating factor 1 (APAF-1) has a critical role in the regulation of apoptosis. In the present study, the mRNA expression analysis of different APAF-1 transcripts (APAF-1S, APAF-1LC, APAF-1LN, and APAF-1XL) was analyzed in bone marrow samples from 37 patients with acute myeloid leukemia (newly diagnosed, with no previous treatment). APAF-1XL and APAF-1LN transcripts (with and without an extra WD-40 repeat region, respectively) were detected in all samples, although the major form expressed was APAF-1XL in 65% of the samples (group 1), while 35% of the samples expressed primarily APAF-1LN (group 2). Only 46% of the patients presented complete remission in response to remission induction therapy (represented by less than 5% marrow blasts and hematological recovery), all but 2 cases being from group 1, 21.6% did not attain complete remission (only 1 case from group 1), and 32.4% of the patients died early. Lower expression of APAF-1XL (APAF-1XL/APAF-1LN ratio <1.2) was associated with a poor response to therapy (P = 0.0005, Fisher exact test). Both groups showed similar characteristics regarding white blood cell counts, cytogenetic data or presence of gene rearrangements associated with good prognosis as AML1-ETO, CBFB-MYH11 and PML/RARA. Since it has been shown that only the isoforms with the extra WD-40 repeat region activate procaspase-9, we suggest that low procaspase-9 activation may also be involved in the deregulation of apoptosis and chemotherapy resistance in acute myeloid leukemia.

Parasympathetic dysfunction is associated with insulin resistance in fructose-fed female rats

The objective of the present study was to identify metabolic, cardiovascular and autonomic changes induced by fructose overload administered in the drinking water of rats for 8 weeks. Female Wistar rats (200-220 g) were divided into 2 groups: control (N = 8) and fructose-fed rats (N = 5; 100 mg/L fructose in drinking water for 8 weeks). The autonomic control of heart rate was evaluated by pharmacological blockade using atropine (3 mg/kg) and propranolol (4 mg/kg). The animals were submitted to an intravenous insulin tolerance test (ITT) and to blood glucose measurement. The fructose overload induced a significant increase in body weight (~10%) and in fasting glycemia (~28%). The rate constant of glucose disappearance (KITT) during ITT was lower in fructose-fed rats (3.25 ± 0.7%/min) compared with controls (4.95 ± 0.3%/min, P < 0.05) indicating insulin resistance. The fructose-fed group presented increased arterial pressure compared to controls (122 ± 3 vs 108 ± 1 mmHg, P < 0.05) and a reduction in vagal tonus (31 ± 9 vs 55 ± 5 bpm in controls, P < 0.05). No changes in sympathetic tonus were observed. A positive correlation, tested by the Pearson correlation, was demonstrable between cardiac vagal tonus and KITT (r = 0.8, P = 0.02). These data provided new information regarding the role of parasympathetic dysfunction associated with insulin resistance in the development of early metabolic and cardiovascular alterations induced by a high fructose diet.

Effect of a cholesterol-rich diet on the metabolism of the free and esterified cholesterol components of a nanoemulsion that resembles LDL in rabbits

We have shown that the free cholesterol (FC) and the cholesteryl ester (CE) moieties of a nanoemulsion with lipidic structure resembling low-density lipoproteins show distinct metabolic fate in subjects and that this may be related to the presence of dyslipidemia and atherosclerosis. The question was raised whether induction of hyperlipidemia and atherosclerosis in rabbits would affect the metabolic behavior of the two cholesterol forms. Male New Zealand rabbits aged 4-5 months were allocated to a control group (N = 17) fed regular chow and to a 1% cholesterol-fed group (N = 13) during a 2-month period. Subsequently, the nanoemulsion labeled with ³H-FC and 14C-CE was injected intravenously for the determination of plasma kinetics and tissue uptake of the radioactive labels. In controls, FC and CE had similar plasma kinetics (fractional clearance rate, FCR = 0.234 ± 0.056 and 0.170 ± 0.038 h-1, respectively; P = 0.065). In cholesterol-fed rabbits, the clearance of both labels was delayed and, as a remarkable feature, FC-FCR (0.089 ± 0.033 h-1) was considerably greater than CE-FCR (0.046 ± 0.010 h-1; P = 0.026). In the liver, the major nanoemulsion uptake site, uptake of the labels was similar in control animals (FC = 0.2256 ± 0.1475 and CE = 0.2135 ± 0.1580%/g) but in cholesterol-fed animals FC uptake (0.0890 ± 0.0319%/g) was greater than CE uptake (0.0595 ± 0.0207%/g; P < 0.05). Therefore, whereas in controls, FC and CE have similar metabolism, the induction of dyslipidemia and atherosclerosis resulted in dissociation of the two forms of cholesterol.

Effect of praziquantel administration on hepatic stereology of mice infected with Schistosoma mansoni and fed a low-protein diet

A study was undertaken to investigate the effect of administering praziquantel (PZQ), focusing on the liver stereological findings of malnourished mice infected with Schistosoma mansoni. Thirty female Swiss Webster mice (age: 21 days; weight: 8-14 g) were fed either a low-protein diet (8%) or standard chow (22% protein) for 15 days. Five mice in each group were infected with 50 cercariae each of the BH strain (Brazil). PZQ therapy (80 mg/kg body weight, per day) was started on the 50th day of infection and consisted of daily administration for 5 days. Volume density (hepatocytes, sinusoids and hepatic fibrosis) was determined by stereology using a light microscope. Body weight gain and total serum albumin levels were always lower in undernourished mice. Our stereological study demonstrated that treatment increased both volume density of hepatocytes in mice fed standard chow (47.56%, treated group and 12.06%, control) and low-protein chow (30.98%, treated group and 21.44%, control), and hepatic sinusoids [standard chow (12.52%, treated group and 9.06%, control), low-protein chow (14.42%, treated group and 8.46%, control)], while hepatic fibrosis was reduced [standard chow (39.92%, treated group and 78.88%, control) and low-protein chow (54.60%, treated group and 70.10%, control)]. On the other hand, mice fed low-protein chow decreased density volume of hepatocytes and hepatic fibrosis. In conclusion, our findings indicate that treatment with PZQ ameliorates hepatic schistosomiasis pathology even in mice fed a low-protein diet.

Mechanism of the transforming growth factor-β induction of fibronectin expression in hepatic stem-like cells

Transforming growth factor-β1 (TGF-β1) plays an important role in the fibrogenic process in the liver. The aim of the present study was to explore the action of TGF-β1 on fibronectin expression in rat hepatic stem-like cells and the underlying mechanisms. The level of fibronectin expression was determined in hepatic stem-like cells (WB cells) before and after TGF-β1 stimulation by RT-PCR and Western blot methods. Using immunogold transmission electron microscopy and the Western blot method, we observed the result of the expression and the distribution of cAMP, phosphorylated Smad3 and Smad7 before and after TGF-β1 treatment. The levels of fibronectin expression in both mRNA and protein increased 4- to 5-fold after TGF-β1 stimulation, reaching an optimum level after 8 h and then gradually falling back. Similarly, TGF-β1 stimulation resulted in an increase of cAMP in WB cells, peaking at 8 h. After treatment with TGF-β1 for 24 h, the expression of cAMP gradually decreased. In addition, we found that TGF-β1 treatment also contributed to the increased expression and to changes in cellular distribution of phosphorylated Smad3 (translocation from the cytoplasm to the nucleus) and Smad7 (translocation from the nucleus to the cytoplasm) in WB cells. The present study demonstrates that TGF-β is involved in the fibrogenic process in hepatic stem cells through up-regulation of fibronectin expression, and the mechanisms underlying this process may be associated with the activation of cAMP and Smad pathways.

Induction of a gloverin-like antimicrobial polypeptide in the sugarcane borer Diatraea saccharalis challenged by septic injury

Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae) is an important pest for Brazilian sugarcane. In the present study, we detected two distinct spots in hemolymph from septic injured larvae (HDs1 and HDs2), which are separated by 2DE gel electrophoresis. Both spots were subjected to in-gel tryptic digestion and MALDI-TOF/TOF analysis, which revealed the sequence VFGTLGSDDSGLFGK present in both HDs1 and HDs2. This sequence had homology and 80% identity with specific Lepidoptera antimicrobial peptides called gloverins. Analyses using the ImageMaster 2D software showed pI 8.94 of the HDs1 spot, which is similar to that described to Hyalophora gloveri gloverin (pI 8.5). Moreover, the 14-kDa molecular mass of the spot HDs1 is compatible to that of gloverins isolated from the hemolymph of Trichoplusia ni, Helicoverpa armigera and H. gloveri. Antimicrobial assays with partially purified fractions containing the HDs1 and HDs2 polypeptides demonstrated activity against Escherichia coli. This is the first report of antimicrobial polypeptides in D. saccharalis, and the identification of these peptides may help in the generation of new strategies to control this pest.