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WT Animals (wt + animals)

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Medical Sciences	90%

Selected Abstracts

Endothelially Derived Nitric Oxide Affects the Severity of Early Acetaminophen-induced Hepatic Injury in Mice

ACADEMIC EMERGENCY MEDICINE, Issue 5 2006
Steven D. Salhanick MD
Abstract Objectives: The precise mechanism of hepatocellular toxicity following acetaminophen (APAP) poisoning remains unclear. Nitric oxide is implicated in APAP toxicity as an inflammatory signaling molecule and as a precursor to the free radical peroxynitrate. The effects of inducible nitric oxide synthase (iNOS)-derived NO in APAP toxicity are known; however, the role of endothelial nitric oxide synthase (eNOS)-derived NO is unknown. The authors sought to evaluate the effect of eNOS-derived NO during APAP toxicity. Methods: C57BL6/J mice deficient in eNOS (eNOS KO) or iNOS (iNOS KO) and wild-type mice (WT) were treated with 300 mg/kg APAP. Alanine aminotransferase levels and plasma nitrate and nitrite levels were measured. Hypoxia inducible factor (HIF)-1, and Glucose Transporter 1 (Glut-1) levels were determined by Western blot. Results: Alanine aminotransferase levels were significantly elevated in all treated animals. Alanine aminotransferase levels were significantly lower in eNOS KO and iNOS KO than in treated WT animals. Plasma nitrate/nitrite levels were significantly higher in WT animals than in iNOS KO and eNOS KO animals. HIF-1, expression was increased in WT mice and decreased in iNOS KO mice. Glut-1 is a downstream, indirect marker of HIF function. Glut-1 expression was increased in WT and eNOS KO mice. Conclusions: Deficiency of either iNOS or eNOS results in decreased NO production and is associated with reduced hepatocellular injury following APAP poisoning. HIF-1, and Glut-1 levels are increased following APAP poisoning, implying that HIF-1, is functional during the pathogenic response to APAP poisoning. [source]

PER2 controls circadian periods through nuclear localization in the suprachiasmatic nucleus

GENES TO CELLS, Issue 11 2007
Koyomi Miyazaki
Molecular circadian clock regulation engages a negative feedback loop comprising components of the negative limb, PERs and CRYs. In addition to the rhythmic transcriptional regulation of clock genes, controlled subcellular localization might contribute to the molecular mechanism of the mammalian circadian clock. To address this issue, we generated transgenic (TG) mice lines harboring either rat PER2 (rPER2) with a deleted nuclear localizing domain [NLD(,)] or intact PER2. In comparison with wild-type (WT) control, the period of the circadian locomotor rhythm in TG mice over-expressing NLD(,) PER2 was longer, while that in TG mice over-expressing intact PER2 was shorter. The nuclear entry of endogenous PER2, CRY1 and CRY2 was delayed in the suprachiasmatic nucleus (SCN) of NLD(,) PER2 TG mice under constant darkness, whereas that of mouse PER2 (mPER2) is accelerated in the SCN of intact PER2 TG mice. Under constant light, the locomotor activity of NLD(,) PER2 TG mice became arrhythmic, whereas WT animals remained rhythmic. These data indicate that PER2 controls circadian periods through nuclear localization in the SCN. In addition, sleep architecture was also affected in intact PER2 TG mice, suggesting PER2 can modulate a sleep molecular mechanism. [source]

Bifidobacterium animalis causes extensive duodenitis and mild colonic inflammation in monoassociated interleukin-10-deficient mice

INFLAMMATORY BOWEL DISEASES, Issue 7 2009
James P. Moran PhD
Abstract Background: We recently showed that Bifidobacterium animalis is more prevalent within the colons of interleukin (IL)-10-deficient (,/,) mice than in wildtype (WT) animals colonized with the same specific pathogen-free (SPF) fecal contents. Here we tested the ability of this organism to cause T-cell-mediated intestinal inflammation by introducing it into germ-free (GF) IL-10,/, mice. Methods: GF IL-10,/, or WT mice were monoassociated with Bifidobacterium animalis subsp. animalis ATCC (American Type Culture Collection, Manassas, VA) 25527T or with B. infantis ATCC 15697T. Inflammation was measured by blinded histologic scores of the duodenum, cecum, and colon and by spontaneous secretion of IL-12/IL-23 p40 from colonic explants. Bacterial antigen-specific CD4+ mesenteric lymph node (MLN) T-cell recall responses were measured in response to antigen-presenting cells (APC) pulsed with bacterial lysates. Results:B. animalis caused marked duodenal inflammation and mild colitis in monoassociated IL-10,/, mice, whereas the intestinal tracts of WT animals remained free of inflammation. B. infantis colonization resulted in mild inflammation in the duodena of IL-10,/, mice. CD4+ MLN T cells from B. animalis monoassociated IL-10,/, mice secreted high levels of IFN-, and IL-17 in response to B. animalis lysate. B. animalis equally colonized the different intestinal regions of WT and IL-10,/, mice. Conclusions:B. animalis, a traditional probiotic species that is expanded in experimental colitis in this model, induces marked duodenal and mild colonic inflammation and TH1/TH17 immune responses when introduced alone into GF IL-10,/, mice. This suggests a potential pathogenic role for this commensal bacterial species in a susceptible host. (Inflamm Bowel Dis 2009) [source]

Intestinal Calcium Transporter Genes Are Upregulated by Estrogens and the Reproductive Cycle Through Vitamin D Receptor-Independent Mechanisms,

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2003
SJ Van Cromphaut
Abstract 1,,25(OH)2 -vitamin D strongly regulates the expression of the epithelial calcium channel CaT1. CaT1 expression is reduced in ERKO, mice and induced by estrogen treatment, pregnancy, or lactation in VDR WT and KO mice. Estrogens and vitamin D are thus independent potent regulators of the expression of this calcium influx mechanism, which is involved in active intestinal calcium absorption. Introduction: Active duodenal calcium absorption consists of three major steps: calcium influx into, transfer through, and extrusion out of the enterocyte. These steps are carried out by the calcium transport protein 1 (CaT1), calbindin-D9K, and the plasma membrane calcium ATPase (PMCA1b), respectively. We investigated whether estrogens or hormonal changes during the female reproductive cycle influence the expression of these genes, and if so, whether these effects are vitamin D-vitamin D receptor (VDR) dependent. Materials and Methods: We evaluated duodenal expression patterns in estrogen receptor (ER), and -, knockout (KO) mice, as well as in ovariectomized, estrogen-treated, pregnant, and lactating VDR wild-type (WT) and VDR KO mice. Results: Expression of calcium transporter genes was not altered in ERKO, mice. CaT1 mRNA expression was reduced by 55% in ERKO, mice, while the two other calcium transporter genes were not affected. Ovariectomy caused no change in duodenal expression pattern of VDR WT and KO mice, whereas treatment with a pharmacologic dose of estrogens induced CaT1 mRNA expression in VDR WT (4-fold) and KO (8-fold) mice. Pregnancy enhanced CaT1 expression equally in VDR WT and KO mice (12-fold). Calbindin-D9K and PMCA1b expression increased to a lesser extent and solely in pregnant VDR WT animals. In lactating VDR WT and KO mice, CaT1 mRNA expression increased 13 times, which was associated with a smaller increase in calbindin-D9K protein content and PMCA1b mRNA expression. Conclusions: Estrogens or hormonal changes during pregnancy or lactation have distinct, vitamin D-independent effects at the genomic level on active duodenal calcium absorption mechanisms, mainly through a major upregulation of the calcium influx channel CaT1. The estrogen effects seem to be mediated solely by ER,. [source]

Pulmonary hypertension is ameliorated in mice deficient in thrombin-activatable fibrinolysis inhibitor

JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 4 2010
L. QIN
Summary.,Background: The fibrinolytic system has been implicated in the pathogenesis of pulmonary hypertension (PH). Thrombin-activatable fibrinolysis inhibitor (TAFI) inhibits fibrinolysis and therefore its absence would be expected to increase fibrinolysis and ameliorate PH. Objective: The objective of the present study was to evaluate the effect of TAFI deficiency on pulmonary hypertension in the mouse. Methods and results: PH was induced in C57/Bl6 wild-type (WT) or TAFI-deficient (KO) mice by weekly subcutaneous treatment with 600 mg kg,1 monocrotaline (MCT) for 8 weeks. PH was inferred from right heart hypertrophy measured using the ratio of right ventricle-to-left ventricle-plus-septum weight [RV/(LV+S)]. Pulmonary vascular remodeling was analyzed by morphometry. TAFI-deficient MCT-treated and wild-type MCT-treated mice suffered similar weight loss. TAFI-deficient MCT-treated mice had reduced levels of total protein and tumor necrosis factor-alpha (TNF-,), interleukin-6 (IL-6), transforming growth factor-, (TGF-,) and monocyte chemoattractant protein-1 (MCP-1) in bronchial alveolar lavage compared with wild-type MCT-treated mice. The ratio of RV to (LV+S) weight was significantly higher in WT/MCT than in KO/MCT mice. The pulmonary artery wall area and vascular stenosis were both greater in MCT-treated WT mice compared with MCT-treated TAFI-deficient mice. Conclusions: TAFI-deficient MCT-treated mice had less pulmonary hypertension, vascular remodeling and reduced levels of cytokines compared with MCT-treated WT animals, possibly as a result of reduced coagulation activation. [source]

Reciprocal regulation of human soluble and particulate guanylate cyclases in vivo

BRITISH JOURNAL OF PHARMACOLOGY, Issue 6 2006
M Madhani
Background & purpose: We demonstrated previously that reciprocal regulation of soluble (sGC) and particulate (pGC) guanylate cyclases by NO and natriuretic peptides coordinates cyclic cGMP-mediated vasodilatation in vitro. Herein, we investigated whether such an interaction contributes to vascular homeostasis in mice and humans in vivo. Experimental approach: Mean arterial blood pressure (MABP) changes in anaesthetized mice were monitored in response to i.v. administration of cGMP- and cAMP-dependent vasodilators in wild-type (WT), endothelial NO synthase (eNOS) and natriuretic peptide receptor (NPR)-A knockout mice. Forearm blood flow (FBF) in response to intra-brachial infusion of ANP (25, 50, 100, 200 pmol min -1) in the absence and presence of the NOS inhibitor NG -methyl-L-arginine (L-NMA; 4 ,mol min -1) and the control constrictor noradrenaline (240 pmol min -1) was assessed in healthy volunteers. Key results: Sodium nitroprusside (SNP; NO-donor) and atrial natriuretic peptide (ANP) produced dose-dependent reductions in MABP in WT animals that were significantly enhanced in eNOS KO mice. In NPR-A K mice, SNP produced a dose-dependent reduction in MABP that was significantly greater than that in WT mice. Responsiveness to the cAMP-dependent vasodilator epoprostenol was similar in WT, eNOS KO and NPR-A KO animals. ANP caused vasodilatation of the forearm resistance vasculature that was significantly greater in individuals lacking endothelium-derived NO (i.e. L-NMA treated). Conclusions & implications: These data demonstrate that crosstalk occurs between the NO-sGC and ANP-pGC pathways to regulate cGMP-dependent vasodilatation in vivo in both mice and humans. These findings have implications for understanding the link between natriuretic peptide activity and cardiovascular risk. British Journal of Pharmacology (2006) 149, 797,801. doi:10.1038/sj.bjp.0706920 [source]

Gene therapy mediates cone rescue and rejuvenation in the R91W mutant form of Rpe65-deficiency mice

ACTA OPHTHALMOLOGICA, Issue 2009
Y ARSENIJEVIC
Purpose Given the advances of gene therapy studies to cure RPE65-derived Leber Congenital Amaurosis (LCA) (clinical trials phase I), it is of prime importance to examine how cones can be rescued in different mutant contexts. Consequently, we evaluated the effect on retinal activity and cone survival of lentivirus-mediated gene therapy in the R91W knock-in mouse model expressing the mutant Rpe65R91W gene. Methods An HIV-1-derived lentiviral vector (LV) expressing either the GFP or the mouse Rpe65 cDNA under the control of a 0.8 kb fragment of the human Rpe65 promoter (R0.8) was produced. LV-R0.8-RPE65 or GFP was injected into 5-days-old (P5) or 1 month-old R91W mice. Functional and morphological retinal rescues were investigated at 4 months of age. Results Increased light sensitivity was detected by ERG and pupillary light responses in animals injected with LV-R0.8-RPE65 at both P5 and 1 month compared to controls. Histological analysis showed improved expression of cone markers and cone outersegment morphology. Furthermore, the density of cones in the region of RPE65 delivery after treatment at P5 reached the wild type level. However, before injection at 1 month of age, only a fraction of the cones (40% of the number found in WT animals) in the Rpe65R91W/R91W mice expressed cone transducin, this fraction increased to 64% after treatment. Moreover, these cones appeared normal. Conclusion We show that lentivirus-mediated Rpe65 gene transfer is very efficacious in early treatments and still efficient during the course of cone degeneration. Moreover, the treatment at 1 month shows a rejuvenation process of the diseased cones. Thus patient suffering from R91W mutation might benefit from a prolonged therapeutic window. [source]

B cells are involved in the modulation of pathogenic gut immune response in food-allergic enteropathy

CLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 2 2008
C. R. Cardoso
Summary Food enteropathies involve uncontrolled or hypersensitivity reactions to ingested nutrients and may result in IgE and T-helper type 2 (Th2) responses as in food allergy. However, the precise role of B cells in the development of food enteropathies remains uncertain. In this work, we used B cell-deficient mice (B KO) and a model of peanut sensitization to examine the involvement of B lymphocytes in the pathogenesis of food allergy. Results showed that priming of wild-type (WT) mice with peanut proteins induced specific IgG1 and IgE responses in serum, with edema, tissue destruction, epithelial exulceration and inflammatory infiltrate in the gut of sensitized and challenged (S + Peanut) WT animals. In contrast, there was no sera immunoglobulin detection and absence of tissue destruction in the gut of B KO mice, which presented moderate inflammatory infiltrate and villous enlargement after peanut challenge. These animals presented marked decrease in IL-4 and TNF-, and high levels of IL-10, TGF-,, IL-12p40 and IFN-, mRNA in the gut. Moreover, the expression of CCL5, CCL11 and CXCL1 was reduced in the gut of B KO mice, in contrast to elevated messages of CCL2 or similar detection of Th1-related chemokines in S + Peanut WT mice. Finally, we provided evidence that B cells are necessary to the development of food-related enteropathies and induction of gut inflammation during allergic reactions to food. [source]

Interferon-gamma is causatively involved in experimental inflammatory bowel disease in mice

CLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 2 2006
R. Ito
Summary Cytokines may be crucially involved in the pathogenesis of inflammatory bowel diseases (IBD), but it remains controversial whether interferon (IFN)-,, a typical proinflammatory cytokine, is an essential mediator to cause the disorders. In the present study, IFN-,,/, and wild-type (WT) C57BL/6 mice were fed 2·5% dextran sodium sulphate (DSS) in drinking water for 7 days, in order to investigate DSS-induced intestinal inflammation. The DSS-treated WT mice exhibited a robust production of IFN-, in the gut, a remarkable loss of body weight, as well as high rate of mortality (60%). In striking contrast, IFN-, deficient mice did not develop DSS-induced colitis, as indicated by the maintenance of body weight and survival rate of 100%. Severe intestinal inflammation was demonstrated exclusively in WT animals in terms of the shortening of the bowel as well as the elevation of the disease activity index, myeloperoxidase (MPO) activity and serum haptoglobin level. Histological study of DSS-treated WT intestine revealed disruption of mucosal epithelium and massive infiltration of inflammatory cells, while the organ from IFN-,,/, mice remained virtually normal in appearance. Enzyme-linked immunosorbent assay (ELISA) analyses indicated abundant production of three chemokines, i.e. monokine induced by interferon-, (MIG), interferon-inducible protein 10 (IP-10) and monocyte chemoattractant protein-1 (MCP-1), in the DSS-irritated intestine of WT but not of IFN-,,/, mice. The present results demonstrate clearly that IFN-, plays indispensable roles in the initiation of DSS colitis, and some chemokines are produced in an IFN-,-dependent fashion. [source]

Protection against Fas-induced liver apoptosis in transgenic mice expressing cyclooxygenase 2 in hepatocytes,

HEPATOLOGY, Issue 3 2007
Marta Casado
Cyclooxygenase-2 (COX-2) is upregulated in many cancers, and the prostanoids synthesized increase proliferation, improve angiogenesis, and inhibit apoptosis in several tissues. To explore the function of COX-2 in liver, transgenic (Tg) mice were generated containing a fusion gene (LIVhCOX-2) consisting of human COX-2 cDNA under the control of the human ApoE promoter. Six lines were developed; all of them expressed the LIVhCOX-2 transgene selectively in hepatocytes. The Tg mice exhibited a normal phenotype, and the increased levels of PGE2 found were due to the constitutively expressed COX-2. Histological analysis of different tissues and macroscopic examination of the liver showed no differences between wild-type (Wt) and Tg animals. However, Tg animals were resistant to Fas-mediated liver injury, as demonstrated by low levels of plasmatic aminotransferases, a lesser caspase-3 activation, and Bax levels and an increase in Bcl-2, Mcl-1, and xIAP proteins, when compared with the Wt animals. Moreover, the resistance to Fas-mediated apoptosis is suppressed in the presence of COX-2,selective inhibitors, which prevented prostaglandin accumulation in the liver of Tg mice. Conclusion: These results demonstrate that expression of COX-2,dependent prostaglandins exerted a protection against liver apoptosis. (HEPATOLOGY 2007;45:631,638.) [source]