Cyclooxygenase-2 Inhibition Attenuates Abdominal Aortic Aneurysm Progression in Hyperlipidemic Mice

Abdominal aortic aneurysms (AAAs) are a chronic inflammatory disease that increase the risk of life-threatening aortic rupture. In humans, AAAs have been characterized by increased expression of cyclooxygenase-2 and the inactivation of COX-2 prior to disease initiation reduces AAA incidence in a mouse model of the disease. The current study examined the effectiveness of selective cyclooxygenase-2 (COX-2) inhibition on reducing AAA progression when administered after the initiation of AAA formation. AAAs were induced in hyperlipidemic apolipoprotein E-deficient mice by chronic angiotensin II (AngII) infusion and the effect of treatment with the COX-2 inhibitor celecoxib was examined when initiated at different stages of the disease. Celecoxib treatment that was started 1 week after initiating AngII infusion reduced AAA incidence by 61% and significantly decreased AAA severity. Mice treated with celecoxib also showed significantly reduced aortic rupture and mortality. Treatment with celecoxib that was started at a late stage of AAA development also significantly reduced AAA incidence and severity. Celecoxib treatment significantly increased smooth muscle alpha-actin expression in the abdominal aorta and did not reduce expression of markers of macrophage-dependent inflammation. These findings indicate that COX-2 inhibitor treatment initiated after formation of AngII-induced AAAs effectively reduces progression of the disease in hyperlipidemic mice

Biotechnology Lab Manual Course BI-453

The purpose of the lab manual is to acquaint undergraduate students with hands-on training on key laboratory experiments needed to gain expertise in Biotechnology, Cell & Molecular Biology

Chylomicrons Promote Intestinal Absorption and Systemic Dissemination of Dietary Antigen (Ovalbumin) in Mice

BACKGROUND:A small fraction of dietary protein survives enzymatic degradation and is absorbed in potentially antigenic form. This can trigger inflammatory responses in patients with celiac disease or food allergies, but typically induces systemic immunological tolerance (oral tolerance). At present it is not clear how dietary antigens are absorbed. Most food staples, including those with common antigens such as peanuts, eggs, and milk, contain long-chain triglycerides (LCT), which stimulate mesenteric lymph flux and postprandial transport of chylomicrons through mesenteric lymph nodes (MLN) and blood. Most dietary antigens, like ovalbumin (OVA), are emulsifiers, predicting affinity for chylomicrons. We hypothesized that chylomicron formation promotes intestinal absorption and systemic dissemination of dietary antigens. METHODOLOGY/PRINCIPAL FINDINGS:Absorption of OVA into MLN and blood was significantly enhanced when OVA was gavaged into fasted mice together with LCT compared with medium-chain triglycerides (MCT), which do not stimulate chylomicron formation. The effect of LCT was blocked by the addition of an inhibitor of chylomicron secretion, Pluronic L-81. Adoptively transferred OVA-specific DO11.10 T-cells proliferated more extensively in peripheral lymph nodes when OVA was gavaged with LCT than with MCT or LCT plus Pluronic L-81, suggesting that dietary OVA is systemically disseminated. Most dietary OVA in plasma was associated with chylomicrons, suggesting that these particles mediate systemic antigen dissemination. Intestinal-epithelial CaCo-2 cells secreted more cell-associated, exogenous OVA when stimulated with oleic-acid than with butyric acid, and the secreted OVA appeared to be associated with chylomicrons. CONCLUSIONS/SIGNIFICANCE:Postprandial chylomicron formation profoundly affects absorption and systemic dissemination of dietary antigens. The fat content of a meal may affect immune responses to dietary antigens by modulating antigen absorption and transport

Calpain 1 Knockdown Improves Tissue Sparing and Functional Outcomes after Spinal Cord Injury in Rats

To evaluate the hypothesis that calpain 1 knockdown would reduce pathological damage and functional deficits after spinal cord injury (SCI), we developed lentiviral vectors encoding calpain 1 shRNA and eGFP as a reporter (LV-CAPN1 shRNA). The ability of LV-CAPN1 shRNA to knockdown calpain 1 was confirmed in rat NRK cells using Northern and Western blot analysis. To investigate the effects on spinal cord injury, LV-CAPN1shRNA or LV-mismatch control shRNA (LV-control shRNA) were administered by convection enhanced diffusion at spinal cord level T10 in Long-Evans female rats (200–250 g) 1 week before contusion SCI, 180 kdyn force, or sham surgery at the same thoracic level. Intraspinal administration of the lentiviral particles resulted in transgene expression, visualized by eGFP, in spinal tissue at 2 weeks after infection. Calpain 1 protein levels were reduced by 54% at T10 2 weeks after shRNA-mediated knockdown (p\u3c0.05, compared with the LV-control group, n=3 per group) while calpain 2 levels were unchanged. Intraspinal administration of LV-CAPN1shRNA 1 week before contusion SCI resulted in a significant improvement in locomotor function over 6 weeks postinjury, compared with LV-control administration (p\u3c0.05, n=10 per group). Histological analysis of spinal cord sections indicated that pre-injury intraspinal administration of LV-CAPN1shRNA significantly reduced spinal lesion volume and improved total tissue sparing, white matter sparing, and gray matter sparing (p\u3c0.05, n=10 per group). Together, results support the hypothesis that calpain 1 activation contributes to the tissue damage and impaired locomotor function after SCI, and that calpain1 represents a potential therapeutic target

Intestinal Epithelial Serum Amyloid A Modulates Bacterial Growth In Vitro and Pro-Inflammatory Responses in Mouse Experimental Colitis

<p>Abstract</p> <p>Background</p> <p>Serum Amyloid A (SAA) is a major acute phase protein of unknown function. SAA is mostly expressed in the liver, but also in other tissues including the intestinal epithelium. SAA reportedly has anti-bacterial effects, and because inflammatory bowel diseases (IBD) result from a breakdown in homeostatic interactions between intestinal epithelia and bacteria, we hypothesized that SAA is protective during experimental colitis.</p> <p>Methods</p> <p>Intestinal SAA expression was measured in mouse and human samples. Dextran sodium sulfate (DSS) colitis was induced in SAA 1/2 double knockout (DKO) mice and in wildtype controls. Anti-bacterial effects of SAA1/2 were tested in intestinal epithelial cell lines transduced with adenoviral vectors encoding the CE/J SAA isoform or control vectors prior to exposure to live <it>Escherichia coli</it>.</p> <p>Results</p> <p>Significant levels of SAA1/SAA2 RNA and SAA protein were detected by in situ hybridization and immunohistochemistry in mouse colonic epithelium. SAA3 expression was weaker, but similarly distributed. SAA1/2 RNA was present in the ileum and colon of conventional mice and in the colon of germfree mice. Expression of SAA3 was strongly regulated by bacterial lipopolysaccharides in cultured epithelial cell lines, whereas SAA1/2 expression was constitutive and not LPS inducible. Overexpression of SAA1/2 in cultured epithelial cell lines reduced the viability of co-cultured <it>E. coli</it>. This might partially explain the observed increase in susceptibility of DKO mice to DSS colitis. SAA1/2 expression was increased in colon samples obtained from Crohn's Disease patients compared to controls.</p> <p>Conclusions</p> <p>Intestinal epithelial SAA displays bactericidal properties in vitro and could play a protective role in experimental mouse colitis. Altered expression of SAA in intestinal biopsies from Crohn's Disease patients suggests that SAA is involved in the disease process..</p

<span style="font-size: 21.5pt;mso-bidi-font-size:14.5pt;font-family:"Times New Roman","serif"">Clastogenic effects of dietary <i>supplement-Spirulina </i>alga, and some medicinal <span style="font-size:22.0pt;mso-bidi-font-size:15.0pt;font-family:"Times New Roman","serif"; mso-bidi-font-weight:bold">plant<b> </b><span style="font-size:21.5pt; mso-bidi-font-size:14.5pt;font-family:"Times New Roman","serif"">products from <i>Boswellia serrata, Withania smnnifera </i>on mice </span></span></span>

1068-1070<span style="font-size: 15.0pt;mso-bidi-font-size:8.0pt;font-family:" times="" new="" roman","serif""="">Pretreatment of aqueous extracts of Zyrulina <span style="font-size:15.5pt; mso-bidi-font-size:8.5pt;font-family:" times="" new="" roman","serif""="">(Spirulina), Aswagandha <span style="font-size:15.5pt;mso-bidi-font-size:8.5pt;font-family: " times="" new="" roman","serif""="">(Withania) <span style="font-size:15.0pt; mso-bidi-font-size:8.0pt;font-family:" times="" new="" roman","serif""="">and Nopane (Boswellia) <span style="font-size:15.0pt;mso-bidi-font-size:8.0pt;font-family: " times="" new="" roman","serif""="">on colchicine induced chromosome damage showed weakness of clastogenic activity in Swiss albino mice. None of the treatments increased significantly the number of chromosome aberrations. </span

Global IP6K1 deletion enhances temperature modulated energy expenditure which reduces carbohydrate and fat induced weight gain

Objective: IP6 kinases (IP6Ks) regulate cell metabolism and survival. Mice with global (IP6K1-KO) or adipocyte-specific (AdKO) deletion of IP6K1 are protected from diet induced obesity (DIO) at ambient (23 °C) temperature. AdKO mice are lean primarily due to increased AMPK mediated thermogenic energy expenditure (EE). Thus, at thermoneutral (30 °C) temperature, high fat diet (HFD)-fed AdKO mice expend energy and gain body weight, similar to control mice. IP6K1 is ubiquitously expressed; thus, it is critical to determine to what extent the lean phenotype of global IP6K1-KO mice depends on environmental temperature. Furthermore, it is not known whether IP6K1 regulates AMPK mediated EE in cells, which do not express UCP1. Methods: Q-NMR, GTT, food intake, EE, QRT-PCR, histology, mitochondrial oxygen consumption rate (OCR), fatty acid metabolism assays, and immunoblot studies were conducted in IP6K1-KO and WT mice or cells. Results: Global IP6K1 deletion mediated enhancement in EE is impaired albeit not abolished at 30 °C. As a result, IP6K1-KO mice are protected from DIO, insulin resistance, and fatty liver even at 30 °C. Like AdKO, IP6K1-KO mice display enhanced adipose tissue browning. However, unlike AdKO mice, thermoneutrality only partly abolishes browning in IP6K1-KO mice. Cold (5 °C) exposure enhances carbohydrate expenditure, whereas 23 °C and 30 °C promote fat oxidation in HFD-KO mice. Furthermore, IP6K1 deletion diminishes cellular fat accumulation via activation of the AMPK signaling pathway. Conclusions: Global deletion of IP6K1 ameliorates obesity and insulin resistance irrespective of the environmental temperature conditions, which strengthens its validity as an anti-obesity target. Keywords: IP6K, Obesity, Diabetes, Energy expenditure, β-oxidatio

COX-2 inhibitor treatment initiated 3 weeks after beginning AngII infusion effectively reduces AAA progression in ApoE-deficient mice.

<p>(A) AAA incidence was determined at necropsy following 3 (3 Wks AngII) or 8 weeks (Control and Celecoxib) of AngII infusion. (B) The maximal external diameter or (C) AAA classification of the suprarenal aorta following 3 (3 Wks AngII) or 8 weeks (Control and Celecoxib) of AngII infusion. For aortic diameter, results depict mean ± SEM. AAA incidence and diameter: 3 Wks AngII <i>n</i> = 12, Control <i>n</i> = 13, Celecoxib <i>n</i> = 14. <i>P</i> values were determined for incidence data using Fisher's exact test and an unpaired two-tailed <i>t</i>-test for aortic diameter. * indicates <i>P</i><0.05.</p