Comparison of the hepatic metabolism of triazolam in wild-type andCyp3a-knockout mice for understanding CYP3A-mediated metabolism inCYP3A-humanised mice in vivo

Xenobiotica ◽  
2019 ◽  
Vol 49 (11) ◽  
pp. 1303-1310 ◽  
Author(s):  
Genki Minegishi ◽  
Yasuhiro Kazuki ◽  
Yuki Yamasaki ◽  
Fuka Okuya ◽  
Hidetaka Akita ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Hepatic Metabolism ◽  
Wild Type ◽  
Humanised Mice

scholarly journals Preoperative administration of the 5-HT4 receptor agonist prucalopride reduces intestinal inflammation and shortens postoperative ileus via cholinergic enteric neurons

Gut ◽  
2018 ◽  
Vol 68 (8) ◽  
pp. 1406-1416 ◽  
Author(s):  
Nathalie Stakenborg ◽  
Evelien Labeeuw ◽  
Pedro J Gomez-Pinilla ◽  
Sebastiaan De Schepper ◽  
Raymond Aerts ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Postoperative Ileus ◽  
Intestinal Inflammation ◽  
Electrical Field Stimulation ◽  
Enteric Neurons ◽  
Postoperative Treatment ◽  
Wild Type ◽  
Clinical Recovery ◽  
Preoperative Administration

ObjectivesVagus nerve stimulation (VNS), most likely via enteric neurons, prevents postoperative ileus (POI) by reducing activation of alpha7 nicotinic receptor (α7nAChR) positive muscularis macrophages (mMφ) and dampening surgery-induced intestinal inflammation. Here, we evaluated if 5-HT4 receptor (5-HT4R) agonist prucalopride can mimic this effect in mice and human.DesignUsing Ca2+ imaging, the effect of electrical field stimulation (EFS) and prucalopride was evaluated in situ on mMφ activation evoked by ATP in jejunal muscularis tissue. Next, preoperative and postoperative administration of prucalopride (1–5 mg/kg) was compared with that of preoperative VNS in a model of POI in wild-type and α7nAChR knockout mice. Finally, in a pilot study, patients undergoing a Whipple procedure were preoperatively treated with prucalopride (n=10), abdominal VNS (n=10) or sham/placebo (n=10) to evaluate the effect on intestinal inflammation and clinical recovery of POI.ResultsEFS reduced the ATP-induced Ca2+ response of mMφ, an effect that was dampened by neurotoxins tetrodotoxin and ω-conotoxin and mimicked by prucalopride. In vivo, prucalopride administered before, but not after abdominal surgery reduced intestinal inflammation and prevented POI in wild-type, but not in α7nAChR knockout mice. In humans, preoperative administration of prucalopride, but not of VNS, decreased Il6 and Il8 expression in the muscularis externa and improved clinical recovery.ConclusionEnteric neurons dampen mMφ activation, an effect mimicked by prucalopride. Preoperative, but not postoperative treatment with prucalopride prevents intestinal inflammation and shortens POI in both mice and human, indicating that preoperative administration of 5-HT4R agonists should be further evaluated as a treatment of POI.Trial registration numberNCT02425774.

scholarly journals Effect of Targeted Deletion of the Heme Oxygenase-2 Gene on Hemoglobin Toxicity in the Striatum

2005 ◽  
Vol 25 (11) ◽  
pp. 1466-1475 ◽  
Author(s):  
Yan Qu ◽  
Jing Chen ◽  
Luna Benvenisti-Zarom ◽  
Xin Ma ◽  
Raymond F Regan
Keyword(s):  
Heme Oxygenase ◽  
Knockout Mice ◽  
Protein Oxidation ◽  
Gene Deletion ◽  
Wild Type ◽  
Cell Culture Study ◽  
Targeted Deletion ◽  
Rate Limiting Step ◽  
Diphenyltetrazolium Bromide

The heme oxygenase (HO) enzymes catalyze the rate-limiting step in the breakdown of heme to iron, carbon monoxide, and biliverdin. A prior cell culture study demonstrated that deletion of HO-2, the isoform constitutively expressed in neurons, attenuated hemoglobin (Hb) neurotoxicity. The present study tested the hypothesis that HO-2 gene deletion is cytoprotective in a model of Hb toxicity in vivo. Stereotactic injection of 6 μL stroma-free Hb (SFHb) into the striatum significantly increased protein oxidation in wild-type mice at 24 to 72 h, as detected by an assay for carbonyl groups. At 72 h, carbonylation was increased 2.5-fold compared with that in the contralateral striatum. In HO-2 knockout mice, protein oxidation was not increased at 24 h, and was increased by only 1.7-fold at 72 h. Similarly, striatal lipid peroxidation, as detected by the malondialdehyde assay, was significantly greater in the SFHb-injected striata of wild-type mice than in knockout mice. Striatal cell viability, determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, was 45.0%±6.3% of that in contralateral striata in wild-type mice at 72 h; it was increased to 85%±8% in knockouts. Heme oxygenase-2 gene deletion did not alter weight loss or mortality after SFHb injection. Baseline striatal HO-1 expression was similar in knockout and wild-type mice; induction after SFHb injection occurred more rapidly in the latter. These results suggest that HO-2 gene deletion protects striatal cells from the oxidative toxicity of Hb in vivo. Pharmacologic or genetic strategies that target HO-2 may be beneficial after central nervous system hemorrhage, and warrant further investigation.

scholarly journals Villin and actin in the mouse kidney brush-border membrane bind to and are degraded by meprins, an interaction that contributes to injury in ischemia-reperfusion

2011 ◽  
Vol 301 (4) ◽  
pp. F871-F882 ◽  
Author(s):  
Elimelda Moige Ongeri ◽  
Odinaka Anyanwu ◽  
W. Brian Reeves ◽  
Judith S. Bond
Keyword(s):  
Brush Border ◽  
Knockout Mice ◽  
Brush Border Membrane ◽  
Ischemia Reperfusion ◽  
Kidney Injury ◽  
Mouse Kidney ◽  
Cytoskeletal Proteins ◽  
Kidney Tubules ◽  
Wild Type

Meprins, metalloproteinases abundantly expressed in the brush-border membranes (BBMs) of rodent proximal kidney tubules, have been implicated in the pathology of renal injury induced by ischemia-reperfusion (IR). Disruption of the meprin β gene and actinonin, a meprin inhibitor, both decrease kidney injury resulting from IR. To date, the in vivo kidney substrates for meprins are unknown. The studies herein implicate villin and actin as meprin substrates. Villin and actin bind to the cytoplasmic tail of meprin β, and both meprin A and B are capable of degrading villin and actin present in kidney proteins as well as purified recombinant forms of these proteins. The products resulting from degradation of villin and actin were unique to each meprin isoform. The meprin B cleavage site in villin was Glu744-Val745. Recombinant forms of rat meprin B and homomeric mouse meprin A had Km values for villin and actin of ∼1 μM (0.6–1.2 μM). The kcat values varied substantially (0.6–128 s−1), resulting in different efficiencies for cleavage, with meprin B having the highest kcat/ Km values (128 M−1·s−1 × 106). Following IR, meprins and villin redistributed from the BBM to the cytosol. A 37-kDa actin fragment was detected in protein fractions from wild-type, but not in comparable preparations from meprin knockout mice. The levels of the 37-kDa actin fragment were significantly higher in kidneys subjected to IR. The data establish that meprins interact with and cleave villin and actin, and these cytoskeletal proteins are substrates for meprins.

scholarly journals IL-17 Activates the IL-6/STAT3 Signal Pathway in the Proliferation of Hepatitis B Virus-Related Hepatocellular Carcinoma

2017 ◽  
Vol 43 (6) ◽  
pp. 2379-2390 ◽  
Author(s):  
Zongqiang Hu ◽  
Ding Luo ◽  
Dongdong Wang ◽  
Linjie Ma ◽  
Yingpeng Zhao ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Knockout Mice ◽  
Hepg2 Cells ◽  
Wild Type ◽  
B Virus ◽  
And Migration ◽  
Proliferation And Migration

Background/Aims: We performed this study to determine the role of IL-17 in the immune microenvironment of hepatitis B virus- (HBV-) related hepatocellular carcinoma (HCC). Methods: HepG2 cells were treated with IL-17, STAT3 inhibitor S31-201 or IL-6 neutralizing monoclonal antibody (IL-6 mAb). Cell proliferation and migration were compared using the Cell Counting kit-8 (CCK-8) and Transwell assays, respectively. Real-time quantitative PCR (RT-qPCR), Western Blot, ELISA, immunofluorescence and histological staining were used for determining the expression levels of IL-17, IL-6, MCP-1, CCL5, VEGF, STAT3 and p-STAT3. HCC xenograft models were constructed in wild type and IL-17 knockout mice to clarify the effects of IL-17 on HCC in vivo. Results: Exogenous IL-17 enhanced the proliferation and migration of HepG2 cells, and it activated the phosphorylation of STAT3. RT-qPCR and ELISA showed that IL-17 promoted the expression of IL-6. The CCK-8 and Transwell assays showed that S31-201 or IL-6 mAb remarkably reversed the promotion effects of proliferation and migration by exogenous IL-17 in HepG2 cells. Additionally, IL-6 could promote the phosphorylation of STAT3, while IL-6 mAb acted as an inhibitor, and exogenous IL-17 could neutralize the inhibitory effects of IL-6 mAb. In vivo, compared to the wild type mice, the tumor volume, weight, density and size were decreased in IL-17 knockout mice. Additionally, the expression levels of p-STAT3, IL-6, MCP-1, CCL5 and VEGF decreased in IL-17 knockout mice. Conclusions: IL-17 can enhance the proliferation of HepG2 cells in vitro and in vivo via activating the IL-6/STAT3 pathway. Therefore, the IL-17/IL-6/STAT3 signaling pathway is a potential therapeutic target for HBV-related HCC.

scholarly journals Ficolins Promote Fungal Clearance in vivo and Modulate the Inflammatory Cytokine Response in Host Defense against Aspergillus fumigatus

2016 ◽  
Vol 8 (6) ◽  
pp. 579-588 ◽  
Author(s):  
Ninette Genster ◽  
Elisabeth Præstekjær Cramer ◽  
Anne Rosbjerg ◽  
Katrine Pilely ◽  
Jack Bernard Cowland ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Host Defense ◽  
Knockout Mice ◽  
Fungal Infections ◽  
Inflammatory Cells ◽  
Lectin Pathway ◽  
Wild Type ◽  
Opportunistic Fungal Pathogen

Aspergillus fumigatus is an opportunistic fungal pathogen that causes severe invasive infections in immunocompromised patients. Innate immunity plays a major role in protection against A. fumigatus. The ficolins are a family of soluble pattern recognition receptors that are capable of activating the lectin pathway of complement. Previous in vitro studies reported that ficolins bind to A. fumigatus, but their part in host defense against fungal infections in vivo is unknown. In this study, we used ficolin-deficient mice to investigate the role of ficolins during lung infection with A. fumigatus. Ficolin knockout mice showed significantly higher fungal loads in the lungs 24 h postinfection compared to wild-type mice. The delayed clearance of A. fumigatus in ficolin knockout mice could not be attributed to a compromised recruitment of inflammatory cells. However, it was revealed that ficolin knockout mice exhibited a decreased production of proinflammatory cytokines in the lungs compared to wild-type mice following A. fumigatus infection. The impaired clearance and cytokine production in ficolin knockout mice was independent of complement, as shown by equivalent levels of A. fumigatus-mediated complement activation in ficolin knockout mice and wild-type mice. In conclusion, this study demonstrates that ficolins are important in initial innate host defense against A. fumigatus infections in vivo.

Vitamin D3 receptor ablation alters mammary gland morphogenesis

Development ◽  
2002 ◽  
Vol 129 (13) ◽  
pp. 3067-3076
Author(s):  
Glendon Zinser ◽  
Kathryn Packman ◽  
JoEllen Welsh
Keyword(s):  
Mammary Gland ◽  
Vitamin D3 ◽  
Stromal Cells ◽  
Knockout Mice ◽  
Wild Type ◽  
Dihydroxyvitamin D3 ◽  
Mammary Gland Morphogenesis ◽  
Gland Morphogenesis ◽  
Gland Development

Postnatal mammary gland morphogenesis is achieved through coordination of signaling networks in both the epithelial and stromal cells of the developing gland. While the major proliferative hormones driving pubertal mammary gland development are estrogen and progesterone, studies in transgenic and knockout mice have successfully identified other steroid and peptide hormones that impact on mammary gland development. The vitamin D3 receptor (VDR), whose ligand 1,25-dihydroxyvitamin D3 is the biologically active form of vitamin D3, has been implicated in control of differentiation, cell cycle and apoptosis of mammary cells in culture, but little is known about the physiological relevance of the vitamin D3 endocrine system in the developing gland. In these studies, we report the expression of the VDR in epithelial cells of the terminal end bud and subtending ducts, in stromal cells and in a subset of lymphocytes within the lymph node. In the terminal end bud, a distinct gradient of VDR expression is observed, with weak VDR staining in proliferative populations and strong VDR staining in differentiated populations. The role of the VDR in ductal morphogenesis was examined in Vdr knockout mice fed high dietary Ca2+ which normalizes fertility, serum estrogen and neonatal growth. Our results indicate that mammary glands from virgin Vdr knockout mice are heavier and exhibit enhanced growth, as evidenced by higher numbers of terminal end buds, greater ductal outgrowth and enhanced secondary branch points, compared with glands from age- and weight-matched wild-type mice. In addition, glands from Vdr knockout mice exhibit enhanced growth in response to exogenous estrogen and progesterone, both in vivo and in organ culture, compared with glands from wild-type mice. Our data provide the first in vivo evidence that 1,25-dihydroxyvitamin D3 and the VDR impact on ductal elongation and branching morphogenesis during pubertal development of the mammary gland. Collectively, these results suggest that the vitamin D3 signaling pathway participates in negative growth regulation of the mammary gland.

Tie2+ Bone Marrow Endothelial Cells Regulate Hematopoietic Reconstitution In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 248-248
Author(s):  
Phuong L. Doan ◽  
J. Lauren Russell ◽  
Sarah K. Meadows ◽  
Heather A Himburg ◽  
Pamela Daher ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Knockout Mice ◽  
Fold Increase ◽  
Chimeric Mice ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Hematopoietic Reconstitution ◽  
Total Body ◽  
Hematopoietic Response

Abstract Abstract 248 Radiation and chemotherapy cause myelosuppression in patients via damage to bone marrow (BM) hematopoietic stem cells (HSCs) and progenitor cells. It has been shown that BM HSCs reside in close association with osteoblasts and that BM osteoblasts regulate the maintenance of quiescent HSCs in vivo. HSCs also reside in proximity to BM sinusoidal vessels but the function of BM endothelial cells (BM ECs) in regulating HSC fate in vivo remains less well understood. We hypothesized that BM ECs play a critical role in regulating BM hematopoietic reconstitution following stress. In order to test this hypothesis, we utilized Cre-LoxP recombination to generate mice bearing targeted deletion of the pro-apoptotic genes, Bak and Bax, in Tie2+ BM ECs (Tie2Cre;Bak−/− ;BaxFl/− mice) and measured their hematopoietic response to total body irradiation (TBI). Tie2Cre;Bak−/−;BaxFl/− mice (EC-Bak;Bax knockouts) were compared with Tie2Cre;Bak−/−;BaxFl/+ mice (EC-Bak;Bax (+) mice) which have constitutive Bak deletion but retain Bax in Tie2+ BM ECs. EC-Bak;Bax knockout mice had no detectable Bak or Bax by Western Blot in vascular tissues. After exposure to 100 cGy total body irradiation (TBI), EC-Bak;Bax knockout mice displayed a 2-fold increase in total viable BM cells (p=0.04), a 3-fold increase in BM ckit+sca+lineage- (KSL) progenitor cells, a 3-fold increase in colony-forming unit-spleen day 12 (CFU-S12) content (p=0.0003) and a 2-fold increase in 4 week competitive repopulating units (CRUs) compared to EC-Bak;Bax (+) mice. After 300 cGy TBI, comparable radioprotection was observed in the EC-Bak;Bax knockout mice, with a 1.9-fold increase in total BM cells, a 2.4-fold increase in CFU-S (p=0.01) and a 3.6-fold increase in 4 week CRU compared to EC-Bak;Bax (+) mice. Taken together, these results suggested that targeted protection of Tie2+ BM ECs from the intrinsic pathway of apoptosis mediated protection of BM hematopoietic stem/progenitor cells following TBI. Since Tie2 is expressed by BM ECs and a small subset of quiescent BM HSCs, we carried out experiments to determine whether the radioprotection we observed in EC-Bak;Bax knockout mice was caused autonomously by protection of Tie2+ BM ECs or Tie2+ HSCs. We transplanted 4 × 106 BM cells from EC-Bak;Bax knockout mice into lethally irradiated (950 cGy) wild type (WT) B6.SJL mice such that, after 12 weeks post-transplant, the recipient mice were chimeric for Bak and Bax deletions only in hematopoietic cells while retaining a wild type BM microenvironment (HSC-Bak;Bax knockout;EC-wild type), verified by qRTPCR. At 16 weeks post-transplant, the chimeric recipient mice were then exposed to 100 and 300 cGy TBI and we compared the hematopoietic response of these mice to that of EC-Bak;Bax knockout mice. Interestingly, after 100 cGy TBI, the hematopoietic response of the chimeric mice was comparable to that of wild type C57Bl6 mice and revealed a significant reduction in total viable BM cells (1.3-fold), BM KSL cells (7.4 fold) and BM CRU-4 weeks (9-fold) compared to 100 cGy-irradiated EC-Bak;Bax knockout mice. Significant reductions in total BM cells and BM CRU content were also observed in chimeric mice compared to EC-Bak;Bax knockout mice following 300 cGy TBI. These results suggest that deletion of Bak and Bax-mediated apoptosis in Tie2+ BM ECs protects BM hematopoietic stem and progenitor cells from ionizing radiation damage and this protection is largely autonomous to Tie2+ BM ECs. More generally, these data demonstrate that protection of BM ECs from the deleterious effects of ionizing radiation results in augmented hematopoietic reconstitution following TBI. This study demonstrates that Tie2+ BM ECs have an essential role in regulating hematopoietic regeneration following injury and reveals that BM ECs are a novel and attractive therapeutic target to augment hematopoietic reconstitution in vivo. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Angiotensin II stimulates superoxide production in the thick ascending limb by activating NOX4

2012 ◽  
Vol 303 (7) ◽  
pp. C781-C789 ◽  
Author(s):  
Katherine J. Massey ◽  
Nancy J. Hong ◽  
Jeffrey L. Garvin
Keyword(s):  
Angiotensin Ii ◽  
Nadph Oxidase ◽  
Knockout Mice ◽  
Thick Ascending Limb ◽  
Ang Ii ◽  
Wild Type ◽  
Outer Medulla ◽  
Catalytic Subunits ◽  
In The Wild

Angiotensin II (ANG II) stimulates production of superoxide (O2−) by NADPH oxidase (NOX) in medullary thick ascending limbs (TALs). There are three isoforms of the catalytic subunit (NOX1, 2, and 4) known to be expressed in the kidney. We hypothesized that NOX2 mediates ANG II-induced O2− production by TALs. To test this, we measured NOX1, 2, and 4 mRNA and protein by RT-PCR and Western blot in TAL suspensions from rats and found three catalytic subunits expressed in the TAL. We measured O2− production using a lucigenin-based assay. To assess the contribution of NOX2, we measured ANG II-induced O2− production in wild-type and NOX2 knockout mice (KO). ANG II increased O2− production by 346 relative light units (RLU)/mg protein in the wild-type mice ( n = 9; P < 0.0007 vs. control). In the knockout mice, ANG II increased O2− production by 290 RLU/mg protein ( n = 9; P < 0.007 vs. control). This suggests that NOX2 does not contribute to ANG II-induced O2− production ( P < 0.6 WT vs. KO). To test whether NOX4 mediates the effect of ANG II, we selectively decreased NOX4 expression in rats using an adenovirus that expresses NOX4 short hairpin (sh)RNA. Six to seven days after in vivo transduction of the kidney outer medulla, NOX4 mRNA was reduced by 77%, while NOX1 and NOX2 mRNA was unaffected. In control TALs, ANG II stimulated O2− production by 96%. In TALs transduced with NOX4 shRNA, ANG II-stimulated O2− production was not significantly different from the baseline. We concluded that NOX4 is the main catalytic isoform of NADPH oxidase that contributes to ANG II-stimulated O2− production by TALs.

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