小异二聚体伴侣与胆固醇7a-羟化酶在肝内胆汁淤积症孕鼠中的表达

目的 建立妊娠期肝内胆汁淤积症孕鼠模型,检测小异二聚体伴侣(SHP)及其靶基因胆固醇7a-羟化酶(CYP7A1)在肝脏的表达,探讨其在妊娠期肝内胆汁淤积症发病机制中的作用.方法 孕15d SD大鼠30只随机分为对照组和雌激素组,每组15只.用药前和用药后第5天分别检测血清中ALT、 AST、碱性磷酸酶、总胆汁酸、总胆红素、直接胆红素水平,同时观察肝脏形态学变化,并应用RT-PCR和Western blot分别检测肝脏SHP和CYP7A1的mRNA和蛋白质的表达.结果 雌激素组用药后ALT、 AST、碱性磷酸酶、总胆汁酸、总胆红素、直接胆红素比用药前明显升高(P<0.01);对照组用药前后各血清生物化学指标无明显变化(P>0.05).雌激素组孕鼠肝脏出现肝内胆汁淤积表现,对照组肝脏形态、结构正常.雌激素组和对照组SHP mRNA分别为0.4865±0.0237和0.3657±0.0317, CYP7A1 mRNA分别为0.4311±0.0157和0.3285±0.0123,差异均有统计学意义(P<0.01).雌激素组和对照组SHP蛋白表达分别为0.5033±0.0274和0.3762±0.0284, CYP7A1蛋白表达分别为0.4802±0.0217和0.3570±0.0175,差异均有统计学意义(P<0.01).结论 雌激素诱导的胆汁淤积促进肝脏SHP及其靶基因CYP7A1表达增加,导致胆汁酸合成进一步增加,从而引发胆汁淤积,这可能是妊娠期肝内胆汁淤积症发病机制之一.     

Progressive familial intrahepatic cholestasis, type 1, is associated with decreased farnesoid X receptor activity

BACKGROUND & AIMS: The mechanisms by which mutations in the familial intrahepatic cholestasis-1 gene cause Byler's disease (progressive familial intrahepatic cholestasis type 1) are unknown. METHODS: Interactions among the apical sodium-dependent bile acid transporter, the farnesoid X receptor (FXR), and familial intrahepatic cholestasis-1 were studied in the ileum of children with progressive familial intrahepatic cholestasis type 1 and in Caco-2 cells. RESULTS: Increased ileal apical sodium-dependent bile acid transporter messenger RNA (mRNA) expression was detected in 3 patients with progressive familial intrahepatic cholestasis type 1. Paradoxically, ileal lipid-binding protein mRNA expression was repressed, suggesting a central defect in bile acid response. Ileal FXR and short heterodimer partner mRNA levels were reduced in the same 3 patients. In Caco-2 cells, antisense-mediated knock-down of endogenous familial intrahepatic cholestasis-1 led to up-regulation of apical sodium-dependent bile acid transporter and down-regulation of FXR, ileal lipid-binding protein, and short heterodimer partner mRNA. In familial intrahepatic cholestasis-1-negative Caco-2 cells, the activity of the human apical sodium-dependent bile acid transporter promoter was enhanced, whereas the human FXR and bile salt excretory pump promoters' activities were reduced. Overexpression of short heterodimer partner but not of the FXR abrogated the effect of familial intrahepatic cholestasis-1 antisense oligonucleotides. FXR cis-element binding and FXR protein were reduced primarily in nuclear but not cytoplasmic extracts from familial intrahepatic cholestasis-1-negative Caco-2 cells. CONCLUSIONS: Loss of familial intrahepatic cholestasis-1 leads to diminished nuclear translocation of the FXR, with the subsequent potential for pathologic alterations in intestinal and hepatic bile acid transporter expression. Marked hypercholanemia and cholestasis are predicted to develop, presumably because of both enhanced ileal uptake of bile salts via up-regulation of the apical sodium-dependent bile acid transporter and diminished canalicular secretion of bile salts secondary to down-regulation of the bile salt excretory pump.     

Integrin alpha2beta1 plays a critical role in osteoblast response to micron-scale surface structure and surface energy of titanium substrates

Efforts to improve bone response to biomaterials have focused on ligands that bind α5β1 integrins. However, antibodies to α5β1 reduce osteoblast proliferation but do not affect differentiation when cells are grown on titanium (Ti). β1-silencing blocks the differentiation stimulus of Ti microtopography, suggesting that other β1 partners are important. Stably α2-silenced MG63 human osteoblast-like cells were used to test whether α2β1 specifically mediates osteoblast response to Ti surface micron-scale structure and energy. WT and α2-silenced MG63 cells were cultured on tissue culture polystyrene (TCPS) and Ti disks with different surface microtopographies: machined pretreatment (PT) surfaces [mean peak to valley roughness (R_a) < 0.02 μm], PT surfaces that were grit-blasted and acid-etched (SLA; R_a = 4 μm), and SLA with high surface energy (modSLA). Alkaline phosphatase (ALP), α2 and β1 mRNA, but not α5, αv, β3, type-I collagen, or osteocalcin, increased on SLA and modSLA at 6 days. α2 increased at 8 days on TCPS and PT, but remained unchanged on SLA and modSLA. α2-protein was reduced 70% in α2-siRNA cells, whereas α5-mRNA and protein were unaffected. α2-knockdown blocked surface-dependent increases in β1 and osteocalcin and decreases in cell number and increases in ALP and local factors typical of MG63 cells grown on SLA and modSLA [e.g., prostaglandin E₂, osteoprotegerin, latent and active TGF-β1, and stimulatory effects of 1α,25(OH)₂D₃ on these parameters]. This finding indicates that α2β1 signaling is required for osteoblastic differentiation caused by Ti microstructure and surface energy, suggesting that conclusions based on cell behavior on TCPS are not predictive of behavior on other substrates or the mechanisms involved.     

The nuclear receptor SHP mediates inhibition of hepatic stellate cells by FXR and protects against liver fibrosis

BACKGROUND AND AIMS: The farnesoid X receptor (FXR) is an endogenous sensor for bile acids and inhibits bile acid synthesis by inducing small heterodimer partner (SHP) gene expression. The aim of this study was to investigate whether FXR is expressed by and modulates function of hepatic stellate cells (HSCs). METHODS: The antifibrotic activity of FXR ligand was tested in 2 rodent models: the porcine serum and bile duct ligation (BDL). RESULTS: Twelve-week administration of 1-10 mg/kg 6-ethyl chenodeoxycholic acid (6-ECDCA), a synthetic FXR ligand, to porcine serum-treated rats prevented liver fibrosis development and reduced liver expression of alpha1(I) collagen, TGF-beta1 and alpha-SMA mRNA by approximately 90%. Therapeutic administration of 6-ECDCA, 3 mg/kg, to BDL rats reduced liver fibrosis and alpha1(I) collagen, transforming growth factor (TGF)-beta1, alpha-SMA, and tissue metalloproteinase inhibitor (TIMP)-1 and 2 messenger RNA (mRNA) by 70%-80%. No protection was observed in BDL rats treated with CDCA, 3 mg/kg, and ursodeoxycholic acid, 15 mg/kg. FXR expression was detected in HSCs. Exposure of HSCs to FXR ligands caused a 3-fold increase of SHP, reduced alpha1(I)collagen and TGF-beta1 by approximately 60%-70% and abrogates alpha1(I) collagen mRNA up-regulation induced by thrombin and TGF-beta1. By retrovirus infection and small interference RNA, we generated SHP overexpressing and SHP-deficient HSC-T6. Using these cell lines, we demonstrated that SHP binds JunD and inhibits DNA binding of adaptor protein (AP)-1 induced by thrombin. CONCLUSIONS: By demonstrating that an FXR-SHP regulatory cascade promotes resolution of liver fibrosis, this study establish that FXR ligands might represent a novel therapeutic option to treat liver fibrosis.     

Functional variants of the central bile acid sensor FXR identified in intrahepatic cholestasis of pregnancy

BACKGROUND AND AIMS: Intrahepatic cholestasis of pregnancy (ICP) is characterized by liver impairment, pruritus, and elevated maternal serum bile acids. It can cause premature delivery and intrauterine death. Bile acid synthesis, metabolism, and transport are regulated by the bile acid sensor FXR, and we hypothesized that genetic variation in FXR confers susceptibility to ICP. METHODS: The coding regions and intron/exon boundaries of FXR were sequenced in 92 British ICP cases of mixed ethnicity. Subsequently, a case-control study of allele frequencies of these variants in 2 independent cohorts of Caucasian ICP patients and controls was performed. Variants were cloned into an FXR expression plasmid and tested in functional assays. RESULTS: We identified 4 novel heterozygous FXR variants (-1g>t, M1V, W80R, M173T) in ICP. W80R was not present in Caucasians and M1V was detected uniquely in 1 British case. M173T and -1g>t occur both in Caucasian cases and controls, and we found a significant association of M173T with ICP (OR, 3.2; 95% confidence interval, 1.1-11.2; P = .02) when the allele frequencies of both Caucasian cohorts were analyzed together. We demonstrate functional defects in either translation efficiency or activity for 3 of the 4 variants (-1g>t, M1V, M173T). CONCLUSIONS: This is the first report of functional variants in FXR. We propose that these variants may predispose to ICP, and because FXR has a central role in regulating bile and lipid homeostasis they may be associated with other cholestatic and dyslipidemic disorders.     

The role of distinct p185neu extracellular subdomains for dimerization with the epidermal growth factor (EGF) receptor and EGF-mediated signaling

The extracellular domain of p185(c-neu) can be viewed as a complex structure of four subdomains, two of which are cysteine-rich subdomains. We have investigated the contribution of these distinct p185(c-neu) extracellular subdomains to p185/epidermal growth factor receptor (EGFR) heteromer formation and EGF-induced heteromeric signaling. Our studies indicate that at least two separate p185 subdomains, a region spanning subdomains I and II and subdomain IV are involved in association of p185 with the EGFR. We also demonstrated that subdomain IV reduced the heteromeric signaling and transforming activities induced by EGF after associating with EGFR. When 126 aa were deleted from subdomain IV, this small subdomain IV-derived fragment could still lead to heterodimers with EGFR and suppress EGF-induced mitogen-activated protein kinase activation and subsequent transformation abilities. These data provide information about trans-inhibitory mechanisms of mutant p185 species and also indicate that both the entire and a part of subdomain IV may represent a therapeutic target for erbB-overexpressing tumors. Finally, these studies define a basic feature of receptor-receptor associations that are determined by cystine-knot containing subdomains.     

Subclinical Inflammation in Heart Failure: A Neutrophil Perspective

Although it is widely recognized that inflammation plays a critical role in the development and pathology of heart failure (HF), very little is known about the involvement of one of the most abundant immune cells in the blood, a primary immune response cell: the neutrophil. This review summarizes the current literature on the role of subclinical inflammation, with a focus on the neutrophil in the pathophysiology of the HF syndrome. Some emerging therapeutic strategies are also discussed.     

Characterization of α,α-trehalase released in the intestinal lumen by the probiotic Saccharomyces boulardii

Objective. Trehalose intolerance due to α,α-trehalase deficiency has scarcely been studied. The purpose of this study was to measure α,α-trehalase activity in intestinal biopsy samples from 200 consecutive patients over a period of 6 months, and to characterize α,α-trehalase released by the probiotic Saccharomyces boulardii (S. boulardii). Material and methods. Enzyme activities were measured in human and rat intestinal mucosal samples using the micromethod of Messer & Dalqvist. α,α-trehalase from S. boulardii was immunoprecipitated and Western blotted using an IgG purified antibody raised against a 23 amino acid peptide of α,α-trehalase of S. cerevisiae. Results. Among 200 patients, most of whom complained of abdominal symptoms and diarrhoea, 18 (9%) had total α,α-trehalase deficiency (0–12 U/g mucosa) and 39 had partial deficiency (3–12 U/g mucosa). Only 4 patients (2%) presented selective α,α-trehalase deficiency with otherwise normal disaccharidases. Expressed per gram of powder, α,α-trehalase from S. boulardii delivered in vitro an activity 175 times higher than that of human trehalase per gram of intestinal mucosa. V_max (22±0.43 µmol) and K_m (5 mM) were close to that of the human enzyme, whereas Western blot revealed a signal of two subunits of 82 kDa. Finally, treatment of rats with S. boulardii resulted in increases in α,α-trehalase activities of 25 to 45% (p<0.01) in endoluminal fluid and intestinal mucosa compared with in controls. Conclusions. Our data suggest that α,α-trehalase deficiency is more common than is believed and that oral administration of S. boulardii could be beneficial in patients with digestive symptoms caused by trehalose intolerance.     

Hockey Games and the Incidence of ST-Elevation Myocardial Infarction

Background

The association between diagnosed acute ST-elevation myocardial infarction (STEMI) and hockey games in the Canadian population is unknown.

Expanding Eligibility for the Ross Procedure: A Reasonable Proposition?

Background

Although the Ross procedure offers potential benefits in young adults, technical complexity represents a significant limitation. Therefore, the safety of expanding its use in more complex settings is uncertain. The aim of this study was to compare early outcomes of standard isolated Ross procedures vs expanding elgibility to higher-risk clinical settings.

Atherosclerotic Heart Disease in Women With Autoimmune Rheumatologic Inflammatory Conditions

Women have a higher prevalence of several inflammatory rheumatologic conditions. These include systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and systemic sclerosis (SSc) to name a few. These conditions are all associated with higher rates of cardiovascular (CV) morbidity and mortality, which is driven primarily by atherosclerotic heart disease. Traditional risk factors are important considerations in the assessment of CV risk in the rheumatologic patient; however, these factors do not appear to impart a similar weight of risk in women with inflammatory autoimmune rheumatologic conditions. In addition, even when controlling for traditional risk factors, patients with RA or SLE continue to have a higher risk of CV events, which has been linked to the burden of systemic inflammation. Currently, the CV risk scoring systems available for the general population underestimate the burden of the problem in these complex patients. The increased CV risk in patients with rheumatologic diseases has been reported in the literature for years but remains underrecognized by internists and cardiologists. Although these conditions themselves are relatively rare, the further study of inflammation and its treatment in CV disease will be beneficial to the general population.