Liver mRNA Levels Research Articles

Analysis of Alpha-2 Macroglobulin from the Long-Lived and Cancer-Resistant Naked Mole-Rat and Human Plasma.

BackgroundThe naked mole-rat (NMR) is a long-lived and cancer resistant species. Identification of potential anti-cancer and age related mechanisms is of great interest and makes this species eminent to investigate anti-cancer strategies and understand aging mechanisms. Since it is known that the NMR expresses higher liver mRNA-levels of alpha 2-macroglobulin than mice, nothing is known about its structure, functionality or expression level in the NMR compared to the human A2M.ResultsHere we show a comprehensive analysis of NMR- and human plasma-A2M, showing a different prediction in glycosylation of NMR-A2M, which results in a higher molecular weight compared to human A2M. Additionally, we found a higher concentration of A2M (8.3±0.44 mg/mL vs. and 4.4±0.20 mg/mL) and a lower total plasma protein content (38.7±1.79 mg/mL vs. 61.7±3.20 mg/mL) in NMR compared to human. NMR-A2M can be transformed by methylamine and trypsin resulting in a conformational change similar to human A2M. NMR-A2M is detectable by a polyclonal antibody against human A2M. Determination of tryptic and anti-tryptic activity of NMR and human plasma revealed a higher anti-tryptic activity of the NMR plasma. On the other hand, less proteolytic activity was found in NMR plasma compared to human plasma.ConclusionWe found transformed NMR-A2M binding to its specific receptor LRP1. We could demonstrate lower protein expression of LRP1 in the NMR liver tissue compared to human but higher expression of A2M. This was accompanied by a higher EpCAM protein expression as central adhesion molecule in cancer progression. NMR-plasma was capable to increase the adhesion in human fibroblast in vitro most probably by increasing CD29 protein expression. This is the first report, demonstrating similarities as well as distinct differences between A2M in NMR and human plasma. This might be directly linked to the intriguing phenotype of the NMR and suggests that A2M might probably play an important role in anti-cancer and the anti-aging mechanisms in the NMR.

Protective effect of bicyclol against bile duct ligation-induced hepatic fibrosis in rats.

To evaluate the protective effect of bicyclol against bile duct ligation (BDL)-induced hepatic fibrosis in rats. Sprague-Dawley male rats underwent BDL and sham-operated animals were used as healthy controls. The BDL rats were divided into two groups which received sterilized PBS or bicyclol (100 mg/kg per day) orally for two consecutive weeks. Serum, urine and bile were collected for biochemical determinations. Liver tissues were collected for histological analysis and a whole genome oligonucleotide microarray assay. Reverse transcription-polymerase chain reaction and Western blotting were used to verify the expression of liver fibrosis-related genes. Treatment with bicyclol significantly reduced liver fibrosis and bile duct proliferation after BDL. The levels of alanine aminotransferase (127.7 ± 72.3 vs 230.4 ± 69.6, P < 0.05) and aspartate aminotransferase (696.8 ± 232.6 vs 1032.6 ± 165.8, P < 0.05) were also decreased by treatment with bicyclol in comparison to PBS. The expression changes of 45 fibrogenic genes and several fibrogenesis-related pathways were reversed by bicyclol in the microarray assay. Bicyclol significantly reduced liver mRNA and/or protein expression levels of collagen 1a1, matrix metalloproteinase 2, tumor necrosis factor, tissue inhibitors of metalloproteinases 2, transforming growth factor-β1 and α-smooth muscle actin. Bicyclol significantly attenuates BDL-induced liver fibrosis by reversing fibrogenic gene expression. These findings suggest that bicyclol might be an effective anti-fibrotic drug for the treatment of cholestatic liver disease.

Hepatic Proliferation and Angiogenesis Markers Are Increased after Portal Deprivation in Rats: A Study of Molecular, Histological and Radiological Changes

Background & AimsTo determine the pathogenesis of liver nodules, and lesions similar to obliterative portal venopathy, observed after portosystemic shunts or portal vein thrombosis in humans.MethodsWe conducted an experimental study comparing portacaval shunt (PCS), total portal vein ligation (PVL), and sham (S) operated rats. Each group were either sacrificed at 6 weeks (early) or 6 months (late). Arterial liver perfusion was studied in vivo using CT, and histopathological changes were noted. Liver mRNA levels were quantified by RT-QPCR for markers of inflammation (Il10, Tnfa), proliferation (Il6st, Mki67, Hgf, Hnf4a), angiogenesis: (Vegfa, Vegfr 1, 2 and 3; Pgf), oxidative stress (Nos2, and 3, Hif1a), and fibrosis (Tgfb). PCS and PVL were compared to the S group.ResultsPeriportal fibrosis and arterial proliferation was observed in late PCS and PVL groups. CT imaging demonstrated increased arterial liver perfusion in the PCS group. RT-QPCR showed increased inflammatory markers in PCS and PVL early groups. Tnfa and Il10 were increased in PCS and PVL late groups respectively. All proliferative markers increased in the PCS, and Hnf4a in the PVL early groups. Mki67 and Hnf4a were increased in the PCS late group. Nos3 was increased in the early and late PCS groups, and Hif1a was decreased in the PVL groups. Markers of angiogenesis were all increased in the early PCS group, and Vegfr3 and Pgf in the late PCS group. Only Vegfr3 was increased in the PVL groups. Tgf was increased in the PCS groups.ConclusionsPortal deprivation in rats induces a sustained increase in intrahepatic markers of inflammation, angiogenesis, proliferation, and fibrosis.

Comparison of the acute effects of benzo-a-pyrene on adult zebrafish (Danio rerio) cardiorespiratory function following intraperitoneal injection versus aqueous exposure

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants. PAH exposure causes developmental toxicity in multiple fish species, while acute adult fish toxicity is thought to be minimal. The literature increasingly suggests sublethal PAH effects may occur, but differences in exposure route may confound conclusions. We hypothesized that acute PAH exposure in adult fish will cause cardiorespiratory impairment that will not differ with exposure route. In order to investigate this hypothesis, adult zebrafish (Danio rerio) were injected intraperitoneal (i.p.) twice with increasing concentrations of the prototypical PAH, benzo-a-pyrene (BaP; 0.1, 10, and 1000μg/kg) or exposed aqueously (static, renewal at 24h; 16.2 and 162μg/L) for 48h and compared to corresponding dimethylsulfoxide controls. No mortalities or significant effects on weight of the fish were noted at any exposure concentration or route. At 48h, fish were subjected to swimming tests with concurrent oxygen consumption measurement (n=10 fish/treatment) or echocardiography (n=12 fish/treatment). Oxygen consumption (MO2) was increased at three swimming speeds in BaP-injected groups compared to control (p<0.01 in Fisher’s LSD tests after two-way ANOVA). In contrast, aqueously BaP-exposed fish showed increased MO2 under only basal conditions. Despite increased oxygen demand, ventricular heart rate was significantly decreased in BaP-exposed fish, both injected and aqueously-exposed. Analysis of BaP body burdens in fish tissue allowed for identification of an overlapping dose group between exposure routes, through which comparisons of cardiorespiratory toxicity were then made. This comparison revealed most effects were similar between the two exposures routes, although minor differences were noted. At similar BaP body burdens, injected fish suffered from more severe bradycardia than aqueously exposed fish and had greater levels of increases in cytochrome P4501A (CYP1A) mRNA levels in liver and heart tissue compared to aqueous exposed fish. In conclusion, acute BaP exposure in adult zebrafish had negative effects on cardiorespiratory function. Differences in effect between exposure routes were attributed primarily to differences in bioavailability, since overall, similar effects were noted between the two exposure routes when similar BaP body burdens were achieved.

247. Characterization of Oversized rAAV Vectors for Human FVIII Gene Transfer

Severe hemophilia A patients with 4 years and consequent reduction or elimination of the need for frequent factor infusions. Similar rAAV-mediated gene transfer for hemophilia A patients is challenging due to the size of the FVIII cDNA that when combined with the necessary transcriptional elements exceeds the rAAV packaging limit of 4.7 kb. To test the feasibility of rAAV-mediated gene therapy for hemophilia A, we generated several oversized FVIII expression cassettes of different lengths and containing human B-domain deleted FVIII cDNA. These constructs were used to produce rAAV vectors by triple transfection and virus quality and yield were analyzed. Characterization of the packaged vector genomes (VGs) by Southern blot showed that the majority of genomes were 4.7 kb in size (with some being larger) and that the vector yield was affected by VG size. The vectors were also administered intravenously into C57BL/6 mice to test in vivo potency. Plasma FVIII levels were measured for 7 months post-treatment and at the end of the study, levels of FVIII mRNA and liver VG copies were quantitated. The data showed intact expression cassettes and comparable number of VG copies in liver among the vectors. Also, the higher liver mRNA levels correlated with higher plasma FVIII levels indicating that expression levels were a limiting factor. Lastly, the rAAV vector ranking was similar to ranking of plasmid expression cassettes (hydrodynamic injections) in vivo. These studies confirmed the viability of oversized rAAV vectors as an approach for the treatment of severe hemophilia A.

255. Simultaneous Administration of Cell Therapy with ADSCs and Gene Therapy Using Ad-huPA Reduces Experimental Liver Fibrosis

BACKGROUND: In experimental models of cirrhosis cell therapy with hADSCs (adipose derived stromal cells) has improved liver function while reduced fibrotic tissue. Also, gene therapy using Ad-huPA has diminished liver fibrosis and increased hepatocyte regeneration. The aim of this study was to evaluate if the simultaneous administration of these therapies presents an enhanced antifibrogenic effect in cirrhotic rats.METHODS: hADSCs were isolated from fat tissue, expanded and characterized by expression of CD105+, CD73+, HLA-ABC+, CD45-, CD34-, HLA-DRII- markers and osteogenic and adipogenic differentiation. Ad-huPA vector was generated by recombination of pMH4-huPA and pJM17ΔE1 Ad-v backbone. CCl4-cirrhotic rats were administered with hADSCs (2X106 cells/rat) or Ad-huPA (3×1011 vp/rat) or both hADSCs/Ad-huPA via ileac vein. 24 hours before initiating treatment all groups were immunosuppressed with 10mg/kg of Cyclosporine A until sacrifice. After 10 days, percentage of fibrotic tissue, collagen fiber staining and mRNA levels of TGF-b1, collagen a1, CTGF, PAI-I and α-SMA were evaluated. Besides, serum levels of ALT, AST and albumin, biodistribution of hADSCs and liver levels of huPA protein were examined.RESULTS: Detection of the DAPI signal revealed that hADSCs mainly homing in liver and few of them were arrested in lung and spleen. Administration of hADSCs, Ad-huPA and Ad-huPA/hADSCs reduces (p<0.01) liver fibrosis in 78.9%, 65.2% and 72% respectively; and diminishes Collagen a1, CTGF and α-SMA mRNA liver levels (p<0.05). Furthermore, TGF-b1 and PAI-I liver mRNA levels (p<0.05) decrease in animals treated with Ad-huPA and hADSCs. Serum levels of albumin increase in the Ad-huPA, hADSCs and Ad-huPA/hADSCs groups (p<0.05) compared with control group. ALT and AST serum levels showed a significant diminish in hADSCs group (p<0.05). huPA protein was expressed in similar levels in liver homogenates of Ad-huPA and Ad-huPA/hADSCs groups. hADSCs, Ad-huPA and hADSC/Ad-huPA administration reduce percentage of collagen staining 4.3, 2.4 and 2.7 fold (p£0.001), respectively.CONCLUSIONS: Xenogenic hADSCs, Ad-huPA and Ad-huPA/hADSCs therapies decreased liver fibrosis and expression of pro-fibrogenic molecules in CCl4-cirrhotic animals. However, simultaneous administration of cell therapy and gene therapy does not appear to improve antifibrogenic effects of individual treatments.

Vitamin B12 and omega-3 fatty acids together regulate lipid metabolism in Wistar rats

Our recent study indicates that maternal vitamin B12 and omega-3 fatty acid status influence plasma and erythrocyte fatty acid profile in dams. The present study examines the effects of prenatal and postnatal vitamin B12 and omega-3 fatty acid status on lipid metabolism in the offspring. Pregnant dams were divided into five groups: Control; Vitamin B12 deficient (BD); Vitamin B12 supplemented (BS); Vitamin B12 deficient group supplemented with omega-3 fatty acids (BDO); Vitamin B12 supplemented group with omega-3 fatty acids (BSO). The offspring were continued on the same diets till 3 month of age. Vitamin B12 deficiency increased cholesterol levels (p<0.01) but reduced docosahexaenoic acid (DHA) (p<0.05), liver mRNA levels of acetyl CoA carboxylase-1 (ACC-1) (p<0.05) and carnitine palmitoyltransferase-1 (CPT-1) (p<0.01) in the offspring. Omega-3 fatty acid supplementation to this group normalized cholesterol but not mRNA levels of ACC-1 and CPT-1. Vitamin B12 supplementation normalized the levels cholesterol to that of control but increased plasma triglyceride (p<0.01) and reduced liver mRNA levels of adiponectin, ACC-1, and CPT-1 (p<0.01 for all). Supplementation of both vitamin B12 and omega-3 fatty acid normalized triglyceride and mRNA levels of all the above genes. Prenatal and postnatal vitamin B12 and omega-3 fatty acids together play a crucial role in regulating the genes involved in lipid metabolism in adult offspring.

Increases in endothelial caveolin-1 and cavins correlate with cirrhosis progression

Background and aimsCaveolin-1 is associated with flat caveolar domains, invaginated smooth plasmalemmal vesicles, and caveolae. Polymerase 1 and transcript release factor (PTRF) (cavin 1) and serum deprivation protein response (SDPR) (cavin 2) are required for the invagination of caveolae, and PRKCDBP (protein kinase C, delta-binding protein; cavin 3) is required for caveolae budding to form caveolar vesicles. To investigate whether cavins are involved in hepatic sinusoidal angiogenesis and remodeling during progression to cirrhosis, normal control liver specimens and early and late cirrhotic liver specimens were studied. Materials and methodsCavin-1, cavin-2, and cavin-3 proteins and their gene expression were examined using immunohistochemistry (IHC), Western blotting, and laser capture microdissection (LCM)-polymerase chain reaction (PCR) during progression of cirrhosis caused by hepatitis C. According to the perfusion, fixation methods were designed to reevaluate the precise ultrastructural localizations and changes of cavin-1 and cavin-2 expression on liver sinusoidal endothelial cells (LSECs) facing the sinusoidal blood flow. ResultsFor IHC, cavin-1 and cavin-2 expressions were found to be upregulated in small angiogenic LSECs with collagen deposition in the perisinusoidal space as well as in the vascular endothelial cells of the remarkably proliferated portal venules, hepatic arterioles, and arterial capillaries within the fibrotic septa of late-stage cirrhotic liver. Cavin-3 was mainly localized in large vessels, and it was detected only scantly on the central vein and hepatic sinusoids in the control liver. In late-stage cirrhotic liver, the intensity of cavin-3 was enhanced mainly on proliferative large vessels in regenerated nodules and in the peripheral regions of nodules and fibrous septa. On conducting immunoelectron microscopy, in the control liver tissue, cavin-1 was found to be localized on the caveolae of hepatic arterial and portal venous endothelial cells, but it was scantly localized on hepatic sinusoidal lining cells, and cavin-2 was found mainly on vesicles in LSECs. In the cirrhotic liver tissue, aberrant cavin-1 and cavin-2 expressions were observed on caveolae-like structures in LSECs. Significant overexpressions of cavin-1 at the protein and messenger RNA (mRNA) levels in a cirrhotic liver were demonstrated by Western blotting and LCM-PCR. ConclusionsCavin-1 and cavin-2 are strongly expressed within caveolae-like structures and associated vesicles within LSECs of the hepatitis C-related cirrhotic liver. Cavin-1 would play a critical role in regulating aspects of caveolin-1 in LSECs. Moreover, these findings suggest a direct association of cavin-1 and cavin-2 with the process of differentiation and transformation of LSECs inducing hepatic sinusoidal capillarization related to the progression of cirrhosis.

Suppression of SAMSN1 Expression is Associated with the Malignant Phenotype of Hepatocellular Carcinoma

Identification of molecular markers for sensitive detection of hepatocellular carcinoma (HCC) is required to achieve efficacious personalized therapy. We focused here on SAM domain, SH3 domain, and nuclear localization signals 1 (SAMSN1) and investigated expression and methylation status of SAMSN1 in HCC cell lines and 144 pairs of surgical specimens. SAMSN1 was expressed at significantly lower levels in tumor tissue compared with the corresponding noncancerous tissues of patients with HCC. Analysis of HCC cell lines revealed that hypermethylation of the SAMSN1 promoter correlated with decreased expression of SAMSN1 mRNA. Furthermore, treating cells with a DNA-demethylating drug increased SAMSN1 transcription. The levels of SAMSN1 mRNA in noncancerous liver were not affected by background liver inflammation or fibrosis. Moreover, the levels of SAMSN1 mRNA in HCC tissues inversely correlated with tumor size and preoperative levels of proteins induced by vitamin K absence. The clinical significance of SAMSN1 was further indicated by the correlation between its decreased expression in patients with HCC and their shorter overall and recurrence-free survival as well as recurrence following initial resection. Moreover, multivariate analysis identified SAMSN1 as an independent prognostic factor of HCC progression. The expression pattern of SAMSN1 correlated significantly with that of SAMSN1 mRNA, making it possible to use PCR techniques to readily quantitate SAMSN1 expression in tumors. Our findings indicate that inhibition of SAMSN1 transcription through DNA hypermethylation may influence the progression of HCC and thus represent a novel biomarker of the phenotype of HCC cells.

Hepatic expression of detoxification enzymes is decreased in human obstructive cholestasis due to gallstone biliary obstruction.

Background & AimsLevels of bile acid metabolic enzymes and membrane transporters have been reported to change in cholestasis. These alterations (e.g. CYP7A1 repression and MRP4 induction) are thought to be adaptive responses that attenuate cholestatic liver injury. However, the molecular mechanisms of these adaptive responses in human obstructive cholestasis due to gallstone biliary obstruction remain unclear.MethodsWe collected liver samples from cholestatic patients with biliary obstruction due to gallstones and from control patients without liver disease (n = 22 per group). The expression levels of bile acid synthetic and detoxification enzymes, membrane transporters, and the related nuclear receptors and transcriptional factors were measured.ResultsThe levels of bile acid synthetic enzymes, CYP7B1 and CYP8B1, and the detoxification enzyme CYP2B6 were increased in cholestatic livers by 2.4-fold, 2.8-fold, and 1.9-fold, respectively (p<0.05). Conversely, the expression levels of liver detoxification enzymes, UGT2B4/7, SULT2A1, GSTA1-4, and GSTM1-4, were reduced by approximately 50% (p<0.05) in human obstructive cholestasis. The levels of membrane transporters, OSTβ and OCT1, were increased 10.4-fold and 1.8-fold, respectively, (p<0.05), whereas those of OSTα, ABCG2 and ABCG8 were all decreased by approximately 40%, (p<0.05) in human cholestatic livers. Hepatic nuclear receptors, VDR, HNF4α, RXRα and RARα, were induced (approximately 2.0-fold, (p<0.05) whereas FXR levels were markedly reduced to 44% of control, (p<0.05) in human obstructive cholestasis. There was a significantly positive correlation between the reduction in FXR mRNA and UGT2B4/7, SULT2A1, GSTA1, ABCG2/8 mRNA levels in livers of obstructive cholestatic patients (p<0.05).ConclusionsThe levels of hepatic detoxification enzymes were significantly decreased in human obstructive cholestasis, and these decreases were positively associated with a marked reduction of FXR levels. These findings are consistent with impaired detoxification ability in human obstructive cholestasis.

Alcohol exposure in utero perturbs retinoid homeostasis in adult rats.

Maternal alcohol exposure and adult alcohol intake have been shown to perturb the metabolism of various micro- and macro-nutrients, including vitamin A and its derivatives (retinoids). Therefore, it has been hypothesized that the well-known detrimental consequences of alcohol consumption may be due to deregulations of the metabolism of such nutrients rather than to a direct effect of alcohol. Alcohol exposure in utero also has long-term harmful consequences on the health of the offspring with mechanisms that have not been fully clarified. Disruption of tissue retinoid homeostasis has been linked not only to abnormal embryonic development, but also to various adult pathological conditions, including cancer, metabolic disorders and abnormal lung function. We hypothesized that prenatal alcohol exposure may permanently perturb tissue retinoid metabolism, predisposing the offspring to adult chronic diseases. Serum and tissues (liver, lung and prostate from males; liver and lung from females) were collected from 60-75 day-old sprague dawley rats born from dams that were: (I) fed a liquid diet containing 6.7% alcohol between gestational day 7 and 21; or (II) pair-fed with isocaloric liquid diet during the same gestational window; or (III) fed ad libitum with regular rat chow diet throughout pregnancy. Serum and tissue retinoid levels were analyzed by reverse-phase high-performance liquid chromatography (HPLC). Serum retinol-binding protein (RBP) levels were measured by western blot analysis, and liver, lung and prostate mRNA levels of lecithin-retinol acyltransferase (LRAT) were measured by qPCR. Retinyl ester levels were significantly reduced in the lung of both males and females, as well as in the liver and ventral prostate of males born from alcohol-fed dams. Tissue LRAT mRNA levels remained unchanged upon maternal alcohol treatment. Prenatal alcohol exposure in rats affects retinoid metabolism in adult life, in a tissue- and sex-dependent manner. We propose that the alcohol-induced perturbations of vitamin A metabolism may predispose to detrimental consequnces on adult health.

Increase in Nociception Threshold and Decrease in Mu-Opioid Receptor Gene Expression in Liver of a Rat Model of Hepatic Encephalopathy

Introduction: Hepatic encephalopathy (HE) is a brain disorder with impairment of cognitive and motor functions caused by liver failure. The aim of this study was to evaluate changes in nociception threshold as well as gene expression of mu-opioid receptor 1 (MOR1) in rat liver after induction of HE. Materials and Methods: The male rats (280-320 g) were randomly divided into three groups of control (non-operated), sham (operated without common bile duct ligation) and a group with a common bile duct ligation (BDL) as an experimental model of HE. Changes in nociception threshold were assessed on day 21 of BDL, using a hotplate test. On day 21 of BDL, rats were weighed, sacrificed and total liver tissue was dissected and weighed, then percentage of liver to body weight was measured. In addition, blood samples were collected for biochemical analysis of serum, and a piece of the liver of each rat was dissected for evaluating the MOR1 gene expression. Results: The results showed that nociception threshold, percentage of liver to body weights, and serum levels of total bilirubin were significantly increased in the HE model group compared to the sham group (P<0.001). Blood urea nitrogen was also increased in this group compared to the sham group (P<0.01). The results also revealed that the MOR1 gene expression at mRNA levels in liver of rats with BDL was significantly decreased compared to the sham group ( P<0.001). Conclusion: Our data indicate that chronic liver failure modulates the MOR1 gene expression in liver. In addition, changes in nociception threshold may confirm a slowness of sensory processing in HE.

Optimization of multifunctional chitosan–siRNA nanoparticles for oral delivery applications, targeting TNF-α silencing in rats

Secretion of tumor necrosis factor-α (TNF-α) by macrophages plays a predominant role in the development and progression of various inflammatory diseases. In the current contribution, multifunctional nanoparticles (NPs) containing TNF-α siRNA targeting macrophages via oral administration were developed to knockdown TNF-α expression against acute hepatic injury in rats. Mannose-modified trimethyl chitosan-cysteine (MTC) NPs were prepared by self-assembly method (sa-MTC NPs), ionic gelation and siRNA entrapment method (en-MTC NPs), and ionic gelation and siRNA adsorption method (ad-MTC NPs). Among them, en-MTC NPs demonstrated the best stability against ionic challenges with desired siRNA integrity against nucleases. By targeting normal enterocytes and M cells that express mannose receptors, en-MTC NPs notably promoted intestinal absorption of siRNA in rats. They further facilitated siRNA internalization by rat peritoneal exudate cells (PECs) via lipid-raft involved endocytosis and macropinocytosis, thus inducing effective in vitro TNF-α knockdown. Orally delivered en-MTC NPs at a low siRNA dose of 50μg/kg inhibited systemic TNF-α production and decreased TNF-α mRNA levels in macrophage-enriched liver, spleen, and lung tissues, which consequently protected rats from acute hepatic injury. Therefore, the en-MTC NPs would provide an effective approach to orally deliver TNF-α siRNA for the anti-inflammatory therapy.

Melatonin reduces lead levels in blood, brain and bone and increases lead excretion in rats subjected to subacute lead treatment

Melatonin, a hormone known for its effects on free radical scavenging and antioxidant activity, can reduce lead toxicity in vivo and in vitro.We examined the effects of melatonin on lead bio-distribution. Rats were intraperitoneally injected with lead acetate (10, 15 or 20mg/kg/day) with or without melatonin (10mg/kg/day) daily for 10 days. In rats intoxicated with the highest lead doses, those treated with melatonin had lower lead levels in blood and higher levels in urine and feces than those treated with lead alone, suggesting that melatonin increases lead excretion. To explore the mechanism underlying this effect, we first assessed whether lead/melatonin complexes were formed directly. Electronic density functional (DFT) calculations showed that a lead/melatonin complex is energetically feasible; however, UV spectroscopy and NMR analysis showed no evidence of such complexes. Next, we examined the liver mRNA levels of metallothioneins (MT) 1 and 2. Melatonin cotreatment increased the MT2 mRNA expression in the liver of rats that received the highest doses of lead. The potential effects of MTs on the tissue distribution and excretion of lead are not well understood. This is the first report to suggest that melatonin directly affects lead levels in organisms exposed to subacute lead intoxication.

Expression and its significance of caveolin–1 in liver and gallbladder of gallstone mice model

Objective To investigate the role and possible mechanism of caveolin–1 (CAV1) in the forming of cholesterol gallstone in mice fed with lithogenic diet. Methods Cholesterol gallstone susceptible C57BL/6 mice were study objects. The mice of control group (n=6) and experiment group (n=6) were fed with normal diet and lithogenic diet for four weeks respectively. The condition of cholesterol gallstone forming, changes of serum lipid and bile composition were measured, and the expressions of CAV1 and scavenger receptor classB member Ⅰ (SR–BⅠ) at mRNA and protein level in the liver and gallbladder were detected by realtime–polymerase chain reaction and Western blot, respectively. The t test was performed for mean comparsion between the two groups. Results The incidence rate of gallstone in experimental group was 100% after fed with lithogenic diet for four weeks, the lipid level significantly increased, and the proportion of cholesterol in bile raised and bile salt decreased. Compared with those of control group, the expressions of CAV1 at mRNA and protein level in the liver and gallbladder tissues siginificantly decreased (in liver tissue, mRNA 0.53±0.13 vs 1.00±0.32, t=3.330, protein level 0.39±0.07 vs 0.92±0.06, t=10.280; in gallbladder tissue, mRNA 0.40±0.22 vs 1.00±0.22, t=3.823, protein level 1.04±0.07 vs 1.34±0.04, t=6.367, all P<0.01). There was no significant difference in the relative expression of SR–BⅠ at mRNA and protein level in the liver and gallbladder tissues between the mice of experiment group and control group. Conclusion The changes of CAV1 expression at mRNA and protein level in liver and gallbladder tissues may affect lipids metabolism and cholesterol transportation in liver and gallbladder tissues of experiment mice, which might play an important role in the formation of cholesterol gallstone. Key words: Cholesterol gallstone disease; Lithogenic diet; Caveolin–1; Scavenger receptor class B member 1

Confined housing system increased abdominal and subcutaneous fat deposition and gene expressions of carbohydrate response element-binding protein and sterol regulatory element-binding protein 1 in chicken.

Free-range production system is increasingly being used in poultry breeding and feed production in many countries. The objective of the current experiment was to evaluate the effects of different raising systems on fat-related traits and mRNA levels of liver lipogenesis genes in Erlang Mountainous chicken. Each of 10 birds (91 day old) from caged, indoor-floor housed, and free-range housing systems was slaughtered, and fat-related traits, live body weight (BW), subcutaneous fat thickness (SFT), abdominal fat weight (AFW), abdominal fat percentage (AFP), and intramuscular fat content were determined. The mRNA levels of liver X receptor α, carbohydrate response element-binding protein (ChREBP), sterol regulatory element-binding protein-1 (SREBP1), and fatty acid synthase were detected. The caged chicken exhibited significantly higher BW, SFT, and AFW than those of free-ranged chicken (P < 0.05). All the 4 genes had a similar expression pattern, and they showed the highest level in caged chicken, while the lowest level was found in free-ranged chicken. Association analysis indicated that there were significant (P < 0.05) or highly significant (P < 0.01) positive correlations between the mRNA levels of ChREBP, SREBP1, and fat traits of SFT, AFW, and AFP. Thus, we deduced that increased fat deposition in caged chicken was probably induced by increased gene expression of ChREBP and SREBP1 in the liver.

Preclinical Development of a Subcutaneous ALAS1 RNAi Therapeutic for Treatment of Hepatic Porphyrias Using Circulating RNA Quantification.

The acute hepatic porphyrias are caused by inherited enzymatic deficiencies in the heme biosynthesis pathway. Induction of the first enzyme 5-aminolevulinic acid synthase 1 (ALAS1) by triggers such as fasting or drug exposure can lead to accumulation of neurotoxic heme intermediates that cause disease symptoms. We have demonstrated that hepatic ALAS1 silencing using siRNA in a lipid nanoparticle effectively prevents and treats induced attacks in a mouse model of acute intermittent porphyria. Herein, we report the development of ALN-AS1, an investigational GalNAc-conjugated RNAi therapeutic targeting ALAS1. One challenge in advancing ALN-AS1 to patients is the inability to detect liver ALAS1 mRNA in the absence of liver biopsies. We here describe a less invasive circulating extracellular RNA detection assay to monitor RNAi drug activity in serum and urine. A striking correlation in ALAS1 mRNA was observed across liver, serum, and urine in both rodents and nonhuman primates (NHPs) following treatment with ALN-AS1. Moreover, in donor-matched human urine and serum, we demonstrate a notable correspondence in ALAS1 levels, minimal interday assay variability, low interpatient variability from serial sample collections, and the ability to distinguish between healthy volunteers and porphyria patients with induced ALAS1 levels. The collective data highlight the potential utility of this assay in the clinical development of ALN-AS1, and in broadening our understanding of acute hepatic porphyrias disease pathophysiology.

Abstract B10: LFA-1/ICAM-1 interaction switches on an orchestrated prometastatic microenvironmental shift during experimental liver metastasis of colon C26 cancer cells.

Abstract Intercellular adhesion molecule (ICAM)-1 is the main counter-receptor for the lymphocyte function associated antigen (LFA)-1. However, the role this interaction plays during liver metastasis of colorectal cancer cells and its underlying mechanisms remain unclear. Previously, we showed that mannose receptor (ManR) stimulation on liver sinusoidal endothelial cells (LSECs) resulted in a reduced cytotoxic potential of liver infiltrating lymphocytes (LIL) towards C26 murine colon carcinoma cells. Using ICAM-1 specific siRNAs in vivo, we aimed to study the role of hepatic ICAM-1 in the prometastatic shift of the liver microenvironment. Livers of control and silenced mice, 24 hours and 14 days after C26 cell inoculation, were compared for: percentage of detained C26 cells, number and volume of metastatic foci, levels of recruited immune, myofibroblastic and inflammatory cells and whole liver expression of pro-inflammatory genes. Detained cancer cells and metastatic foci number were reduced in silenced mice compared to controls, along with a decrease in myeloid suppressor cells and recruited macrophages and myofibroblasts. On the contrary, significant higher numbers of lymphocytic cells were observed in mice with reduced ICAM-1. Finally, silencing of ICAM1 resulted in altered total liver mRNA levels of proinflammatory factors in the tumor-bearing mice. Therefore, an orchestrated prometastatic program might be switched on in the liver microenvironment by LFA-1/ICAM-1 interaction which, in turn, could lead not only to a reduced antitumor citotoxicity but, also, to activation of the stromal compartment favoring tumor progression, and, opening liver's doors to tumor cells to aggressively metastasize. Based on these results, LFA-1/ICAM-1 interaction arises as a potential therapeutic target in cancer treatment. Citation Format: Aitor Benedicto, Joana Marquez, Elvira Olaso, Beatriz Arteta. LFA-1/ICAM-1 interaction switches on an orchestrated prometastatic microenvironmental shift during experimental liver metastasis of colon C26 cancer cells.. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B10. doi:10.1158/1538-7445.CHTME14-B10

Insights into molecular profiles and genomic evolution of an IRAK4 homolog from rock bream (Oplegnathus fasciatus): Immunogen- and pathogen-induced transcriptional expression

As a pivotal signaling mediator of toll-like receptor (TLR) and interleukin (IL)-1 receptor (IL-1R) signaling cascades, the IL-1R-associated kinase 4 (IRAK4) is engaged in the activation of host immunity. This study investigates the molecular and expressional profiles of an IRAK4-like homolog from Oplegnathus fasciatus (OfIRAK4). The OfIRAK4 gene (8.2 kb) was structured with eleven exons and ten introns. A putative coding sequence (1395bp) was translated to the OfIRAK protein of 464 amino acids. The deduced OfIRAK4 protein featured a bipartite domain structure composed of a death domain (DD) and a kinase domain (PKc). Teleost IRAK4 appears to be distinct and divergent from that of tetrapods in terms of its exon-intron structure and evolutionary relatedness. Analysis of the sequence upstream of translation initiation site revealed the presence of putative regulatory elements, including NF-κB-binding sites, which are possibly involved in transcriptional control of OfIRAK4. Quantitative real-time PCR (qPCR) was employed to assess the transcriptional expression of OfIRAK4 in different juvenile tissues and post-injection of different immunogens and pathogens. Ubiquitous basal mRNA expression was widely detected with highest level in liver. In vivo flagellin (FLA) challenge significantly intensified its mRNA levels in intestine, liver and head kidney indicating its role in FLA-induced signaling. Meanwhile, up-regulated expression was also determined in liver and head kidney of animals challenged with potent immunogens (LPS and poly I:C) and pathogens (Edwardsiella tarda and Streptococcus iniae and rock bream iridovirus (RBIV)). Taken together, these data implicate that OfIRAK4 might be engaged in antibacterial and antiviral immunity in rock bream.

Effects of Long-Term Cobalamin Treatment on the Expression of the Epidermal Growth Factor System in Mouse Kidney

Background: The epidermal growth factor (EGF) system plays an important role in the development and function of the kidneys. Studies have suggested this system to protect against acute renal injury and to prevent the progression of chronic kidney disease. We have demonstrated that cobalamin (Cbl) modulates the expression of the EGF system in mouse spinal cord. Here, we explore the effects of Cbl treatment on the mouse kidney expression of members of the EGF system comparing these with the expression in the submaxillary gland and liver. Methods: Following four weeks of administrating different doses of Cbl, we analysed kidney, submaxillary gland, and liver mRNA levels of the four EGF system receptors and the activating ligands EGF and heparin-binding EGF-like growth factor using quantitative reverse transcription-PCR. In addition, we analysed mRNA expression of the renal tubular injury markers, kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin. Results: In controls, kidney expression of EGF system receptors and ligands was similar to or more than twice the levels in the submaxillary gland. A relatively high expression of the EGF receptor was observed in liver tissue, but only low or no expression of other EGF related molecules. Treatment with Cbl decreased expression of the EGF system in the kidneys in a dose-dependent manner, while no or little change was observed in other organs. No alterations were observed in the markers of kidney tubular injury after Cbl administration. Conclusions: Our results show modulation of the renal EGF system expression in mice following long term Cbl administration. This may have implications for prevention of progressive kidney disease. In addition, differences in the expression of the EGF receptors and ligands in liver, submaxillary gland and kidney were demonstrated.