A BALB/c mouse model shows that liver involvement in dengue disease is immune-mediated

In the present study, BALB/c mice were used to develop a model for the hepatic injury associated to dengue infection. Histological analysis after subcutaneous inoculation with a low viral dose of dengue-2 virus showed Kupffer cell hyperplasia and an increased inflammatory cellular infiltrate next to the bile ducts on days 5, 7 and 14 post-inoculation, mainly characterized by the presence of mononuclear cells. The liver mRNA transcription level of IL-1 beta was highest on the 5th day post-infection (p.i.) and decreased by the 21st day, TNF-alpha showed a peak of mRNA transcription after 14 days p.i. coinciding with the regression of cellular infiltrates and elevated expression of TGF-beta mRNA. Serum AST and ALT levels were slightly elevated at 7 and 14 days post-infection. Dengue-2 RNA levels were undetectable in the liver on any of the days following inoculation. Our observations suggest that, as it is true for humans, the animals undergo a transient and slight liver inflammation, probably due to local cytokine production and cellular infiltration in the liver. (C) 2010 Elsevier Inc. All rights reserved.

Host genetic factors in mouse malaria liver stage infection

Fundação para a Ciência e a Tecnologia

Genetic analysis of c-Myc in mouse bone marrow and liver

1. Summary The transcription factor and proto-oncogene c-myc plays an important role in integrating many mitogenic signals within the cell. The consequences are both broad and varied and include the regulation of apoptosis, cellular differentiation, cellular growth and cell cycle progression. It is found to be mis-regulated in over 70% of all cancers, however, our knowledge about c-Myc remains limited and very little is known about its physiological role in mammalian development and in adulthood. We have addressed the physiological role of c-Myc in both the bone marrow and the liver of mice by generating adult c-myc flox/flox mice that lacked c-myc in either the bone marrow or the liver after conversion of the c-myc flox alleles into null alleles by the inducible Mx¬Cre transgene with polyI-polyC. In investigating the role of c-Myc in the haematopoietic system, we concentrated on the aspects of cellular proliferation, cellular differentiation and apoptosis. Mice lacking c-Myc develop anaemia between 3-8 weeks and all more differentiated cell types are severely depleted leading to death. However in addition to its role in driving proliferation in transient amplifying cells, we unexpectedly discovered a new role for c-Myc in controlling haematopoietic stem cell (HSC) differentiation. c-Myc deficient HSCs are able to proliferate normally in vivo. In addition, their differentiation into more committed progenitors is blocked. These cells expressed increased adhesion molecules, which possibly prevent HSCs from being released from the special stem cell supporting stromal niche cells with which they closely associate. Secondly we used the liver as a model system to address the role of c-Myc in cellular growth, meaning the increase in cell size, and also cellular proliferation. Our results revealed c-Myc to play no role in metabolic cellular growth following a period of fasting. Following treatment with the xenobiotic TCPOBOP, c-Myc deficient hepatocytes increased in cell size as control hepatocytes and could surprisingly proliferate albeit at a reduced rate demonstrating a c-Myc independent proliferation pathway to exist in parenchymal cells. However, following partial hepatectomy, in which two-thirds of the liver was removed, mutant livers were severely restricted in their regeneration capacity compared to control livers demonstrating that c-Myc is essential for liver regeneration. Résumé Le facteur de transcription et proto-oncogène c-myc joue un rôle important dans l'intégration de nombreux signaux mitogéniques dans la cellule. Les conséquences de son activation sont étendues et variées et incluent la régulation de l'apoptose, de la différenciation, de la croissance et de la progression du cycle cellulaire. Même si plus de 20% des cancers montrent une dérégulation de c-myc, les connaissances sur ce facteur de transcription restent limitées et ses rôles physiologiques au cours du développement et chez l'adulte sont très peu connus. Nous avons étudié le rôle physiologique de c-Myc dans la molle osseuse et le foie murin en générant des souris adultes c-myc flox/flox. Dans ces souris, les allèles c-myc flox sont convertis en allèles nuls par le transgène Mx-Cre après induction avec du Poly-I.C. Pour notre étude du rôle de c-Myc dans le système hématopoiétique, nous nous sommes concentrés sur les aspects de la prolifération et de la différenciation cellulaire, ainsi que sur l'apoptose. Les souris déficientes pour c-Myc développent une anémie 3 à 8 semaines après la délétion du gène; tous les différents types cellulaires matures sont progressivement épuisés ce qui entraîne la mort des animaux. Néanmoins, outre sa capacité à induire la prolifération des cellules transitoires de la molle osseuse, nous avons inopinément découvert un nouveau rôle pour c-Myc dans le contrôle de la différenciation des cellules souches hématopoiétiques (HSC). Les HSC déficientes pour c-Myc prolifèrent normalement in vivo mais leur différenciation en progéniteurs plus engagés dans une voie de différenciation est bloquée. Ces cellules surexpriment certaines molécules d'adhésion ce qui empêcherait les HSC d'être relachées du stroma spécialisé, ou niche, auquel elles sont étroitement associées. D'autre part, nous avons utilisé le foie comme système modèle pour étudier le rôle de c-Myc dans la prolifération et dans la croissance cellulaire, c'est à dire l'augmentation de taille des cellules. Nos résultats ont révélé que c-Myc ne joue pas de rôle dans le métabolisme cellulaire qui suit une période de jeûne. L'augmentation de la taille cellulaire des hépatocytes déficients pour c-Myc suite au traitement avec l'agent xénobiotique TCPOBOP est identique à celle observée pour les cellules de contrôle. Le taux de prolifération des hépatocytes mutants est par contre réduit, indiquant qu'une voie de différenciation indépendante de c-Myc existe dans les cellules parenchymales. Néanmoins, après hépatectomie partielle, où deux-tiers du foie sont éliminés chirurgicalement, les foies mutants sont sévèrement limités dans leur capacité de régénération par rapport aux foies de contrôle, montrant ainsi que c-Myc est essentiel pour la régénération hépatique.

No development of hypertension in the hyperuricemic liver-Glut9 knockout mouse.

Urate is the metabolic end point of purines in humans. Although supra-physiological plasma urate levels are associated with obesity, insulin resistance, dyslipidemia, and hypertension, a causative role is debated. We previously established a mouse model of hyperuricemia by liver-specific deletion of Glut9, a urate transporter that provides urate to the hepatocyte enzyme uricase. These LG9 knockout mice show mild hyperuricemia (120 μmol/l), which can be further increased by the urate precursor inosine. Here, we explored the role of progressive hyperuricemia on the cardiovascular function. Arterial blood pressure and heart rate were periodically measured by telemetry over 6 months in LG9 knockout mice supplemented with incremental amounts of inosine in a normal chow diet. This long-term inosine treatment elicited a progressive increase in uricemia up to 300 μmol/l; however, it did not modify heart rate or mean arterial blood pressure in LG9 knockout compared with control mice. Inosine treatment did not alter cardiac morphology or function measured by ultrasound echocardiography. However, it did induce mild renal dysfunction as revealed by higher plasma creatinine levels, lower glomerular filtration rate, and histological signs of chronic inflammation and fibrosis. Thus, in LG9 knockout mice, inosine-induced hyperuricemia was not associated with hypertension despite partial renal deficiency. This does not support a direct role of urate in the control of blood pressure.

Identification of noninvasive biomarkers for alcohol-induced liver disease using urinary metabolomics and the Ppara-null mouse

Alcohol-induced liver disease (ALD) is a leading cause of nonaccident-related deaths in the United States. Although liver damage caused by ALD is reversible when discovered at the earlier stages, current risk assessment tools are relatively nonspecific. Identification of an early specific signature of ALD would aid in therapeutic intervention and recovery. In this study, the metabolic changes associated with ALD were examined using alcohol-fed male Ppara-null mouse as a model of ALD. Principal components analysis of the mass spectrometry-based urinary metabolic profile showed that alcohol-treated wild-type and Ppara-null mice could be distinguished from control animals without information on history of alcohol consumption. The urinary excretion of ethyl-sulfate, ethyl-beta-d-glucuronide, 4-hydroxyphenylacetic acid, and 4-hydroxyphenylacetic acid sulfate was elevated and that of the 2-hydroxyphenylacetic acid, adipic acid, and pimelic acid was depleted during alcohol treatment in both wild-type and the Ppara-null mice albeit to different extents. However, indole-3-lactic acid was exclusively elevated by alcohol exposure in Ppara-null mice. The elevation of indole-3-lactic acid is mechanistically related to the molecular events associated with development of ALD in alcohol-treated Ppara-null mice. This study demonstrated the ability of a metabolomics approach to identify early, noninvasive biomarkers of ALD pathogenesis in Ppara-null mouse model.

UPLC-MS-based urine metabolomics reveals indole-3-lactic acid and phenyllactic acid as conserved biomarkers for alcohol-induced liver disease in the Ppara-null mouse model

Since the development and prognosis of alcohol-induced liver disease (ALD) vary significantly with genetic background, identification of a genetic background-independent noninvasive ALD biomarker would significantly improve screening and diagnosis. This study explored the effect of genetic background on the ALD-associated urinary metabolome using the Ppara-null mouse model on two different backgrounds, C57BL/6 (B6) and 129/SvJ (129S), along with their wild-type counterparts. Reversed-phase gradient UPLC-ESI-QTOF-MS analysis revealed that urinary excretion of a number of metabolites, such as ethylsulfate, 4-hydroxyphenylacetic acid, 4-hydroxyphenylacetic acid sulfate, adipic acid, pimelic acid, xanthurenic acid, and taurine, were background-dependent. Elevation of ethyl-β-d-glucuronide and N-acetylglycine was found to be a common signature of the metabolomic response to alcohol exposure in wild-type as well as in Ppara-null mice of both strains. However, increased excretion of indole-3-lactic acid and phenyllactic acid was found to be a conserved feature exclusively associated with the alcohol-treated Ppara-null mouse on both backgrounds that develop liver pathologies similar to the early stages of human ALD. These markers reflected the biochemical events associated with early stages of ALD pathogenesis. The results suggest that indole-3-lactic acid and phenyllactic acid are potential candidates for conserved and pathology-specific high-throughput noninvasive biomarkers for early stages of ALD.

Granulocyte colony-stimulating factor supports liver regeneration in a small-for-size liver remnant mouse model

Experimental partial hepatectomy of more than 80% of the liver weight bears an increased mortality in rodents, due to impaired hepatic regeneration in small-for-size liver remnants. Granulocyte colony-stimulating factor (G-CSF) promotes progenitor cell expansion and mobilization and also has immunomodulatory properties. The aim of this study was to determine the effect of systemically administered G-CSF on liver regeneration and animal survival in a small-for-size liver remnant mouse model. Mice were preconditioned daily for 5 days with subcutaneous injections of 5 microg G-CSF or aqua ad injectabile. Subsequently, 83% partial hepatectomy was performed by resecting the median, the left, the caudate, and the right inferior hepatic lobes in all animals. Daily sham or G-CSF injection was continued. Survival was significantly better in G-CSF-treated animals (P < 0.0001). At 36 and 48 h after microsurgical hepatic resection, markers of hepatic proliferation (Ki67, BrdU) were elevated in G-CSF-treated mice compared to sham injected control animals (P < 0.0001) and dry liver weight was increased (P < 0.05). G-CSF conditioning might prove to be useful in patients with small-for-size liver remnants after extended hepatic resections due to primary or secondary liver tumors or in the setting of split liver transplantation.

Granulocyte colony stimulating factor supports liver regeneration in a surgical small for size liver remnant mouse model

ntense liver regeneration and almost 100% survival follows partial hepatectomy of up to 70% of liver mass in rodents. More extensive resections of 70 to 80% have an increased mortality and partial hepatectomies of >80% constantly lead to acute hepatic failure and death in mice. The aim of the study was to determine the effect of systemically administered granulocyte colony stimulating factor (G-CSF) on animal survival and liver regeneration in a small for size liver remnant mouse model after 83% partial hepatectomy (liver weight <0.8% of mouse body weight). Methods: Male Balb C mice (n=80, 20-24g) were preconditioned daily for five days with 5μg G-CSF subcutaneously or sham injected (aqua ad inj). Subsequently 83% hepatic resection was performed and daily sham or G-CSF injection continued. Survival was determined in both groups (G-CSF n=35; Sham: n=33). In a second series BrdU was injected (50mg/kg Body weight) two hours prior to tissue harvest and animals euthanized 36 and 48 hours after 83% liver resection (n=3 each group). To measure hepatic regeneration the BrdU labeling index and Ki67 expression were determined by immunohistochemistry by two independent observers. Harvested liver tissue was dried to constant weight at 65 deg C for 48 hours. Results: Survival was 0% in the sham group on day 3 postoperatively and significantly better (26.2% on day 7 and thereafter) in the G-CSF group (Log rank test: p<0.0001). Dry liver weight was increased in the G-CSF group (T-test: p<0.05) 36 hours after 83% partial hepatectomy. Ki67 expression was elevated in the G-CSF group at 36 hours (2.8±2.6% (Standard deviation) vs 0.03±0.2%; Rank sum test: p<0.0001) and at 48 hours (45.1±34.6% vs 0.7±1.0%; Rank sum test: p<0.0001) after 83% liver resection. BrdU labeling at 48 hours was 0.1±0.3% in the sham and 35.2±34.2% in the G-CSF group (Rank sum test: p<0.0001) Conclusions: The surgical 83% resection mouse model is suitable to test hepatic supportive regimens in the setting of small for size liver remnants. Administration of G-CSF supports hepatic regeneration after microsurgical 83% partial hepatectomy and leads to improved long-term survival in the mouse. G-CSF might prove to be a clinically valuable supportive substance in small for size liver remnants in humans after major hepatic resections due to primary or secondary liver tumors or in the setting of living related liver donation.

Inhibition of neoplastic development in the liver by hepatocyte growth factor in a transgenic mouse model.

Overexpression of the c-myc oncogene is associated with a variety of both human and experimental tumors, and cooperation of other oncogenes and growth factors with the myc family are critical in the evolution of the malignant phenotype. The interaction of hepatocyte growth factor (HGF) with c-myc during hepatocarcinogenesis in a transgenic mouse model has been analyzed. While sustained overexpression of c-myc in the liver leads to cancer, coexpression of HGF and c-myc in the liver delayed the appearance of preneoplastic lesions and prevented malignant conversion. Furthermore, tumor promotion by phenobarbital was completely inhibited in the c-myc/HGF double transgenic mice, whereas phenobarbital was an effective tumor promoter in the c-myc single transgenic mice. The results indicate that HGF may function as a tumor suppressor during early stages of liver carcinogenesis, and suggest the possibility of therapeutic application for this cytokine.

Apolipoprotein E competitively inhibits receptor-dependent low density lipoprotein uptake by the liver but has no effect on cholesterol absorption or synthesis in the mouse.

This study examines the question of whether apolipoprotein E (apoE) alters steady-state concentrations of plasma cholesterol carried in low density lipoproteins (LDL-C) by acting as a competitive inhibitor of hepatic LDL uptake or by altering the rate of net cholesterol delivery from the intestinal lumen to the liver. To differentiate between these two possibilities, rates of cholesterol absorption and synthesis and the kinetics of hepatic LDL-C transport were measured in vivo in mice with either normal (apoE+/+) or zero (apoE-/-) levels of circulating apoE. Rates of cholesterol absorption were essentially identical in both genotypes and equaled approximately 44% of the daily dietary load of cholesterol. This finding was consistent with the further observation that the rates of cholesterol synthesis in the liver (approximately 2,000 nmol/h) and extrahepatic tissues (approximately 3,000 nmol/h) were also essentially identical in the two groups of mice. However, the apparent Michaelis constant for receptor-dependent hepatic LDL-C uptake was markedly lower in the apoE-/- mice (44 +/- 4 mg/dl) than in the apoE+/+ animals (329 +/- 77 mg/dl) even though the maximal transport velocity for this uptake process was essentially the same (approximately 400 micrograms/h per g) in the two groups of mice. These studies, therefore, demonstrate that apoE-containing lipoproteins can act as potent competitive inhibitors of hepatic LDL-C transport and so can significantly increase steady-state plasma LDL-C levels. This apolipoprotein plays no role, however, in the regulation of cholesterol absorption, sterol biosynthesis, or hepatic LDL receptor number, at least in the mouse.

A New Mouse Model That Spontaneously Develops Chronic Liver Inflammation and Fibrosis

Here we characterize a new animal model that spontaneously develops chronic inflammation and fibrosis in multiple organs, the non-obese diabetic inflammation and fibrosis (N-IF) mouse. In the liver, the N-IF mouse displays inflammation and fibrosis particularly evident around portal tracts and central veins and accompanied with evidence of abnormal intrahepatic bile ducts. The extensive cellular infiltration consists mainly of macrophages, granulocytes, particularly eosinophils, and mast cells. This inflammatory syndrome is mediated by a transgenic population of natural killer T cells (NKT) induced in an immunodeficient NOD genetic background. The disease is transferrable to immunodeficient recipients, while polyclonal T cells from unaffected syngeneic donors can inhibit the disease phenotype. Because of the fibrotic component, early on-set, spontaneous nature and reproducibility, this novel mouse model provides a unique tool to gain further insight into the underlying mechanisms mediating transformation of chronic inflammation into fibrosis and to evaluate intervention protocols for treating conditions of fibrotic disorders.

Mesenchymal progenitor cells from canine fetal tissues: yolk sac, liver, and bone marrow

During fetal development, mesenchymal progenitor (MP) cells are co-localized in major hematopoietic territories, such as yolk sac (YS), bone marrow (BM), liver (LV), and others. Studies using mouse and human MP cells isolated from fetus have shown that these cells are very similar but not identical to adult mesenchymal stem cells (MSC). Their differentiation potential is usually restricted to production of highly committed osteogenic and chondrogenic precursors. Such properties of fetal MP cells can be very useful for tissue regeneration, when a great number of committed precursors are required. The objectives of this study were to isolate and characterize MP cells from canine YS, BM, and LV in early and late stages of fetal development. Gestational stage was identified, and cell culture conditions were evaluated for efficient isolation of canine MP cells. All canine fetal MP cells expressed vimentin, nestin, and CD44 proteins. Cytokeratin 18 expression was observed in BM-and LV-MP cells, and vascular endothelial (VE)-cadherin expression was observed only in YS-MP cells. A small number of MP cells (5%) from LV and YS expressed Oct3/4 protein. The differentiation potential of canine fetal MP cells varied significantly: YS- and BM-MP cells differentiated into bone and cartilage, whereas LV-MP cells differentiation was limited to osteogenic fate. None of the canine fetal MP cells were able to differentiate into adipose cells. Our data suggest that canine fetal MP cells are an appropriate in vitro model to study MP biology from hematopoietic territories and they are a source of committed osteogenic and chondrogenic precursors for regenerative medicine.