NADPH oxidase 4 (Nox4) deletion accelerates liver regeneration in mice.

Liver is a unique organ in displaying a reparative and regenerative response after acute/chronic damage or partial hepatectomy, when all the cell types must proliferate to re-establish the liver mass. The NADPH oxidase NOX4 mediates Transforming Growth Factor-beta (TGF-ß) actions, including apoptosis in hepatocytes and activation of stellate cells to myofibroblasts. Aim of this work was to analyze the impact of NOX4 in liver regeneration by using two mouse models where Nox4 was deleted: 1) general deletion of Nox4 (NOX4-/-) and 2) hepatocyte-specific deletion of Nox4 (NOX4hepKO). Liver regeneration was analyzed after 2/3 partial hepatectomy (PH). Results indicated an earlier recovery of the liver-to-body weight ratio in both NOX4-/- and NOX4hepKO mice and an increased survival, when compared to corresponding WT mice. The regenerative hepatocellular fat accumulation and the parenchyma organization recovered faster in NOX4 deleted livers. Hepatocyte proliferation, analyzed by Ki67 and phospho-Histone3 immunohistochemistry, was accelerated and increased in NOX4 deleted mice, coincident with an earlier and increased Myc expression. Primary hepatocytes isolated from NOX4 deleted mice showed higher proliferative capacity and increased expression of Myc and different cyclins in response to serum. Transcriptomic analysis through RNA-seq revealed significant changes after PH in NOX4-/- mice and support a relevant role for Myc in a node of regulation of proliferation-related genes. Interestingly, RNA-seq also revealed changes in the expression of genes related to activation of the TGF-ß pathway. In fact, levels of active TGF-ß1, phosphorylation of Smads and levels of its target p21 were lower at 24 h in NOX4 deleted mice. Nox4 did not appear to be essential for the termination of liver regeneration in vivo, neither for the in vitro hepatocyte response to TGF-ß1 in terms of growth inhibition, which suggest its potential as therapeutic target to improve liver regeneration, without adverse effects.

Investigation of vitamin D receptor polymorphisms in amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) patients manifest aberrations in the vitamin D endocrine system, with a vitamin D deficiency. Genetic investigations have identified those proteins which link vitamin D to ALS pathology: major histocompatibility complex class II molecules, toll-like receptors, poly(ADP ribose) polymerase-1, haeme oxygenase-1, the reduced form of nicotinamide adenine dinucleotide phosphate and calcium-binding proteins. Vitamin D additionally impacts ALS through cell-signalling mechanisms: glutamate, matrix metalloproteinases, the Wnt/ß-catenin signalling pathway, mitogen-activated protein kinase pathways, prostaglandins, reactive oxygen species and nitric oxide synthase, but its role has been only poorly investigated. OBJECTIVE: Our aim was to investigate vitamin D receptor (VDR) gene single nucleotide polymorphisms (SNPs) in an ALS population. This gene encodes the nuclear hormone receptor for vitamin D3. MATERIALS AND METHODS: A total of 75 consecutive sporadic ALS patients (~20% of the Hungarian ALS population) and 97 healthy controls were enrolled to investigate the possible effects of the different VDR alleles. A restriction fragment length polymorphism technique was utilized for allele discrimination. RESULTS: One of the four investigated SNPs was associated with the disease, but none of the alleles of these SNPs influenced the age at disease onset. The ApaI A allele was more frequent in the ALS group than in the control group and may be an ALS risk factor. CONCLUSIONS: This is the first verification of the genetic link between ALS and VDR. However, further studies are needed to confirm these findings.

Cavitary lung lesions in systemic lupus: an unusual presentation.

Lupus is a multisystem disease with varied clinical manifestations. Cavitary lung lesions in lupus could be secondary to infections, embolism, or underlying vasculitis. We report a man who presented with lung cavitations and was diagnosed as lupus, secondary antiphospholipid syndrome, and plasma cell dyscrasia. The lesions resolved after the initiation of immunosuppressive therapy. All the previous case reports have been reviewed and the possible mechanisms underlying this association are discussed.

MetS and cardiovascular risk factors among Palestinians of East Jerusalem.

In a cross-sectional survey conducted in 2005, we determined the prevalence metabolic syndrome (MetS) and other atherosclerotic cardiovascular disease risk factors among a sample of 342 Palestinians > or = 20 years in East Jerusalem. Participants were interviewed and anthropometric measurements and blood testing were done. MetS was found in 115 (33.6%) participants, with no significant difference between the sexes. The prevalence of obesity, diabetes and other cardiovascular risk factors was also high, with central obesity and obesity (BMI > or = 30 kg/m2) being significantly higher in women (P <0.01). With the exception of low-density lipoprotein cholesterol and haemoglobin, there was a significantly higher prevalence of atherosclerotic markers among the MetS group.

MetS and cardiovascular risk factors among Palestinians of East Jerusalem

In a cross-sectional survey conducted in 2005, we determined the prevalence metabolic syndrome [MetS] and other atherosclerotic cardiovascular disease risk factors among a sample of 342 Palestinians >/= 20 years in East Jerusalem. Participants were interviewed and anthropometric measurements and blood testing were done. MetS was found in 115 [33.6%] participants, with no significant difference between the sexes. The prevalence of obesity, diabetes and other cardiovascular risk factors was also high, with central obesity and obesity [BMI >/= 30 kg/m[2]] being significantly higher in women [P < 0.01]. With the exception of low-density lipoprotein cholesterol and haemoglobin, there was a significantly higher prevalence of atherosclerotic markers among the MetS group

Alterations in vesicle transport and cell polarity in rat hepatocytes subjected to mechanical or chemical cholestasis.

BACKGROUND & AIMS: The molecular mechanisms that contribute to the cholestatic condition in hepatocytes are poorly defined. It has been postulated that a disruption of normal vesicle-based protein trafficking may lead to alterations in hepatocyte polarity. METHODS: To determine if vesicle motility is reduced by cholestasis, hepatocytes cultured from livers of bile duct ligation (BDL)- or ethinyl estradiol (EE)-injected rats, were viewed and recorded by high-resolution video microscopy. Cholestatic hepatocytes were analyzed by phalloidin staining and electron microscopy. Functional analysis was done by the sodium fluorescein sequestration assay. RESULTS: In cholestatic hepatocytes, there was a significant decrease in the number of motile cytoplasmic vesicles observed compared with control cells. Further examination of cells from BDL- or EE-treated livers revealed the presence of numerous large intracellular lumina. More than 24% of cells in BDL-treated livers and 19% of cells in EE-treated livers displayed these structures, compared with 1.1% found in control hepatocytes. Phalloidin staining of hepatocytes showed a prominent sheath of actin surrounding the lumina, reminiscent of those seen about bile canaliculi. Electron microscopy revealed that these structures were lined by actin-filled microvilli. Further, these pseudocanaliculi perform many of the functions exhibited by bona fide canaliculi, such as sequestering sodium fluorescein. CONCLUSIONS: Both mechanically and chemically induced cholestasis have substantial effects on vesicle-based transport, leading to marked disruption of hepatocellular polarity.

Cholangiocarcinoma.

Chronic inflammatory disease of the biliary tract predispose to the development of cholangiocarcinoma. For example, the prevalence of cholangiocarcinoma is between 7% to 14% in patients with primary sclerosing cholangitis. The diagnosis of cholangiocarcinoma is challenging because it is difficult to distinguish benign from malignant strictures. Recently, several advances have helped in the diagnosis of cholangiocarcinoma. The serum Ca 19-9 value is a useful adjunct for the diagnosis of malignant stictures in the absence of bacterial cholangitis. The recent development of digital image analysis for assessing biopsy specimens and brush cytology is also useful for the diagnosis of malignant strictures. Positron emission tomography is a new imaging technique that uses 18F fluoro-2-deoxy-D-glucose to noninvasively assess metabolism in human tissues. Early studies suggest that this technique is sensitive in identifying small bile duct cancers. Thus, the combination of a Ca 19-9 value, digital image analysis, and positron emission tomography scanning have greatly helped in the differential diagnosis of benign from malignant strictures. Management of cholangiocarcinoma is also challenging with limited survival after surgical resection. Recently, we have shown that preoperative chemoirradiation followed by liver transplantation results in prolonged disease-free survival in highly selected patients with early bile duct cancers. Successful treatment outcome of these patients highlights the need for an early diagnosis of cholangiocarcinoma using the approaches described above.

Ethanol-induced alterations of the microtubule cytoskeleton in hepatocytes.

Ethanol has been predicted to alter vesicle-based protein traffic in hepatocytes, in part, via a disruption of the microtubule (MT) cytoskeleton. However, information on the effects of chronic ethanol exposure on MT function in vivo is sparse. Therefore the goal of this study was to test for ethanol-induced changes in rat liver tubulin expression, assembly, and cellular organization, using molecular, biochemical and morphological methods. The results of this study showed that tubulin mRNA and protein levels were not altered by ethanol. Tubulin, isolated from control and ethanol-fed rats, showed similar polymerization characteristics as assessed by calculation of the critical concentration for assembly and morphological structure. In contrast, the total amount of assembly-competent tubulin was reduced in livers from ethanol-fed rats compared with control rats when assessed by quantitative immunoblot analysis using a tubulin antibody. In addition, we observed that MT regrowth and organization in cultured hepatocytes treated with cold and nocodazole was markedly impaired by chronic ethanol exposure. In summary, these results indicate that tubulin levels in liver are not reduced by ethanol exposure. While there is a substantial amount of tubulin protein capable of assembling into functional MTs in ethanol-damaged livers, a marked portion of this tubulin is polymerization incompetent. This may explain why these hepatocytes exhibit a reduced number of MTs with an altered organization.

Vesicle movement in rat hepatocytes is reduced by ethanol exposure: alterations in microtubule-based motor enzymes.

BACKGROUND & AIMS: Ethanol is known to alter vesicle-mediated protein trafficking in hepatocytes by undefined mechanisms. In this study, the effects of long- and short-term ethanol exposure on vesicle movements were measured in isolated hepatocytes, and alterations in the function of the microtubule-associated motor enzymes dynamin, kinesin, and dynein, which are believed to support the transport and/or budding of vesicles along microtubules, were tested. METHODS: Vesicular movements in isolated hepatocytes exposed to short- and long-term ethanol treatment were measured. Motor adenosine triphosphatase activities and their association with specific membrane organelles were assessed in response to long-term administration of ethanol in vivo or acetaldehyde in vitro. RESULTS: Hepatocytes exposed to short- or long-term ethanol treatment showed a significant reduction in the number of motile vesicles. No alterations in the levels of motor messenger RNA, protein, or enzymatic activity were observed. Interestingly, ethanol had no effect on the association of dynein and kinesin with membranes, whereas there was a significant increase in the amount of dynamin associated specifically with Golgi membranes. CONCLUSIONS: Long- and short-term ethanol exposure markedly reduces hepatocellular vesicle transport by a mechanism apparently independent of any alteration in the enzymatic activity of molecular motors, possibly involving a change in the function of dynamin.

Upregulation of molecular motor-encoding genes during hepatocyte growth factor- and epidermal growth factor-induced cell motility.

Hepatocyte growth factor (HGF) and epidermal growth factor (EGF) are known to stimulate the locomotion of epithelial cells in culture. However, the molecular mechanisms which mediate these important changes are poorly understood. Here we have determined the effects of HGF and EGF on hepatocyte morphology, cytoskeletal organization, and the expression of molecular motor-encoding genes. Primary cultures of hepatocytes were treated with 10 ng/ml of HGF or EGF and observed with phase and fluorescence microscopy at 10, 24, and 48 h after treatment. We found that, over time, treated cells spread and became elongated after 24 h of treatment while forming long processes with dramatic alterations in the microtubule and actin cytoskeletons by 48 h. Quantitative Northern blot analysis was performed to measure expression of cytoskeletal-(beta-actin, alpha-tubulin) and molecular motor-(dynein, kinesin, and myosin I alpha and II) encoding genes which may contribute to this change in form. We observed the highest increase in levels of expression for myosin II (3.3-fold), kinesin (2.7-fold), myosin I alpha (2.2-fold), and alpha-tubulin (1.9-fold) after only 2 h of treatment with HGF. In contrast, EGF upregulated the expression of myosin I alpha (2.4-fold), kinesin (1.5-fold), and dynein (1.5-fold) at 10 h. The expression of the beta-actin gene remained constant in HGF-treated cells, while EGF induced a slight upregulation after 10 h of treatment. These results show for the first time that a selective upregulation of molecular motor-encoding genes correlates with alterations in cell shape and motility induced by HGF and EGF.

Keratin 19 as a biochemical marker of skin stem cells in vivo and in vitro: keratin 19 expressing cells are differentially localized in function of anatomic sites, and their number varies with donor age and culture stage.

This study was undertaken to evaluate keratin 19 (K19) as a biochemical marker for skin stem cells in order to address some long standing questions concerning these cells in the field of cutaneous biology. We first used the well-established mouse model enabling us to identify skin stem cells as [3H]thymidine-label-retaining cells. A site directed antibody was raised against a synthetic peptide of K19. It reacted specifically with a 40 kDa protein (K19) on immunoblotting. It labelled the bulge area of the outer root sheath on mouse skin by immunohistochemistry. Double-labelling revealed that K19-positive-cells were also [3H]thymidine-label-retaining cells, suggesting that K19 is a marker for skin stem cells of hair follicles. K19-expression was then used to investigate the variation in mouse and human skin stem cells as a function of body site, donor age and culture time. K19 was expressed in the hair follicle and absent from the interfollicular epidermis at hairy sites (except for some K18 coexpressing Merkel cells). In contrast, at glabrous sites, K19-positive-cells were in deep epidermal rete ridges. K19 expressing cells also contained high levels of alpha 3 beta 1 integrin. The proportion of K19-positive-cells was greater in newborn than older foreskins. This correlated with keratinocyte culture lifespan variation with donor age. Moreover, it could explain clinical observations that children heal faster than adults. In conclusion, K19 expression in skin provides an additional tool to allow further characterization of skin stem cells under normal and pathological conditions in situ and in vitro.

The economic impact of clinical pharmacists' unsolicited recommendations.

Clinical pharmacists in a 580-bed teaching hospital reported all targeted recommendations that occurred during a 5-month evaluation period. Five types of clinical recommendations were identified: (1) to start drug therapy, (2) to stop drug therapy, (3) to increase drug dose, (4) to decrease drug dose, and (5) to suggest alternative drug therapy. Two thousand sixty-four unsolicited, accepted recommendations were submitted to the investigator by approximately eight holders of full-time equivalent positions dedicated to clinical pharmacy services during the evaluation period. Three hundred forty-four recommendations were selected (every sixth submitted recommendation) and evaluated for their economic impact. The costs of drugs, monitoring, and treatment for the drug regimens were compared before and after each recommendation. Three classes of drugs (antineoplastics, anti-infectives, and gastrointestinal agents) contributed more than 90% of the economic impact. The net economic impact of the evaluated recommendations was a cost saving of $4636.06; this extrapolated to a net cost saving of $34.10 per pharmacist-day.

Vestibular and auditory function in Usher's syndrome.

Usher's syndrome is a recessive hereditary disorder in which a congenital hearing loss is combined with nyctalopia, retinal degeneration, and restriction of visual fields. The results of a comprehensive ophthalmic and neurotologic study on 70 patients are reported. Two distinct clinical and presumed genetic types were discernible on the basis of hearing impairment and vestibular sensitivity and, to a lesser extent, deterioration of retinal photoreceptor function. Such a classification has proved valuable in diagnosis, prognosis, and genetic counselling.

Usher's syndrome. Ophthalmic and neuro-otologic findings suggesting genetic heterogeneity.

The conditions of 70 patients with Usher's syndrome were studied by ophthalmic and neuro-otologic examinations. Two distinct clinical and presumed genetic types were discernible on the basis of differences in hearing impairment, vestibular sensitivity, and, to a lesser extent, deterioration in retinal photoreceptor function. Distinguishing these two types has relevance for both diagnosis and genetic counseling of patients with Usher's syndrome.

Anatomic specificity of central vestibular signs in posterior fossa lesions.

The reliability of central vestibular signs such as decruitment in signifying retrolabyrinthine lesions has been well established. This study was initiated to determine if this and other central vestibular signs, individually or collectively, indicate involvement of specific anatomic structures in the posterior fossa. Forty-six patients with morphologic lesions defined by computerized tomography (CT), pneumo-CT autopsy were studied. Oculomotor signs showed a statistically significant correlation with lesions of the archicerebellum. The other central vestibular signs did not show any correlation.

Central vestibular signs, posterior fossa pathology and computerized tomography-regional blood brain circulation.

Central vestibular signs are sensitive indicators of lesions in the posterior fossa. With the availability of computerized tomography and computerized tomography-regional blood brain circulation, the identification of suspected retrolabyrinthine lesions has improved considerably. This is a report about the reliability of central vestibular signs in predicting posterior fossa lesions.