Investigation of the genetic overlap between rheumatoid arthritis and psoriatic arthritis in a Greek population.

OBJECTIVES: Several rheumatoid arthritis (RA) susceptibility loci have also been found to be associated with psoriatic arthritis (PsA), demonstrating that there is a degree of genetic overlap between various autoimmune diseases. We sought to investigate whether single nucleotide polymorphisms (SNPs) mapping to previously reported RA and/or PsA susceptibility loci, including PLCL2, CCL21, REL, STAT4, CD226, PTPN22, and TYK2, are associated with risk for the two diseases in a genetically homogeneous Greek population. METHOD: This study included 392 RA patients, 126 PsA patients, and 521 healthy age- and sex-matched controls from Greece. Genotyping of the SNPs was performed with Taqman primer/probe sets. Bioinformatic analysis was performed using BlastP, PyMOL, and Maestro and Desmond. RESULTS: A significant association was detected between the GC genotype of rs34536443 (TYK2) in both the PsA and RA cohorts. The C allele of this SNP was associated with PsA only. Evidence for association with PsA was also found for the GG genotype and G allele of the rs10181656 SNP of STAT4. The TC genotype of the rs763361 SNP of CD226 was associated with PsA only. CONCLUSIONS: Genetic overlap between PsA and RA was detected for the rs34536443 SNP of the TYK2 gene within a Greek population. An association of STAT4 (rs10181656) with PsA was confirmed whereas CD226 (rs763361) was associated with PsA but not with RA, in contrast to previous reports. The different findings of this study compared to previous ones highlights the importance of comparative studies that include various ethnic or racial populations.

Apolipoprotein A-I inhibits experimental colitis and colitis-propelled carcinogenesis.

In both humans with long-standing ulcerative colitis and mouse models of colitis-associated carcinogenesis (CAC), tumors develop predominantly in the distal part of the large intestine but the biological basis of this intriguing pathology remains unknown. Herein we report intrinsic differences in gene expression between proximal and distal colon in the mouse, which are augmented during dextran sodium sulfate (DSS)/azoxymethane (AOM)-induced CAC. Functional enrichment of differentially expressed genes identified discrete biological pathways operating in proximal vs distal intestine and revealed a cluster of genes involved in lipid metabolism to be associated with the disease-resistant proximal colon. Guided by this finding, we have further interrogated the expression and function of one of these genes, apolipoprotein A-I (ApoA-I), a major component of high-density lipoprotein. We show that ApoA-I is expressed at higher levels in the proximal compared with the distal part of the colon and its ablation in mice results in exaggerated DSS-induced colitis and disruption of epithelial architecture in larger areas of the large intestine. Conversely, treatment with an ApoA-I mimetic peptide ameliorated the phenotypic, histopathological and inflammatory manifestations of the disease. Genetic interference with ApoA-I levels in vivo impacted on the number, size and distribution of AOM/DSS-induced colon tumors. Mechanistically, ApoA-I was found to modulate signal transducer and activator of transcription 3 (STAT3) and nuclear factor-κB activation in response to the bacterial product lipopolysaccharide with concomitant impairment in the production of the pathogenic cytokine interleukin-6. Collectively, these data demonstrate a novel protective role for ApoA-I in colitis and CAC and unravel an unprecedented link between lipid metabolic processes and intestinal pathologies.

Metabolism of HDL and its regulation.

Epidemiological studies have shown that low plasma levels of High Density Lipoprotein Cholesterol (HDL-C) are associated with an increased risk for myocardial infarction. These studies suggested that by increasing HDL-C levels one could reduce cardiovascular risk. However, emerging evidence from studies in animals and humans indicate that high levels of HDL-C are not sufficient to confer atheroprotection but that the functionality of the HDL particles is equally important. The picture is complicated further by the finding that HDL functionality is compromised in patients with chronic inflammatory diseases such as Coronary Artery Disease (CAD), diabetes and rheumatoid arthritis. Despite these obstacles, HDL raising is still a promising strategy for the reduction of CAD risk. Low HDL-C can be caused by inactivating mutations in apoA-I, ATP Binding Cassette Transporter A1 (ABCA1) or Lecithin-Cholesterol Acyl Transferase (LCAT) which affect HDL biogenesis and maturation whereas high HDL-C can be caused by mutations in Cholesteryl Ester Transfer Protein (CETP) or Scavenger receptor Class B Type I (SR-BI). Recent studies suggest that heterogeneity in HDL levels in the population is polygenic in origin. One approach to raise plasma HDL-C is to increase the rate of HDL biosynthesis by capitalizing on the mechanisms that control the transcription of genes that play key roles in HDL biogenesis. We review some of the genetic and non-genetic factors that affect plasma HDL levels and functions and discuss the mechanisms that regulate HDL metabolism at the level of gene transcription in the liver focusing on apoA-I, ABCA1 and apoM.

Carotid artery remodelling in relation to body fat distribution, inflammation and sustained weight loss in obesity.

BACKGROUND: Obesity is known to be associated with carotid artery remodelling, but less is known about how body fat distribution, inflammation and weight loss may affect this relation. METHODS: Ultrasonography, dual-energy X-ray absorptiometry and computed tomography were performed to evaluate carotid artery intima-media thickness (IMT), body composition and fat distribution, respectively. Participants were divided into three matched study groups (n = 44 per group): obese patients with sustained weight loss 10 years after bariatric surgery [surgery group, body mass index (BMI) 31.5 kg m(-2)]; obese patients who maintained stable weight during the same time period (obese group, BMI 42.5 kg m(-2)); and normal weight subjects (lean group, BMI 24.4 kg m(-2)). RESULTS: Patients in the surgery group, compared with those in the obese group, had slightly lower common carotid artery (CCA) IMT (0.75 ± 0.18 vs. 0.78 ± 0.17 mm) and common carotid bulb (CCB) IMT (0.92 ± 0.32 vs. 0.97 ± 0.32 mm); however, these differences were not statistically significant. Lean individuals, compared with those in the surgery group, had significantly lower CCA and CCB IMT values (P < 0.001). In forward stepwise multiple regression analyses including all subjects (n = 132), CCA IMT was predicted mainly by visceral adipose tissue, but was also related to blood pressure and levels of triglycerides and high-sensitivity C-reactive protein. Carotid lumen diameter was primarily influenced by lean body mass. CONCLUSION: Visceral adiposity was the main determinant of premature carotid artery atherosclerosis, possibly through elevated blood pressure, dyslipidaemia and inflammation. Lean body mass predicted carotid artery lumen diameter. Obese patients with long-term sustained weight loss did not have thinner carotid artery walls compared with their weight-stable obese counterparts.

Duration of the thoracic epidural catheter in a fast-track recovery protocol may decrease the length of stay after a major hepatectomy: a case control study.

BACKGROUND: Fast-track recovery protocols are applied to major surgeries, including hepatectomies. The optimal duration of thoracic epidural catheter has not yet been defined. OBJECTIVE: To determine the ideal time to remove the epidural catheter after major hepatectomy. PATIENTS-METHODS: Forty-eight consecutive patients who underwent major hepatectomy over 4 years were studied. The data from laparoscopic hepatectomy were not included. Patients who underwent hepaticojejunostomy were included. A modified protocol of rapid postoperative recovery was implemented. In the first 24 patients, an epidural catheter was maintained for 4 days (group A), while in the next 24, the catheter was maintained for 2 days (group B). The length of hospital stay, time of functional recovery, and use of opioids and laxatives were recorded. RESULTS: There was no postoperative mortality. The average length of hospital stay was 6.92 ± 1.79 and 6.09 ± 2.08 days for groups A and B, respectively. The mean functional recovery was 5.46 ± 0.3 and 5.26 ± 0.91 days for groups A and B, respectively. However, in group B, more opioid analgesics by 50% and more laxatives by 17% were used. CONCLUSIONS: After major hepatectomy, a reduction from 4 to 2 days' duration of the epidural catheter may lead to a reduction in the length of hospital stay.

Investigation of juvenile idiopathic arthritis susceptibility loci: results from a Greek population.

The strategy of studying the putative role of RA susceptibility genetic factors in the development of juvenile idiopathic arthritis (JIA), an autoimmune disease characterized by persistent chronic arthritis, has been proven highly successful so far. Moreover, accumulated evidence indicates that an ethnic heterogeneity of genetic factors exists for rheumatic disorders. We investigated whether five single nucleotide polymorphisms (SNPs), previously found to be associated with JIA in various populations so far, are also associated with JIA in Greece. The sample set consisted of 128 Caucasian JIA patients and 221 healthy controls from Northern Greece. Five Single Nucleotide Polymorphisms (SNPs) markers, namely TRAF1/C5 rs10818488, PTPN22 rs2476601, STAT4 rs7574865, CD247 rs1773560 and PTPN2 rs7234029 SNPs were genotyped in a case-control study with Restriction Fragment Length Polymorphisms (RFLPs) or Taqman primer-probe sets. This study demonstrated for the first time in a Greek population that the PTPN22, TRAF1/C5 and CD247 polymorphisms examined are associated with an increased susceptibility to JIA, thus suggesting that the respective risk alleles may confer susceptibility to clinically distinct disorders. However, our results did not demonstrate any association of STAT4 and PTPN2 SNPs with the disease in our population, thus highlighting the importance of comparative studies in different ethnic populations.

Acoustic detection of DNA conformation in genetic assays combined with PCR.

Application of PCR to multiplexing assays is not trivial; it requires multiple fluorescent labels for amplicon detection and sophisticated software for data interpretation. Alternative PCR-free methods exploiting new concepts in nanotechnology exhibit high sensitivities but require multiple labeling and/or amplification steps. Here, we propose to simplify the problem of simultaneous analysis of multiple targets in genetic assays by detecting directly the conformation, rather than mass, of target amplicons produced in the same PCR reaction. The new methodology exploits acoustic wave devices which are shown to be able to characterize in a fully quantitative manner multiple double stranded DNAs of various lengths. The generic nature of the combined acoustic/PCR platform is shown using real samples and, specifically, during the detection of SNP genotyping in Anopheles gambiae and gene expression quantification in treated mice. The method possesses significant advantages to TaqMan assay and real-time PCR regarding multiplexing capability, speed, simplicity and cost.

Differential regulation of the two RhoA-specific GEF isoforms Net1/Net1A by TGF-ß and miR-24: role in epithelial-to-mesenchymal transition.

In the present study we analyzed the regulation of the two isoforms of the RhoA-specific guanine nucleotide exchange factor Net1 by transforming growth factor-ß (TGF-ß) in keratinocytes. We report that short-term TGF-ß treatment selectively induced Net1 isoform2 (Net1A) but not Net1 isoform1. This led to upregulation of cytoplasmic Net1A protein levels that were necessary for TGF-ß-mediated RhoA activation. Smad signaling and the MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway were involved in Net1A upregulation by TGF-ß. Interestingly, long-term TGF-ß treatment resulted in Net1 mRNA downregulation and Net1A protein degradation by the proteasome. Furthermore, we identified the microRNA miR-24 as a novel post-transcriptional regulator of Net1A expression. Silencing of Net1A resulted in disruption of E-cadherin- and zonula occludens-1 (ZO-1)-mediated junctions, as well as expression of the transcriptional repressor of E-cadherin, Slug and the mesenchymal markers N-cadherin, plasminogen activator inhibitor-1 (PAI-1) and fibronectin, indicating that late TGF-ß-induced downregulation of Net1A is involved in epithelial-to-mesenchymal transition (EMT). Finally, miR-24 was found to be implicated in the regulation of the EMT program in response to TGF-ß and was shown to be directly involved in the TGF-ß-induced breast cancer cell invasiveness through Net1A regulation. Our results emphasize the importance of Net1 isoform2 in the short- and long-term TGF-ß-mediated regulation of EMT.

Mutation in APOA1 predicts increased risk of ischaemic heart disease and total mortality without low HDL cholesterol levels.

OBJECTIVES: To determine whether mutations in APOA1 affect levels of high-density lipoprotein (HDL) cholesterol and to predict risk of ischaemic heart disease (IHD) and total mortality in the general population. BACKGROUND: Epidemiologically, risk of IHD is inversely related to HDL cholesterol levels. Mutations in apolipoprotein (apo) A-I, the major protein constituent of HDL, might be associated with low HDL cholesterol and predispose to IHD and early death. DESIGN: We resequenced APOA1 in 190 individuals and examined the effect of mutations on HDL cholesterol, risk of IHD, myocardial infarction (MI) and mortality in 10 440 individuals in the prospective Copenhagen City Heart Study followed for 31 years. Results were validated in an independent case-control study (n = 16 035). Additionally, we determined plasma ratios of mutant to wildtype (WT) apoA-I in human heterozygotes and functional effects of mutations in adenovirus-transfected mice. RESULTS: We identified a new mutation, A164S (1 : 500 in the general population), which predicted hazard ratios for IHD, MI and total mortality of 3.2 [95% confidence interval (CI): 1.6-6.5], 5.5 (95% CI: 2.6-11.7) and 2.5 (95% CI: 1.3-4.8), respectively, in heterozygotes compared with noncarriers. Mean reduction in survival time in heterozygotes was 10 years (P < 0.0001). Results for IHD and MI were confirmed in the case-control study. Furthermore, the ratio of mutant S164 to WT A164 apoA-I in plasma of heterozygotes was reduced. In addition, A164S heterozygotes had normal plasma lipid and lipoprotein levels, including HDL cholesterol and apoA-I, and this finding was confirmed in adenovirus-transfected mice. CONCLUSIONS: A164S is the first mutation in APOA1 to be described that predicts an increased risk of IHD, MI and total mortality without low HDL cholesterol levels.

The evolution of the role of liver transplantation in treating alcoholic cirrhosis in Greece.

BACKGROUND: Liver transplantation represents the main treatment for alcoholic cirrhosis. The goal of this article is to review the results of liver transplantation for alcoholic cirrhosis in Greece over the last 2 decades. METHODS: Among 247 patients who underwent liver transplantation between 1991 and 2007, 34 (13.7%) experienced alcoholic cirrhosis as the primary diagnosis. We reviewed their demographic data, stage of liver disease, and outcomes regarding survival via a Kaplan-Meier curve. Also we analyzed the causes of death and the postoperative complications. RESULTS: Mean Model for End-Stage Liver Disease (MELD) score was 18.4. Other diagnoses included hepatitis C virus (HCV; 23.5%), hepatitis B virus (HBV; 14.7%), and hepatocellular carcinoma (8.8%). Eleven patients died the most frequent causes being primary graft nonfunction (n = 3), hepatic artery thrombosis (n = 2), sepsis (n = 2), and portal vein thrombosis (n = 2). Complications included rejection (32.4%), infection (26.5%), hepatic graft dysfunction (11.8%), and recurrent HCV, recurrent HBV, and renal failure (8.8% each). Recurrence of alcoholism was observed in 3 patients (8.8%) with mild effects on liver function tests. There has been a significant increase in the number of liver transplantations for alcoholic cirrhosis in the last 6 years, namely 25 patients versus 9 in the previous 10 years. CONCLUSIONS: We observed a significant increase in the frequency of alcoholic cirrhosis leading to liver transplantation in the last several years in Greece.

Clinical extracorporeal hybrid liver support--phase I study with primary porcine liver cells.

The objective of this study was to evaluate the feasibility and safety of a hybrid liver support system with extracorporeal plasma separation and bioreactor perfusion in patients with acute liver failure (ALF) who had already fulfilled the criteria for high urgency liver transplantation (LTx). Eight patients (one male, seven female) were treated in terms of bridging to transplantation. The mean age was 36.5 yr (range 20 to 58). Etiology of liver failure was drug-related in two patients, hepatitis B infection in three patients, and unknown for three patients. The bioreactors were charged with primary liver cells from specific pathogen-free pigs. Cell viability varied between 91 and 98%. Continuous liver support treatment over a period of 8 to 46 h (mean 27.3 h) was safely performed and well-tolerated by all patients. No complications associated with the therapy were observed during the follow-up period. Thrombocytopenia was considered to be an effect of the plasma separation. Subsequently, all patients were transplanted successfully and were observed over at least 3 yr with an organ and patient survival rate of 100%. Screening of patient's sera for antibodies specific for porcine endogenous retroviruses (PERVs) showed no reactivity--either prior to application of the system, or after extracorporeal treatment. The results encourage us to continue the development of the technology, and further studies appear to be justified. The bioreactor technology has been integrated into a modular extracorporeal liver support (MELS) system, combining biologic liver support with artificial detoxification technology.

Primary human liver cells as source for modular extracorporeal liver support--a preliminary report.

Cell-based extracorporeal liver support is an option to assist or replace the failing organ until regeneration or until transplantation can be performed. The use of porcine cells or tumor cell lines is controversial. Primary human liver cells, obtained from explanted organs found to be unsuitable for transplantation, are a desirable cell source as they perform human metabolism and regulation. The Modular Extracorporeal Liver Support (MELS) concept combines different extracorporeal therapy units, tailored to suit the individual and intra-individual clinical needs of the patient. A multi-compartment bioreactor (CellModule) is loaded with human liver cells obtained by 5-step collagenase liver perfusion. A cell mass of 400 g - 600 g enables the clinical application of a liver lobe equivalent hybrid organ. A detoxification module enables single pass albumin-dialysis via a standard high-flux dialysis filter, and continuous veno-venuous hemodiafiltration may be included if required. Cells from 54 human livers have been isolated (donor age: 56 +/- 13 years, liver weight: 1862 +/- 556 g resulting in a viability of 55.0 +/- 15.9%). These grafts were not suitable for LTx, due to steatosis (54%), cirrhosis (15%), fibrosis (9%), and other reasons (22%). Out of 36 prepared bioreactors, 10 were clinically used to treat 8 patients with liver failure. The overall treatment time was 7-144 hours. No adverse events were observed. Initial clinical applications of the bioreactor evidenced the technical feasibility and safety of the system.

Sp1 plays a critical role in the transcriptional activation of the human cyclin-dependent kinase inhibitor p21(WAF1/Cip1) gene by the p53 tumor suppressor protein.

In the present study we present evidence for the critical role of Sp1 in the mechanism of transactivation of the human cell cycle inhibitor p21(WAF1/Cip1) (p21) gene promoter by the tumor suppressor p53 protein. We found that the distal p53-binding site of the p21 promoter acts as an enhancer on the homologous or heterologous promoters in hepatoma HepG2 cells. In transfection experiments, p53 transactivated the p21 promoter in HaCaT cells that express Sp1 but have a mutated p53 form. In contrast, p53 could not transactivate the p21 promoter in the Drosophila embryo-derived Schneider's SL2 cells that lack endogenous Sp1 or related factors. Cotransfection of SL2 cells with p53 and Sp1 resulted in a synergistic transactivation of the p21 promoter. Synergistic transactivation was greatly decreased in SL2 cells and HaCaT cells by mutations in either the p53-binding site or in the -82/-77 Sp1-binding site indicating functional cooperation between Sp1 and p53 in the transactivation of the p21 promoter. Synergistic transactivation was also decreased by mutations in the transactivation domain of p53. Physical interactions between Sp1 and p53 proteins were established by glutathione S-transferase pull-down and coimmunoprecipitation assays. By using deletion mutants we found that the DNA binding domain of Sp1 is required for its physical interaction with p53. In conclusion, Sp1 must play a critical role in regulating important biological processes controlled by p53 via p21 gene activation such as DNA repair, cell growth, differentiation, and apoptosis.

Transcriptional regulatory mechanisms of the human apolipoprotein genes in vitro and in vivo.

The present review summarizes recent advances in the transcriptional regulation of the human apolipoprotein genes, focusing mostly, but not exclusively, on in-vivo studies and signaling mechanisms that affect apolipoprotein gene transcription. An attempt is made to explain how interactions of transcription factors that bind to proximal promoters and distal enhancers may bring about gene transcription. The experimental approaches used and the transcriptional regulatory mechanisms that emerge from these studies may also be applicable in other gene systems that are associated with human disease. Understanding extracellular stimuli and the specific mechanisms that underlie apolipoprotein gene transcription may in the long run allow us to selectively switch on antiatherogenic genes, and switch off proatherogenic genes. This may have beneficial effects and may confer protection from atherosclerosis to humans.

Transcriptional regulation of the human apolipoprotein genes.

This review provides experiments and putative mechanisms which underlie the transcription of the human apolipoprotein genes in vitro and in vivo. Summarized below are the key findings for individual genes and gene clusters. ApoA-II. 1- The -911/+29 promoter is sufficient to direct expression of a reporter gene exclusively in the liver and thus represents a liver-specific promoter. 2- Important factors for the activity of this promoter are hormone nuclear receptors and the ubiquitous factor USF. 3. SREBP-1 and SREBP-2 bind to five and four sites respectively and transactivate the apoA-II promoter. Their role in the in vivo transcription of the apoA-II gene has not been established. ApoB. 1. Regulatory sequence extending 5 Kb upstream and 1.5 Kb downstream of the apoB promoter are sufficient to direct hepatic expression of the apoB gene. The intestinal expression of the apoB gene requires in addition a 315 bp intestinal enhancer located 56 Kb upstream of the apoB gene. 2. Important factors for apoB gene transcription appear to be C/EBP, HNF-3, HNF-4 and other nuclear receptors which bind both on the proximal promoter and the intestinal enhancer. ApoE/ApoCI/ApoCIV/ApoCII Cluster. 1. The expression of the genes of the apoE/apoCI/apoCII/apoE cluster are controlled by two homologous hepatic control regions designated HCR-1 and HCR-2 of approximately 600 bp located 15 and 27 Kb 3? of the apoE gene. Either region is sufficient to direct gene expression in vivo, although HCR-1 appears to have a dominant effect on apoE and apoCI and HCR-2 has a dominant effect on apoCIV and apoCII gene expression. 2. Two other homologous regulatory regions designated ME-1 and ME-2 located 3.3 and 15.9 Kb downstream of the apoE gene can direct independently the expression of the apoE gene in macrophages and adipocytes. 3. Important factors for apoE gene regulation appear to be SP1 on the proximal promoter, and possibly HNF-3, C/EBP and hormone nuclear receptors on the enhancers. 4. Important factors for apoCII gene transcription appear to be HNF-4 and RXR-alpha/T3R-beta which binds to a thyroid response element of the proximal promoter. ApoA-I/ApoCIII/ApoA-IV Gene Cluster. 1. The transcription of the apoA-I/apoCIII/apoA-IV gene cluster is controlled by a common enhancer located 590 to 790 nucleotides upstream of the apoCIII gene. 2. Important factors for the activity of the enhancer are SP1, HNF-4 and possibly other nuclear receptors. Important factors for the activity of the proximal promoters are HNF-4, and possibly other nuclear receptors. 3. The HNF-4 binding site of the apoCIII enhancer is required for the intestinal expression of apoA-I and apoCIII gene and enhances synergistically the hepatic transcription of the two genes and possibly of apoA-IV in vivo. The three SP1 sites of the enhancer are also required for the intestinal expression of apoA-I and apoCIII genes in vivo and for the enhancement of the hepatic transcription. 4. Pro-inflammatory cytokines such as TNF-alpha and IL-1 repress, and TGF-beta stimulates the apoCIII promoter activity. The TGF-beta pathway activates SMAD3/4 proteins which interact with HNF-4 bound to the apoCIII promoter and enhancer and increase its activity. 5. It appears that other factors activated by different signaling pathways (NF-kappa-B, Jun and others) interact with HNF-4 bound to the enhancer and thus repress the activity of apoCIII promoter. Understanding the transcriptional regulatory mechanism of the apolipoprotein genes may allow, in the long run, selective increase of anti-atherogenic lipoproteins and thus reduce the risk of cardiovascular disease.

Development of a hybrid liver support system.

Hybrid liver systems are being developed as temporary extracorporeal liver support therapy. The overview given here emphasizes the development of both hepatocyte culture models for bioreactors and of systems for clinical therapy. In vitro studies demonstrate long term external metabolic function in isolated primary hepatocytes within bioreactors. These systems are capable of supporting essential liver functions. Animal experiments verify the possibility of upscaling bioreactors for clinical treatment. However, since there is no reliable animal model for investigating the treatment of acute liver failure, the promising results obtained from these studies have limited relevance to human beings. The small number of clinical studies performed thus far are not sufficient to enable any conclusions concerning improvements in the therapy of acute liver failure. Although important progress has been made in the development of these systems, multiple hepatocyte culture models and bioreactor constructions are being discussed in the literature, indicating competition in this field of medical research. For the use of hepatocytes and sinusoidal endothelial cells in coculture, a bioreactor has been designed. The construction is based on capillaries for hepatocyte aggregate immobilization. Four separate capillary membrane systems, each permitting a different function, are woven in order to create a three-dimensional network. Cells are perfused via independent capillary membrane compartments. Decentralized oxygen supply and carbon dioxide removal with low gradients is possible. The parallel use of identical units enables easy upscaling. Initial studies on the use of discarded organs that are unsuitable for transplantation as a source for primary human liver cells seem to be promising.

Experimental evaluation of a cell module for hybrid liver support.

Aim of the study was to evaluate a hybrid liver support system in a porcine model of acute liver failure, after hepatectomy. Pigs with a body weight of 70+/-18 kg underwent total hepatectomy and porto-cavo-caval shunting as well as ligation of the bile duct and the hepatic artery. Control animals were connected to the system (including capillary membrane plasma separation) containing a four compartment bioreactor with integral oxygenation and decentralized mass exchange but without liver cells. The treatment group received hybrid liver support with the same system including 370+/-42 g primary isolated porcine parenchymal liver cells in co-culture with hepatocyte nursing cells, tissue engineered to liver- like structures at high density. Treatment started after complete recovery from anesthesia and was performed continuously. A positive influence on peripheral vascular resistance and a reduced need of catecholamine dosage was observed in the treatment group. Hybrid liver support with a cell module upscaled for clinical application significantly prolonged survival time in animals after hepatectomy with the longest survival being 26 hours in the control group an 57 hours in the treatment group.

SMAD proteins transactivate the human ApoCIII promoter by interacting physically and functionally with hepatocyte nuclear factor 4.

Cotransfection of HepG2 cells with SMADs established that SMAD3 and SMAD3-SMAD4 transactivated (15-70-fold) the -890/+24 apoCIII promoter and shorter promoter segments, whereas cotransfection with a dominant negative SMAD4 mutant repressed the apoCIII promoter activity by 50%, suggesting that SMAD proteins participate in apoCIII gene regulation. Transactivation required the presence of a hormone response element, despite the fact that SMADs could not bind directly to it. Cotransfection of SMAD3-SMAD4 along with hepatocyte nuclear factor-4 resulted in a strong synergistic transactivation of the -890/+24 apoCIII promoter, proximal promoter segments, or synthetic promoters containing either the apoCIII enhancer or the proximal apoCIII hormone response element. Inhibition of endogenous hepatocyte nuclear factor-4 synthesis by an antisense ribozyme construct reduced the constitutive activity of the apoCIII promoter in HepG2 cells to 10% and abolished the SMAD-mediated transactivation. Co-immunoprecipitation and GST pull-down assays provided evidence for physical interactions between SMAD3, SMAD4, and hepatic nuclear factor-4. Our findings indicate that transforming growth factor beta and its signal transducer SMAD proteins can modulate gene transcription by novel mechanisms that involve their physical and functional interaction with hepatocyte nuclear factor-4, suggesting that SMAD proteins may play an important role in apolipoprotein gene expression and lipoprotein metabolism.

Role of Smad proteins and transcription factor Sp1 in p21(Waf1/Cip1) regulation by transforming growth factor-beta.

Transforming growth factor-beta (TGF-beta) inhibits cell cycle progression, in part through up-regulation of gene expression of the p21(WAF1/Cip1) (p21) cell cycle inhibitor. Previously we have reported that the intracellular effectors of TGF-beta, Smad3 and Smad4, functionally cooperate with Sp1 to activate the human p21 promoter in hepatoma HepG2 cells. In this study we show that Smad3 and Smad4 when overexpressed in HaCaT keratinocytes lead to activation of the p21 promoter. Activation requires the binding sites for the ubiquitous transcription factor Sp1 on the proximal promoter. Induction of the endogenous HaCaT p21 gene by TGF-beta1 is further enhanced after overexpression of Smad3 and Smad4, whereas dominant negative mutants of Smad3 and Smad4 and the inhibitory Smad7 all inhibit p21 induction by TGF-beta1 in a dose-dependent manner. We show that Sp1 expressed in the Sp1-deficient Drosophila SL-2 cells binds to the proximal p21 promoter sequences, whereas Smad proteins do not. In support of this finding, we show that DNA-binding domain mutants of Smad3 and Smad4 are capable of transactivating the p21 promoter as efficiently as wild type Smads. Co-expression of Smad3 with Smad4 and Sp1 in SL-2 cells or co-incubation of phosphorylated Smad3, Smad4, and Sp1 in vitro results in enhanced binding of Sp1 to the p21 proximal promoter sequences. We demonstrate that Sp1 physically and directly interacts with Smad2, Smad3, and weakly with Smad4 via their amino-terminal (Mad-Homology 1) domain. Finally, by using GAL4 fusion proteins we show that the glutamine-rich sequences in the transactivation domain of Sp1 contribute to the cooperativity with Smad proteins. In conclusion, Smad proteins play important roles in regulation of the p21 gene by TGF-beta, and the functional cooperation of Smad proteins with Sp1 involves the physical interaction of these two types of transcription factors.

c-Jun transactivates the promoter of the human p21(WAF1/Cip1) gene by acting as a superactivator of the ubiquitous transcription factor Sp1.

The cell cycle inhibitor protein p21(WAF1/Cip1) (p21) is a critical downstream effector in p53-dependent mechanisms of growth control and p53-independent pathways of terminal differentiation. We have recently reported that the transforming growth factor-beta pathway-specific Smad3 and Smad4 proteins transactivate the human p21 promoter via a short proximal region, which contains multiple binding sites for the ubiquitous transcription factor Sp1. In the present study we show that the Sp1-occupied promoter region mediates transactivation of the p21 promoter by c-Jun and the related proteins JunB, JunD, and ATF-2. By using gel electrophoretic mobility shift assays we show that this region does not contain a binding site for c-Jun. In accordance with the DNA binding data, c-Jun was unable to transactivate the p21 promoter when overexpressed in the Sp1-deficient Drosophila-derived SL2 cells. Coexpression of c-Jun and Sp1 in these cells resulted in a strong synergistic transactivation of this promoter. In addition, a chimeric promoter consisting of six tandem high affinity Sp1-binding sites fused with the CAT gene was transactivated by overexpressed c-Jun in HepG2 cells. The above data propose functional cooperation between c-Jun and Sp1. Physical interactions between the two factors were demonstrated in vitro by using GST-Sp1 hybrid proteins expressed in bacteria and in vitro transcribed-translated c-Jun. The region of c-Jun mediating interaction with Sp1 was mapped within the basic region leucine zipper domain. In vivo, functional interactions between c-Jun and Sp1 were demonstrated using a GAL4-based transactivation assay. Overexpressed c-Jun transactivated a chimeric promoter consisting of five tandem GAL4-binding sites only when coexpressed with GAL4-Sp1-(83-778) fusion proteins in HepG2 cells. By utilizing the same assay, we found that the glutamine-rich segment of the B domain of Sp1 (Bc, amino acids 424-542) was sufficient for c-Jun-induced transactivation of the p21 promoter. In conclusion, our data support a mechanism of superactivation of Sp1 by c-Jun, which is based on physical and functional interactions between these two transcription factors on the human p21 and possibly other Sp1-dependent promoters.