JNK signaling prevents biliary cyst formation through a CASPASE-8-dependent function of RIPK1 during aging.

The c-Jun N-terminal kinase (JNK) signaling pathway mediates adaptation to stress signals and has been associated with cell death, cell proliferation, and malignant transformation in the liver. However, up to now, its function was experimentally studied mainly in young mice. By generating mice with combined conditional ablation of Jnk1 and Jnk2 in liver parenchymal cells (LPCs) (JNK1/2LPC-KO mice; KO, knockout), we unraveled a function of the JNK pathway in the regulation of liver homeostasis during aging. Aging JNK1/2LPC-KO mice spontaneously developed large biliary cysts that originated from the biliary cell compartment. Mechanistically, we could show that cyst formation in livers of JNK1/2LPC-KO mice was dependent on receptor-interacting protein kinase 1 (RIPK1), a known regulator of cell survival, apoptosis, and necroptosis. In line with this, we showed that RIPK1 was overexpressed in the human cyst epithelium of a subset of patients with polycystic liver disease. Collectively, these data reveal a functional interaction between JNK signaling and RIPK1 in age-related progressive cyst development. Thus, they provide a functional linkage between stress adaptation and programmed cell death (PCD) in the maintenance of liver homeostasis during aging.

Glycogen storage disease type I patients with hyperlipidemia have no signs of early vascular dysfunction and premature atherosclerosis.

BACKGROUND AND AIMS: Glycogen storage disease type I (GSD I) is associated with hyperlipidemia, a known risk factor for premature atherosclerosis. Few studies have addressed endothelial dysfunction in patients with GSD I, and these studies yielded controversial results. METHODS AND RESULTS: We investigated vascular dysfunction in a cohort of 32 patients with GSD I (26 GSD Ia, 6 GSD Ib, mean age 20.7 (4.8-47.5) years) compared to 32 age-, gender-, and BMI-matched healthy controls using non-invasive techniques such as quantification of carotid intima media thickness, retinal vessel analysis and 24 h-blood pressure measurements. In addition, early biomarkers of inflammatory and oxidative endothelial stress were assessed in blood. Although GSD I patients had a clearly proatherogenic lipid profile, increased oxidative stress, higher levels of high sensitivity C-reactive protein and increased lipoprotein associated phospholipase A2 activity, functional and structural parameters including carotid intima media thickness and retinal vessel diameters did not indicate premature atherosclerosis in this patient cohort. Blood pressure values and pulse wave velocity were comparable in patients and healthy controls, while central blood pressure and augmentation index were higher in GSD patients. CONCLUSION: Our data suggest that GSD I is not associated with early vascular dysfunction up to the age of at least 20 years. Further studies are needed to elucidate the possibly protective mechanisms that prevent early atherosclerosis is GSD I. Longer follow-up studies are required to assess the long-term risk of vascular disease with increased oxidative stress being present in GSD I patients.

Elevated oxysterol levels in human and mouse livers reflect nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH), a primary cause of liver disease, leads to complications such as fibrosis, cirrhosis, and carcinoma, but the pathophysiology of NASH is incompletely understood. Epstein-Barr virus-induced G protein-coupled receptor 2 (EBI2) and its oxysterol ligand 7α,25-dihydroxycholesterol (7α,25-diHC) are recently discovered immune regulators. Several lines of evidence suggest a role of oxysterols in NASH pathogenesis, but rigorous testing has not been performed. We measured oxysterol levels in the livers of NASH patients by LC-MS and tested the role of the EBI2-7α,25-diHC system in a murine feeding model of NASH. Free oxysterol profiling in livers from NASH patients revealed a pronounced increase in 24- and 7-hydroxylated oxysterols in NASH compared with controls. Levels of 24- and 7-hydroxylated oxysterols correlated with histological NASH activity. Histological analysis of murine liver samples demonstrated ballooning and liver inflammation. No significant genotype-related differences were observed in Ebi2-/- mice and mice with defects in the 7α,25-diHC synthesizing enzymes CH25H and CYP7B1 compared with wild-type littermate controls, arguing against an essential role of these genes in NASH pathogenesis. Elevated 24- and 7-hydroxylated oxysterol levels were confirmed in murine NASH liver samples. Our results suggest increased bile acid synthesis in NASH samples, as judged by the enhanced level of 7α-hydroxycholest-4-en-3-one and impaired 24S-hydroxycholesterol metabolism as characteristic biochemical changes in livers affected by NASH.

Farnesoid X receptor-dependent and -independent pathways mediate the transcriptional control of human fibroblast growth factor 19 by vitamin A.

Fibroblast growth factor 19 (FGF19) is a gut-derived hormone that controls bile acid (BA), carbohydrate and lipid metabolism. Whereas strong evidence supports a key role of BAs and farnesoid X receptor (FXR) for the control of FGF19 expression, information on other regulators is limited. In mice, FGF15 expression (ortholog of human FGF19) is induced by vitamin A (VitA) in an FXR-dependent manner. However, the significance of this finding for human FGF19 is currently unclear. Here, we demonstrate that VitA derivatives induce FGF19 in human intestinal cell lines by a direct transcriptional mechanism. In contrast to mouse FGF15, however, this direct regulation is not dependent on FXR but mediated by retinoic acid receptors (RARs) and their interaction with a novel DR-5 element in the human FGF19 gene. In addition to this direct effect, VitA derivatives impacted on the BA-mediated control of FGF19 by regulation of FXR protein levels. In conclusion, VitA regulates human FGF19 expression through FXR-dependent and -independent pathways. Moreover, we suggest that considerable mechanistic differences exist between humans and mice with regard to the nuclear receptors controlling the VitA-FGF15/19 axis. These findings may implicate a clinical relevance of RAR-activating VitA derivatives for the regulation of FGF19 levels in humans.

Analysis of meiosis in SUN1 deficient mice reveals a distinct role of SUN2 in mammalian meiotic LINC complex formation and function.

LINC complexes are evolutionarily conserved nuclear envelope bridges, composed of SUN (Sad-1/UNC-84) and KASH (Klarsicht/ANC-1/Syne/homology) domain proteins. They are crucial for nuclear positioning and nuclear shape determination, and also mediate nuclear envelope (NE) attachment of meiotic telomeres, essential for driving homolog synapsis and recombination. In mice, SUN1 and SUN2 are the only SUN domain proteins expressed during meiosis, sharing their localization with meiosis-specific KASH5. Recent studies have shown that loss of SUN1 severely interferes with meiotic processes. Absence of SUN1 provokes defective telomere attachment and causes infertility. Here, we report that meiotic telomere attachment is not entirely lost in mice deficient for SUN1, but numerous telomeres are still attached to the NE through SUN2/KASH5-LINC complexes. In Sun1(-/-) meiocytes attached telomeres retained the capacity to form bouquet-like clusters. Furthermore, we could detect significant numbers of late meiotic recombination events in Sun1(-/-) mice. Together, this indicates that even in the absence of SUN1 telomere attachment and their movement within the nuclear envelope per se can be functional.

The meiotic nuclear lamina regulates chromosome dynamics and promotes efficient homologous recombination in the mouse.

The nuclear lamina is the structural scaffold of the nuclear envelope and is well known for its central role in nuclear organization and maintaining nuclear stability and shape. In the past, a number of severe human disorders have been identified to be associated with mutations in lamins. Extensive research on this topic has provided novel important clues about nuclear lamina function. These studies have contributed to the knowledge that the lamina constitutes a complex multifunctional platform combining both structural and regulatory functions. Here, we report that, in addition to the previously demonstrated significance for somatic cell differentiation and maintenance, the nuclear lamina is also an essential determinant for germ cell development. Both male and female mice lacking the short meiosis-specific A-type lamin C2 have a severely defective meiosis, which at least in the male results in infertility. Detailed analysis revealed that lamin C2 is required for telomere-driven dynamic repositioning of meiotic chromosomes. Loss of lamin C2 affects precise synapsis of the homologs and interferes with meiotic double-strand break repair. Taken together, our data explain how the nuclear lamina contributes to meiotic chromosome behaviour and accurate genome haploidization on a mechanistic level.

Protective effects of farnesoid X receptor (FXR) on hepatic lipid accumulation are mediated by hepatic FXR and independent of intestinal FGF15 signal.

BACKGROUND & AIMS: There is a growing evidence that bile acids are involved in the regulation of triglyceride-, cholesterol-homoeostasis and fat absorption. In this study organ-specific Fxr knockout mice were used to further investigate the influence of farnesoid X receptor FXR in lipogenesis. METHODS: Liver- and intestine-specific Fxr knockout mice were fed a 1% cholesterol diet for 28 days. Histological examination of frozen tissue sections included Sudan III/H&E, BODIPY staining and liver X receptor (LXR) immunohistochemistry. Liver triglycerides, serum cholesterol, serum bile acids and nuclear LXR protein were measured. mRNA expression of several genes involved in bile acid-, cholesterol-homoeostasis and lipogenesis was quantified by real-time PCR. RESULTS: Hepatic FXR deficiency contributes to lipid accumulation under 1% cholesterol administration which is not observed in intestinal Fxr knockout mice. Strong lipid accumulation, characterized by larger vacuoles could be observed in hepatic Fxr knockout sections, while intestinal Fxr knockout mice show no histological difference to controls. In addition, these mice have the ability to maintain normal serum cholesterol and bile acid levels. Hepatic Fxr knockouts were characterized by elevated triglycerides and bile acid levels. Expression level of LXR was significantly elevated under control and 1% cholesterol diet in hepatic Fxr knockout mice and was followed by concomitant lipogenic target gene induction such as Fas and Scd-1. This protective FXR effect against hepatic lipid accumulation was independent of intestinal Fgf15 induction. CONCLUSION: These results show that the principal site of protective bile acid signalling against lipid accumulation is located in the liver since the absence of hepatic but not intestinal FXR contributes to lipid accumulation under cholesterol diet.

Adaptation of iron transport and metabolism to acute high-altitude hypoxia in mountaineers.

UNLABELLED: Human iron homeostasis is regulated by intestinal iron transport, hepatic hepcidin release, and signals from pathways that consume or supply iron. The aim of this study was to characterize the adaptation of iron homeostasis under hypoxia in mountaineers at the levels of (1) hepatic hepcidin release, (2) intestinal iron transport, and (3) systemic inflammatory and erythropoietic responses. Twenty-five healthy mountaineers were studied. Blood samples and duodenal biopsies were taken at baseline of 446 m as well as on day 2 (MG2) and 4 (MG4) after rapid ascent to 4559 m. Divalent metal-ion transporter 1 (DMT-1), ferroportin 1 (FP-1) messenger RNA (mRNA), and protein expression were analyzed in biopsy specimens by quantitative reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. Serum hepcidin levels were analyzed by mass spectrometry. Serum iron, ferritin, transferrin, interleukin (IL)-6, and C-reactive protein (CRP) were quantified by standard techniques. Serum erythropoietin and growth differentiation factor 15 (GDF15) levels were measured by enzyme-linked immunosorbent assay (ELISA). Under hypoxia, erythropoietin peaked at MG2 (P < 0.001) paralleled by increased GDF15 on MG2 (P < 0.001). Serum iron and ferritin levels declined rapidly on MG2 and MG4 (P < 0.001). Duodenal DMT-1 and FP-1 mRNA expression increased up to 10-fold from baseline on MG2 and MG4 (P < 0.001). Plasma CRP increased on MG2 and MG4, while IL-6 only increased on MG2 (P < 0.001). Serum hepcidin levels decreased at high altitude on MG2 and MG4 (P < 0.001). CONCLUSION: This study in healthy volunteers showed that under hypoxemic conditions hepcidin is repressed and duodenal iron transport is rapidly up-regulated. These changes may increase dietary iron uptake and allow release of stored iron to ensure a sufficient iron supply for hypoxia-induced compensatory erythropoiesis.

Free fatty acids repress small heterodimer partner (SHP) activation and adiponectin counteracts bile acid-induced liver injury in superobese patients with nonalcoholic steatohepatitis.

UNLABELLED: Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in industrialized countries and may proceed to steatohepatitis (NASH). Apoptosis and free fatty acid (FFA)-induced lipotoxicity are important features of NASH pathogenesis. We have shown a hepatoprotective effect of adiponectin in steatotic livers of hepatitis C virus (HCV) patients and recent data links bile acid (BA) metabolism to the pathogenesis of NAFLD. The aim of this study was to identify potential interactions between BA and FFA metabolism in NAFLD. Liver biopsies and serum samples from 113 morbidly obese patients receiving bariatric surgery, healthy individuals, and moderately obese NAFLD patients were studied. Serum FFA, BA, and M30 were increased in NASH versus simple steatosis, while adiponectin was significantly decreased. The NAFLD activity score (NAS) score correlated with BA levels and reversely with adiponectin. Adiponectin reversely correlated with CD95/Fas messenger RNA (mRNA) and hepatocellular apoptosis. The BA transporter high-affinity Na+ /taurocholate cotransporter (NTCP) and the BA synthesizing enzyme cholesterol 7 alpha-hydroxylase (CYP7A1) were significantly up-regulated in obese patients and hepatoma cells exposed to FFA. Up-regulation of NTCP and CYP7A1 indicate failure to activate small heterodimer partner (SHP) upon farnesoid X receptor (FXR) stimulation by increasing BA concentrations. In line with the NAS score, adiponectin levels were reversely correlated with BA levels. Adiponectin correlated with NTCP and affects Cyp7A1 expression both in vivo and in vitro. CONCLUSION: BA synthesis and serum BA levels correlated with disease severity in NAFLD, while adiponectin is reversely correlated. FFA exposure prevented SHP-mediated repression of NTCP and Cyp7A1 expression, which lead to increased BA synthesis and uptake. In NASH, BA accumulation induced hepatocyte cell death and late FXR activation failed to prevent hepatocyte injury due to decreased adiponectin levels. Early treatment with FXR ligands and/or adiponectin-receptor agonists might prevent NASH.

Radiofrequency ablation suppresses distant tumour growth in a novel rat model of multifocal hepatocellular carcinoma.

RFA (radiofrequency ablation) is an established therapy for HCC (hepatocellular carcinoma). The multikinase inhibitor sorafenib prolongs survival in advanced HCC. We examined the effects of RFA alone and in combination with sorafenib on a bystanding tumour in a two-tumour rat model of HCC. A total of 80 rats were implanted with two liver tumours and randomized to four treatment groups: vehicle and sham operation (control), sorafenib and sham operation (Sora/Sham), vehicle and RFA (Vh/RFA), and sorafenib and RFA (Sora/RFA) (n=10/group per time point). RFA or sham-operation was performed on the left lobe tumour on day 15. Animals were killed at day 18 and day 30. Non-RFA-targeted right lobe tumours were analysed for angiogenesis, growth factors [HGF (hepatocyte growth factor), EGF (epidermal growth factor) and VEGF (vascular endothelial growth factor)] and infiltrating immune cells (CD3 and CD68). At day 30, the non-RFA-targeted tumours were significantly smaller in all three treatment groups compared with control (Sora/Sham P≤0.0001, Vh/RFA P=0.005 and Sora/RFA P≤0.0001). The smallest tumours were observed in animals treated with a combination of sorafenib and RFA, whereas the size reduction seen in the RFA-only group indicated an RFA-mediated distant suppression of tumour growth. Growth factor measurement revealed transiently decreased EGF levels after RFA (P=0.008), whereas sorafenib treatment decreased HGF levels (P=0.001). MVD (microvessel density) was reduced by sorafenib (P=0.002) despite increased VEGF levels (P≤0.0001). The immune parameters revealed augmented T-cells and IL-10 (interleukin 10) levels in all three treatment groups; sorafenib additionally increased macrophage numbers (P≤0.0001). RFA and sorafenib alone resulted in significant volume reduction of the non-RFA-targeted tumour; this effect was enhanced when both modalities were combined.

Elderly age is not a negative predictive factor for virological response to therapy with pegylated interferon-α and ribavirin in chronic hepatitis C virus patients.

BACKGROUND & AIMS: Age is frequently discussed as negative host factor to achieve a sustained virological response (SVR) to antiviral therapy of chronic hepatitis C. However, elderly patients often show advanced fibrosis/cirrhosis as known negative predictive factor. The aim of this study was to assess age as an independent predictive factor during antiviral therapy. METHODS: Overall, 516 hepatitis C patients were treated with pegylated interferon-α and ribavirin, thereof 66 patients ≥60 years. We analysed the impact of host factors (age, gender, fibrosis, haemoglobin, previous hepatitis C treatment) and viral factors (genotype, viral load) on SVR per therapy course by performing a generalized estimating equations (GEE) regression modelling, a matched pair analysis and a classification tree analysis. RESULTS: Overall, SVR per therapy course was 42.9 and 26.1%, respectively, in young and elderly patients with hepatitis C virus (HCV) genotypes 1/4/6. The corresponding figures for HCV genotypes 2/3 were 74.4 and 84%. In the GEE model, age had no significant influence on achieving SVR. In matched pair analysis, SVR was not different in young and elderly patients (54.2 and 55.9% respectively; P = 0.795 in binominal test). In classification tree analysis, age was not a relevant splitting variable. CONCLUSIONS: Age is not a significant predictive factor for achieving SVR, when relevant confounders are taken into account. As life expectancy in Western Europe at age 60 is more than 20 years, it is reasonable to treat chronic hepatitis C in selected elderly patients with relevant fibrosis or cirrhosis but without major concomitant diseases, as SVR improves survival and reduces carcinogenesis.

Impact of genetic SLC28 transporter and ITPA variants on ribavirin serum level, hemoglobin drop and therapeutic response in patients with HCV infection.

BACKGROUND & AIMS: In the last decade, pegylated interferon-α (PegIFN-α) plus ribavirin (RBV) was the standard treatment of chronic hepatitis C for genotype 1, and it remains the standard for genotypes 2 and 3. Recent studies reported associations between RBV-induced anemia and genetic polymorphisms of concentrative nucleoside transporters such as CNT3 (encoded by SLC28A3) and inosine triphosphatase (encoded by ITPA). We aimed at studying genetic determinants of RBV kinetics, efficacy and treatment-associated anemia. METHODS: We included 216 patients from two Swiss study cohorts (61% HCV genotype 1, 39% genotypes 2 or 3). Patients were analyzed for SLC28A2 single nucleotide polymorphism (SNP) rs11854484, SLC28A3 rs56350726, and SLC28A3 rs10868138 as well as ITPA SNPs rs1127354 and rs7270101, and followed for treatment-associated hemoglobin changes and sustained virological response (SVR). In 67 patients, RBV serum levels were additionally measured during treatment. RESULTS: Patients with SLC28A2 rs11854484 genotype TT had higher dosage- and body weight-adjusted RBV levels than those with genotypes TC or CC (p=0.02 and p=0.06 at weeks 4 and 8, respectively). ITPA SNP rs1127354 was associated with hemoglobin drop ≥3 g/dl during treatment, in genotype (relative risk (RR)=2.1, 95% CI 1.3-3.5) as well as allelic analyses (RR=2.0, 95%CI 1.2-3.4). SLC28A3 rs56350726 was associated with SVR in genotype (RR=2.2; 95% CI 1.1-4.3) as well as allelic analyses (RR=2.0, 95% CI 1.1-3.4). CONCLUSIONS: The newly identified association between RBV serum levels and SLC28A2 rs11854484 genotype, as well as the replicated association of ITPA and SLC28A3 genetic polymorphisms with RBV-induced anemia and treatment response, may support individualized treatment of chronic hepatitis C and warrant further investigation in larger studies.

Influence of attachment and bone loss on the mobility of incisors and canine teeth.

OBJECTIVE: The purpose of this clinical study was to evaluate the correlation between tooth mobility (TM), crown-to-root ratio (CRR) and clinical attachment loss (CAL) in periodontally-compromised participants. MATERIALS AND METHODS: While slowly biting on a load cell, the mobility of the upper incisors and canine teeth of 20 volunteers was measured using a photogrammetric measurement technique. An automated software program recorded the force-related three-dimensional TM at 3-N intervals. CAL was assessed clinically and CRR values were assessed radiographically. For each contralateral pair of teeth (central, lateral incisor, canine) and for each main level of force, the Pearson product-moment correlation coefficient between TM and CRR and between TM and CAL was computed. Correlations were considered statistically significant at p < 0.05. RESULTS: Statistically significant positive correlations were found between TM and CRR for incisors and canines for each main level of force, whereas canines had the lowest correlation. Statistically significant positive correlations were also found between TM and CAL for the central and lateral incisors at each main level of force. Canines showed no significant correlation between CAL and TM, regardless of force level. CONCLUSION: The loss of attachment and bone seem to have more influence on the mobility of incisors than canines.

Complement factor C5 deficiency significantly delays the progression of biliary fibrosis in bile duct-ligated mice.

Fibrogenesis represents the universal response of the liver to chronic liver injury. Complement factor C5 has been linked to fibrosis in murine toxic liver injury and human chronic hepatitis C. C5 may also play a central role in chronic cholestatic disorders, since the BA receptor FXR has been characterized as an activator of the C3 gene. We aimed to investigate, whether C5 deficiency is able to prevent biliary fibrosis in the mouse bile-duct-ligation model. BDL for 1-4 weeks was performed in either Hc(0)/Hc(0) mice (deficient for C5) or WT controls. BA levels were measured by RIA. Histological examination included H&E, sirius-red and immunohistochemistry. mRNA expression was quantified by RT-PCR. Protein expression levels were determined by Western blotting or ELISA. Enzymatic MMP-activity was analysed by zymography. One week BDL leads to fibrosis in WT (F2.0 ± 0), while it is almost absent in Hc(0)/Hc(0) mice (F0.5 ± 0.5). No differences in fibrosis can be detected at week-4. Together with delayed fibrogenesis at week-1, fibrotic markers are decreased in Hc(0)/Hc(0) mice. Expression of the inflammatory cytokine TNF-α is decreased in Hc(0)/Hc(0) mice. In parallel C5 deficiency leads to an attenuated peribiliary infiltration of CD45(+) cells in fibrotic areas together with decreased MMP-9 expression and gelatinase activity. The present study proves a functional role of C5 during biliary fibrogenesis. C5 deficiency leads to attenuated inflammation and normalized MMP-9 activity concomitantly with a significant reduction of fibrosis. C5 appears to be an attractive target for future therapeutic intervention in chronic cholestatic liver disease.

Genetic variations in bile acid homeostasis are not overrepresented in alcoholic cirrhosis compared to patients with heavy alcohol abuse and absent liver disease.

Increased serum bile salt levels have been associated to a single-nucleotide polymorphism in the bile salt export pump (BSEP; ABCB11) in several acquired cholestatic liver diseases but there is little evidence in alcoholic liver disease (ALD). Furthermore, a crosstalk between vitamin D and bile acid synthesis has recently been discovered. Whether this crosstalk has an influence on the course of ALD is unclear to date. Our aim was to analyse the role of genetic polymorphisms in BSEP and the vitamin D receptor gene (NR1I1) on the emergence of cirrhosis in patients with ALD. Therefore, 511 alcoholic patients (131 with cirrhosis and 380 without cirrhosis) underwent ABCB11 genotyping (rs2287622). Of these, 321 (131 with cirrhosis and 190 without cirrhosis) were also tested for NR1I1 polymorphisms (bat-haplotype: BsmI rs1544410, ApaI rs7975232 and TaqI rs731236). Frequencies of ABCB11 and NR1I1 genotypes and haplotypes were compared between alcoholic patients with and without cirrhosis and correlated to serum bile salt, bilirubin and aspartate aminotransferase levels in those with cirrhosis. Frequencies of ABCB11 and NR1I1 genotypes and haplotypes did not differ between the two subgroups and no significant association between genotypes/haplotypes and liver function tests could be determined for neither polymorphism. We conclude that ABCB11 and NR1I1 polymorphisms are obviously not associated with development of cirrhosis in patients with ALD.

Combined effect of 25-OH vitamin D plasma levels and genetic vitamin D receptor (NR 1I1) variants on fibrosis progression rate in HCV patients.

BACKGROUND: Decreased vitamin D levels have been described in various forms of chronic liver disease and associated with advanced fibrosis. Whether this association is a cause or consequence of advanced fibrosis remains unclear to date. AIMS: To analyse combined effects of 25-OH vitamin D plasma levels and vitamin D receptor gene (VDR; NR1I1) polymorphisms on fibrosis progression rate in HCV patients. METHODS: 251 HCV patients underwent VDR genotyping (bat-haplotype: BsmI rs1544410 C, ApaI rs7975232 A and TaqI rs731236 A). Plasma 25-OH vitamin D levels were quantified in a subgroup of 97 patients without advanced fibrosis. The VDR haplotype and genotypes as well as plasma 25-OH vitamin D levels were associated with fibrosis progression. RESULTS: The bAt[CCA]-haplotype was significantly associated with fibrosis progression >0.101 U/year (P = 0.007; OR = 2.02) and with cirrhosis (P = 0.022; OR = 1.84). Forty-five percent of bAt[CCA]-haplotype patients were rapid fibrosers, 21.1% were cirrhotic. Likewise, ApaI rs7975232 CC genotype was significantly associated with fibrosis progression and cirrhosis. Lower plasma 25-OH vitamin D levels were significantly associated with fibrosis progression >0.101 U/year in F0-2 patients (P = 0.013). Combined analysis of both variables revealed a highly significant additive effect on fibrosis progression with 45.5% rapid fibrosers for bAt[CCA]-haplotype and 25-OH vitamin D < 20 µg/L compared with only 9.1% for the most favourable combination (P = 0.006). In multivariate analysis, the bAt-haplotype was an independent risk factor for fibrosis progression (P = 0.001; OR = 2.83). CONCLUSION: Low 25-OH vitamin D plasma levels and the unfavourable VDR bAt[CCA]-haplotype are associated with rapid fibrosis progression in chronic HCV patients. In combination, both variables exert significant additive effects on fibrosis progression.

Correlations between photogrammetric measurements of tooth mobility and the Periotest method.

OBJECTIVE: The purpose of the present study was to investigate whether or not the quantitative Periotest values of anterior teeth correlate with quantitative metric values of tooth mobility under vertical (VL) and horizontal load (HL) in periodontally healthy subjects. MATERIALS AND METHODS: Thirty-one subjects with good periodontal conditions were included and subjected to two different tooth mobility measurement techniques. Periotest values were measured at reproducible measurement points in the vertical (vPT) and horizontal (hPT) dimensions of upper central and lateral incisors and canine teeth. Using the optical measurement technique (photogrammetry), tooth mobility was measured under load in the horizontal (HL) and vertical loading directions (VL) at different load forces. Pearson's correlation coefficients were used to determine exploratory associations. RESULTS: The comparison between hPT and HL showed no correlations between the two measurements except for 'weak' and 'moderate' correlations for teeth 21 and 23. The analysis of correlations between vPT and VL data showed statistically significant correlations for both the left and right canine teeth that ranged from 'weak' to 'high'. Comparisons between hPT values and VL and between vPT and HL showed significant correlations at a few loading forces only. CONCLUSION: Quantitative Periotest values cannot be used to draw conclusions about the metric assessment of tooth mobility. For this purpose, the photogrammetric technique could be an additional tool for scientific questions.

The Impact of Drug Interactions in Patients with Community-Acquired Pneumonia on Hospital Length of Stay.

(1) Background: An aging society is frequently affected by multimorbidity and polypharmacy, which, in turn, leads to an increased risk for drug interaction. The aim of this study was to evaluate the influence of drug interactions on the length of stay (LOS) in hospitals. (2) Methods: This retrospective, single-centre study is based on patients treated for community-acquired pneumonia in the hospital. Negative binomial regression was used to analyse the association between drug interactions and the LOS in the hospital. (3) Results: The total cohort contained 503 patients, yet 46 inpatients (9%) that died were not included in the analyses. The mean age was 74 (±15.3) years, 35% of patients older than 65 years were found to have more than two drug interactions, and 55% had a moderate, severe, or contraindicated adverse drug reaction. The regression model revealed a significant association between the number of drug interactions (rate ratio (RR) 1.02; 95%-CI 1.01-1.04) and the severity of drug interactions (RR 1.22; 95%-CI 1.09-1.37) on the LOS for the overall cohort as well as for the subgroup of patients aged 80 years and older. (4) Conclusion: Drug interactions are an independent risk factor for prolonged hospitalisation. Standardised assessment tools to avoid drug interactions should be implemented in clinical routines.

Downregulation of breast cancer resistance protein in colon adenomas reduces cellular xenobiotic resistance and leads to accumulation of a food-derived carcinogen.

Several molecular changes in colorectal adenomas provide the basis of the adenoma-carcinoma sequence. We investigated the expression of xenobiotic ATP-binding cassette (ABC) transporters in humans and in ApcMin mice and conducted functional studies estimating the importance of the expression changes. Twenty-nine adenomas from 21 patients and eight adenomas from four ApcMin mice were analyzed using Western blotting and quantitative Real-time polymerase chain reaction (RT-PCR). Adjacent healthy tissue served as control for each polyp. Breast cancer resistance protein (BCRP) was significantly downregulated in human colorectal adenomas (to 28 ± 35% of adjacent healthy tissue). This was in line with data from ApcMin mice adenomas, where downregulation was significant as well (to 58 ± 34%). In parallel, quantitative RT-PCR showed BCRP mRNA downregulation in human adenomas (to 17 ± 31%). Basal multidrug resistance-associated protein 2 expression was low and did not change in adenomas; multidrug resistance transporter 1 expression also did not differ between adenomas and healthy tissue. In a functional study, ApcMin mice received radioactively labelled 2-amino-1-methyl-6-phenylimidazo[4,5-ß] pyridine (PhIP), a food colon carcinogen and substrate of BCRP, by oral gavage with analysis of PhIP accumulation and DNA adduct formation 48 hr later. In this setting, we could demonstrate a higher carcinogen concentration in adenomas of ApcMin mice (181 ± 113% of normal tissue) including immunohistochemical detection of PhIP-DNA adducts. We conclude that significant transcriptional downregulation of BCRP/Bcrp leads to higher carcinogen concentrations in colorectal adenomas of mice and men. This might promote the adenoma-carcinoma sequence by higher genotoxic effects. The results indicate a possible role of transporter deficiencies in susceptibility for colon carcinoma.

A common polymorphism in the ABCB11 gene is associated with advanced fibrosis in hepatitis C but not in non-alcoholic fatty liver disease.

Chronic HCV (hepatitis C virus)-associated cirrhosis represents a major indication for liver transplantation. Bile acids contribute to hepatic stellate cell activation as a key event in fibrogenesis. The aim of the present study was to investigate the role of bile acids and polymorphisms in bile acid level-regulating genes on fibrosis progression. A total of 206 subjects with chronic HCV infection were included for ABCB11 (ATP-binding cassette, subfamily B, member II) 1331T>C and NR1H4 (nuclear receptor) -1G>T genotyping, 178 of which were analysed for fibrosis stage. Exclusion criteria were HBV (hepatitis B virus) or HIV coinfection, alcohol >40 g/day and morbid obesity. A total of 358 patients with NAFLD (non-alcoholic fatty liver disease) were genotyped for comparison with a non-viral liver disease. Caucasian individuals (n = 110), undergoing liver resection for focal hepatic metastasis, served as controls. The ABCB11 1331C allele was significantly overrepresented in HCV patients compared with controls {allelic frequency 62.9%; OR (odds ratio), 1.41 [95% CI (confidence interval), 1.012-1.965]}. Median plasma bile acid levels were not significantly increased in the CC compared with TT genotype [7.2 (1-110) µmol/l compared with 3.5 (1-61) µmol/l; values are medians (range). A significant association between the presence of cirrhosis and ABCB11 genotype (CC compared with CT or TT, P=0.047) was observed in the χ2 test and independent of other risk factors of age, gender, body mass index and disease duration in multivariate analysis (P = 0.010). No such association could be observed in fatty liver patients with regard to advanced fibrosis (F ≥ 2). The common ABCB11 1331CC genotype, which is present in 40% of HCV patients and renders the carrier susceptible to increased bile acid levels, is associated with cirrhosis.