Endogenous ursodeoxycholic acid and cholic acid in liver disease due to cystic fibrosis

Focal biliary cirrhosis causes significant morbidity and mortality in cystic fibrosis (CF). Although the mechanisms of pathogenesis remain unclear, bile acids have been proposed as potential mediators of liver injury. This study examined bile acid composition in CF and assessed altered bile acid profiles to determine if they are associated with incidence and progression of liver injury in CF-associated liver disease (CFLD). Bile acid composition was determined by gas-liquid chromatography/mass spectrometry in bile, urine, and serum samples from 30 children with CFLD, 15 children with CF but without liver disease (CFnoLD)), and 43 controls. Liver biopsies from 29 CFLD subjects were assessed histologically by grading for fibrosis stage, inflammation, and disruption of the limiting plate. A significantly greater proportion of endogenous biliary ursodeoxycholic acid (UDCA) was demonstrated in CFnoLD subjects vs. both CFLD subjects and controls (2.4- and 2.2-fold, respectively; ANOVA, P = .04), and a 3-4 fold elevation in endogenous serum UDCA concentration was observed in both CFLD subjects and CFnoLD subjects vs. controls (ANOVA, P < .05). In CFLD, there were significant correlations between serum cholic acid and hepatic fibrosis, inflammation, and limiting plate disruption as well as the ratio of serum cholic acid/chenodeoxycholic acid to hepatic fibrosis, inflammation, and limiting plate disruption. In conclusion, elevated endogenous UDCA in CFnoLD suggests a possible protective role against liver injury in these patients. The correlation between both cholic acid and cholic acid/chenodeoxycholic acid levels with histological liver injury and fibrosis progression suggests a potential monitoring role for these bile acids in CFLD.

Rapid genotyping of Pseudomonas aeruginosa isolates harboured by adult and paediatric patients with cystic fibrosis using repetitive-element-based PCR assays

In this study, the suitability of two repetitive-element-based PCR (rep-PCR) assays, enterobacterial repetitive intergenic consensus (ERIC)-PCR and BOX-PCR, to rapidly characterize Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis (CF) was examined. ERIC-PCR utilizes paired sequence-specific primers and BOX-PCR a single primer that target highly conserved repetitive elements in the P. aeruginosa genome. Using these rep-PCR assays, 163 P. aeruginosa isolates cultured from sputa collected from 50 patients attending an adult CF clinic and 50 children attending a paediatric CF clinic were typed. The results of the rep-PCR assays were compared to the results of PFGE. All three assays revealed the presence of six major clonal groups shared by multiple patients attending either of the CF clinics, with the dominant clonal group infecting 38% of all patients. This dominant clonal group was not related to the dominant clonal group detected in Sydney or Melbourne (pulsotype 1), nor was it related to the dominant groups detected in the UK. In all, PFGE and rep-PCR identified 58 distinct clonal groups, with only three of these shared between the two clinics. The results of this study showed that both ERIC-PCR and BOX-PCR are rapid, highly discriminatory and reproducible assays that proved to be powerful surveillance screening tools for the typing of clinical P. aeruginosa isolates recovered from patients with CF.

Cardiac expression of the cystic fibrosis transmembrane conductance regulator involves novel Exon 1 usage to produce a unique amino-terminal protein

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel present in many cells. In cardiomyocytes, we report that multiple exon 1 usage and alternative splicing produces four CFTR transcripts, with different 5'-untranslated regions, CFTRTRAD-139, CFTR-1C/-1A, CFTR-1C, and CFTR-1B. CFTR transcripts containing the novel upstream exons (exons -1C, -1B, and -1A) represent more than 90% of cardiac expressed CFTR mRNA. Regulation of cardiac CFTR expression, in response to developmental and pathological stimuli, is exclusively due to the modulation of CFTR-1C and CFTR-1C/-1A expression. Upstream open reading frames have been identified in the 5'-untranslated regions of all CFTR transcripts that, in conjunction with adjacent stem-loop structures, modulate the efficiency of translation initiation at the AUG codon of the main CFTR coding region in CFTRTRAD-139 and CFTR-1C/-1A transcripts. Exon(-1A), only present in CFTR-1C/-1A transcripts, encodes an AUG codon that is in-frame with the main CFTR open reading frame, the efficient translation of which produces a novel CFTR protein isoform with a curtailed amino terminus. As the expression of this CFTR transcript parallels the spatial and temporal distribution of the cAMP-activated whole-cell current density in normal and diseased hearts, we suggest that CFTR-1C/-1A provides the molecular basis for the cardiac cAMP-activated chloride channel. Our findings provide further insight into the complex nature of in vivo CFTR expression, to which multiple mRNA transcripts, protein isoforms, and post-transcriptional regulatory mechanisms are now added.

Post-transcriptional regulation of the cystic fibrosis gene in cardiac development and hypertrophy

Eukaryotic gene expression, reflected in the amount of steady-state mRNA, is regulated at the post-transcriptional level. The 5'-untranslated regions (5'-UTRs) of some transcripts contain cis-acting elements, including upstream open reading frames (uORFs), that have been identified as being fundamental in modulating translation efficiency and mRNA stability. Previously, we demonstrated that uORFs present in the 5'-UTR of cystic fibrosis transmembrane conductance regular (CFTR) transcripts expressed in the heart were able to modulate translation efficiency of the main CFTR ORF. Here, we show that the same 5'-UTR elements are associated with the differential stability of the 5'-UTR compared to the main coding region of CFTR transcripts. Furthermore, these post-transcriptional mechanisms are important factors governing regulated CFTR expression in the heart, in response to developmental and pathophysiological stimuli. (C) 2004 Elsevier Inc. All rights reserved.

Clinical presentation of metabolic alkalosis in an adult patient with cystic fibrosis

In subtropical and tropical climates, dehydration is common in cystic fibrosis patients with respiratory exacerbations. This may lead to a clinical presentation of metabolic alkalosis with associated hyponatraemia and hypochloraemia. An adult cystic fibrosis patient who presented with a severe respiratory exacerbation accompanied by metabolic alkalosis is presented and the effects of volume correction are reported.

Integrated actions of transforming growth factor-beta(1) and connective tissue growth factor in renal fibrosis

Matrix accumulation in the renal tubulointerstitium is predictive of a progressive decline in renal function. Transforming growth factor-beta(1) (TGF-beta(1)) and, more recently, connective tissue growth factor (CTGF) are recognized to play key roles in mediating the fibrogenic response, independently of the primary renal insult. Further definition of the independent and interrelated effects of CTGF and TGF-beta(1) is critical for the development of effective antifibrotic strategies. CTGF (20 ng/ml) induced fibronectin and collagen IV secretion in primary cultures of human proximal tubule cells (PTC) and cortical fibroblasts (CF) compared with control values (P < 0.005 in all cases). This effect was inhibited by neutralizing antibodies to either TGF-beta or to the TGF-beta type II receptor (TbetaRII). TGF-beta(1) induced a greater increase in fibronectin and collagen IV secretion in both PTC (P < 0.01) and CF (P < 0.01) compared with that observed with CTGF alone. The combination of TGF-beta(1) and CTGF was additive in their effects on both PTC and CF fibronectin and collagen IV secretion. TGF-beta(1) (2 ng/ml) stimulated CTGF mRNA expression within 30 min, which was sustained for up to 24 h, with a consequent increase in CTGF protein (P < 0.05), whereas CTGF had no effect on TGF-beta(1) mRNA or protein expression. TGF-beta(1) (2 ng/ml) induced phosphorylated (p)Smad-2 within 15 min, which was sustained for up to 24 h. CTGF had a delayed effect on increasing pSmad-2 expression, which was evident at 24 h. In conclusion, this study has demonstrated the key dependence of the fibrogenic actions of CTGF on TGF-beta. It has further uniquely demonstrated that CTGF requires TGF-beta, signaling through the TbetaRII in both PTCs and CFs, to exert its fibrogenic response in this in vitro model.

Neonatal calyceal dilation and renal fibrosis resulting from loss of Adamts-1 in mouse kidney is due to a developmental dysgenesis

Background. A disintegrin and metalloproteinase with thrombospondin motifs 1, Adamts-1, is important for the development and function of the kidney. Mice lacking this protein present with renal lesions comprising enlarged calyces, and reduced cortex and medulla layers. Our current findings are consistent with the defect occurring due to a developmental dysgenesis. Methods. We generated Adamts-1 null mice, and further investigated their kidney phenotype in a time course study ranging from E18.5 to 12 months of age. Immunohistochemistry was used to assess the localization of type IV collagen, TGF-beta and F4/80-positive macrophages in the kidneys of Adcants-1 null mice compared to wild-type control animals. The expression of Adamts-1 mRNA was determined in metanephric kidney explants by in situ hybridization. Results. Adamts-1 null mice have a gross kidney defect. At day 18.5 of gestation, the Adcants-1 null kidney has a normal appearance but at birth when the kidney begins to function, the defect becomes evident. During development of the kidney Adamts-1 expression was specifically detected in the developing loops of Henle, as well as in the proximal and distal convoluted tubules. Expression was not detected in the ureter, ureteric bud or its derivatives as had been previously suggested. At 6 months and I year of age, the Adamts-1 null mice displayed interstitial fibrosis in the cortical and medullary regions of the kidney. At I year of age, the Adamts-1 null mice displayed mild interstitial matrix expansion associated with increased collagen type IV expression, without apparent tubular dilatation, compared to wild-type animals. Immunohistochemical analysis demonstrated TGF-beta protein localized to infiltrating macrophages and glomeruli of Adamts-1 null mice. Conclusions. Adamts-1 is required for the normal development of the kidney. The defect observed in its absence results from a dysgenic malformation affecting the medulla that becomes apparent at birth, once the kidneys start to function.

Change in physiotherapy management of children with cystic fibrosis in a large urban hospital

A retrospective audit was conducted in 1998 and 2000 to review the physiotherapy management of hospitalized children with cystic fibrosis (CF) at the Brisbane Royal Children's Hospital (RCH). The objective was to detect and explore possible changes in patient management in this time period and investigate whether these changes reflected changes in the current theory of CF management. All children over two years of age with CF admitted during 1998 and 2000 with pulmonary manifestation and who satisfied set criteria were included (n = 249). Relative frequency of each of six treatment modalities used were examined on two occasions, revealing some degree of change in practice reflecting the changes in current theory. There was a significant decrease in the frequency of usage of postural drainage with head-down tilt (p < 0.001), and autogenic drainage (p < 0.001) between 1998 and 2000. Modified postural drainage without head-down tilt (p < 0.001), and positive expiratory pressure devices (p < 0.001) were used more frequently in 2000 (p < 0.001). No significant changes were identified in the use of Flutter VRP1 (p = 0.145) and exercise (p = 0.763). No significant differences were found in population demographics or occurrence of concomitant factors that may influence patient management.

Optimal design for model discrimination and parameter estimation for itraconazole population pharmacokinetics in cystic fibrosis patients

Optimal sampling times are found for a study in which one of the primary purposes is to develop a model of the pharmacokinetics of itraconazole in patients with cystic fibrosis for both capsule and solution doses. The optimal design is expected to produce reliable estimates of population parameters for two different structural PK models. Data collected at these sampling times are also expected to provide the researchers with sufficient information to reasonably discriminate between the two competing structural models.

Fibrosis correlates with a ductular reaction in hepatitis C: Roles of impaired replication, progenitor cells and steatosis

The mechanisms for progressive fibrosis and exacerbation by steatosis in patients with chronic hepatitis C (HCV) are still unknown. We hypothesized that proliferative blockade in HCV-infected and steatotic hepatocytes results in the default activation of hepatic progenitor cells (HPC), capable of differentiating into both biliary and hepatocyte lineages, and that the resultant ductular reaction promotes portal fibrosis. To study this concept, 115 liver biopsy specimens from subjects with HCV were scored for steatosis, inflammation, and fibrosis. Biliary epithelium and HPC were decorated by cytokeratin 7 immunoperoxidase, and the replicative state of hepatocytes was assessed by p21 and Ki-67 immunohistochemistry. A ductular reaction at the portal interface was common. There was a highly significant correlation between the area of ductular reaction and fibrosis stage (r = 0.453, P < .0001), which remained independently associated after multivariate analysis. HPC numbers also correlated with fibrosis (r = 0.544, P < .0001) and the ductular area (r = 0.624, P < .0001). Moreover, steatosis correlated with greater HPC proliferation (r = 0.372, P = .0004) and ductular reaction (r = 0.374, P < .0001) but was not an obligate feature. Impaired hepatocyte replication by p21 expression was independently associated with HPC expansion (P = .002) and increased with the body mass index (P < .001) and lobular inflammation (P = .005). In conclusion, the strong correlation between portal fibrosis and a periportal ductular reaction with HPC expansion, the exacerbation by steatosis, and the associations with impaired hepatocyte replication suggest that an altered regeneration pathway drives the ductular reaction. We believe this triggers fibrosis at the portal tract interface. This may be a stereotyped response of importance in other chronic liver diseases.