The molecular basis of partial penetrance of splicing mutations in cystic fibrosis

Biotin-labelled DNA probes and restriction-endonuclease digestion (RED) with HindIII were used to study the diversity of resistance plasmids (R-plasmids) from 414 Escherichia coli isolates: 168 from children living in close contact with antibiotic-fed poultry and 246 from the chickens. Full sensitivity to all 10 antimicrobials tested was more common in the isolates from poultry than in those from the children (36.2% v. 9.5%; P < 0.001). Multi-drug resistance, to at least two of the antimicrobials, was relatively common in the isolates from the children (85.5% v. 26.00%; P < 0.001). Overall, 31% of the poultry isolates were resistant to tetracycline alone. Resistance to amoxycillin was due to production of TEM-1 (89%) and TEM-2 (11%). In > 71% of the isolates from children and 79% of those from poultry, resistance was encoded on a 100-110-kb transferable plasmid belonging to incompatibility group FII. However, RED patterns of R-plasmids from the two groups of isolates were highly diverse and not indicative of any close relatedness. This difference in patterns and in the levels of multi-drug resistance indicate that the isolates from the children and those from the poultry represent two distinct pools of resistance plasmids.     

The pancreas in cystic fibrosis: chemical composition and comparative morphology

Sections of pancreas from 16 individuals who died with cystic fibrosis (CF) were classified by morphometric criteria into four categories in increasing order of pancreatic involvement. The concentration of acini, islets, main ducts, lobular ducts, connective tissue, and fat was compared with control levels. The results show that in the least involved pancreases, from neonates who died under 5 months of age, acini were reduced to 33% of control levels and the following were increased: islets, to 410%, lobular ducts, to 250%; and main ducts, to 1700% of controls. With increasing severity of the pancreatic disease the acini were further reduced to 5% and lobular ducts to 37% of control levels, respectively. Main ducts increased by 19-fold, and fatty infiltration accounted for more than 25% of the fresh weight of the pancreas in 9 of the 16 specimens. Comparative biochemical studies of 35 fibrocystic pancreases were quantitatively related to the severity of the pancreatic involvement as follows. Water and volatile matter, normally accounting for 80 +/-% of the weight of the fresh pancreas, was reduced to less than 30% in the most affected organs. The concentration of zinc diminished from near normal mean levels of 193 mugZn/g dry pancreas to 10% of this amount in the severely involved pancreas. Elevated concentrations of calcium, amounting to over 10 times control level, were found in obstructed ductal structures. Calcium was depleted from pancreatic sections adjacent to the obstructions. The following biochemical indicators were significantly different in their mean levels in the 35 fibrocystic pancreases when compared with the 17 controls: (P less than or equal to 0.001) fat, water, zinc, calcium, copper, magnesium, potassium, and sodium (P less than or equal to 0.01).     

Progress in understanding the molecular pathogenesis of human lung cancer

We review the molecular pathogenesis of lung cancer including alterations in dominant oncogenes, recessive oncogenes/tumor suppressor genes, alterations in growth regulatory signaling pathways, abnormalities in other pathways, such as apoptosis, autocrine and paracrine growth stimulatory loops, angiogenesis, and host immune responses, other mechanisms of genetic changes, such as microsatellite and methylation alterations, and the potential for inherited predisposition to lung cancer. These changes are related to multistage carcinogenesis involving preneoplastic lesions, and lung development and differentiation. The translational applications of these findings for developing new ways of early detection, prevention, treatment, and prognosis of lung cancer are discussed.     

Role of heat shock proteins in the pathogenesis of cystic fibrosis arthritis

BACKGROUND: Arthritis is a well recognised complication of cystic fibrosis. The cause of this arthritis is not yet clear but it is likely to be an immunological reaction to one of the many bacterial antigens to which the lungs are exposed. One such group, the heat shock proteins, (hsp), was investigated. These are immunodominant antigens of a wide variety of infectious microorganisms and have varying amino acid chain sequences, some of which are similar to those found in human tissues. METHODS: Antibodies to human hsp 27 and hsp 90 in the serum of patients with cystic fibrosis, with and without arthritis, and in normal age and sex matched healthy controls were measured. The severity of the cystic fibrosis was assessed by lung function tests and chest radiographs. The nature of the organisms colonising the lungs was determined by bacteriological examination of sputum. RESULTS: Higher mean titres of serum IgG anti-human hsp 27 and hsp 90 antibodies were found in 50 patients with cystic fibrosis than in healthy controls (hsp 27, 0.25 (95% CI 0.19 to 0.33) versus 0.05 (95% CI 0.04 to 0.07); hsp 90, 0.27 (95% CI 0.22 to 0.32) versus 0.11 (95% CI 0.08 to 0.14)). These antibodies were higher in patients in whom the lungs were colonised with Pseudomonas aeruginosa than in those without infection (hsp 27, 0.41 (95% CI 0.17 to 0.63) versus 0.18 (95% CI 0.10 to 0.27); hsp 90, 0.37 (95% CI 0.18 to 0.57) versus 0.22 (95% CI 0.16 to 0.29)). The eight patients with cystic fibrosis with arthritis had higher anti-hsp 27 antibodies (0.48 (95% CI 0.13 to 0.92)) than the 42 patients without arthritis (0.22 (95% CI 0.17 to 0.30)). CONCLUSIONS: These findings suggest that the arthritis associated with cystic fibrosis, despite being seronegative for rheumatoid factor, was associated with more severe lung disease and with a greater inflammatory response to heat shock proteins.     

Cystic-fibrosis-like disease unrelated to the cystic fibrosis transmembrane conductance regulator

OBJECTIVE: The effect of bleaching agents on bacterial adherence to polished surfaces of composite resin restorations was assessed in vitro. STUDY DESIGN: Samples of light-curing composite resins were treated with either 10% carbamide peroxide or 10% hydrogen peroxide for 1, 3, or 7 days. Bacterial adherence of Streptococcus mutans, Streptococcus sobrinus, and Actinomyces viscosus to the treated resin samples was analyzed and compared with adherence to nonbleached controls. RESULTS: A 10% solution of carbamide peroxide caused a significant increase in surface adherence of Streptococcus mutans and Streptococcus sobrinus after 3 days (P < .01). A 10% solution of hydrogen peroxide caused a significant increase in surface adherence of Streptococcus mutans and Streptococcus sobrinus after 3 and 7 days (P < .01). A decrease in adherence of Actinomyces viscosus was found after treatment with 10% hydrogen peroxide for 7 days (P < .05). CONCLUSIONS: It appears that bleaching agents may affect adherence of certain cariogenic microorganisms to the outer surfaces of composite resin restorations.     

High incidence of cystic fibrosis on the Faroe Islands: a molecular and genealogical study

We have studied the genetics of cystic fibrosis (CF) in The Faroe Islands. Based on the number of affected children born during the period 1954-1993, the incidence of CF at birth is 1:1775, which is more than twice the incidence in the rest of Denmark. We have tested all known CF patients and/or their parents for the presence of delta F508 and found it to be the only CF mutation in this population. Based on testing 881 unrelated control individuals, the carrier frequency was estimated to be 1:24, given a calculated incidence of 1:2300. Genealogical studies enabled us to trace several of the families over seven generations. Haplotype investigations within the families suggest that delta F508 was introduced by two founders, probably from the Celtic population in Brittany, Ireland, Wales or the North West of Scotland.     

Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease

The pathogenesis of cystic fibrosis (CF) airways infection is unknown. Two hypotheses, "hypotonic [low salt]/defensin" and "isotonic volume transport/mucus clearance," attempt to link defects in cystic fibrosis transmembrane conductance regulator-mediated ion transport to CF airways disease. We tested these hypotheses with planar and cylindrical culture models and found no evidence that the liquids lining airway surfaces were hypotonic or that salt concentrations differed between CF and normal cultures. In contrast, CF airway epithelia exhibited abnormally high rates of airway surface liquid absorption, which depleted the periciliary liquid layer and abolished mucus transport. The failure to clear thickened mucus from airway surfaces likely initiates CF airways infection. These data indicate that therapy for CF lung disease should not be directed at modulation of ionic composition, but rather at restoring volume (salt and water) on airway surfaces.     

Myasthenia gravis: pathogenesis and therapeutic approach on the basis of molecular structure and immunogenicity of acetylcholine receptor

Recent research for myasthenia gravis has been advanced by the availability of primary sequence of acetylcholine receptor (AChR) precursor and AChR transmembrane topography. (1) Synthetic peptides of AChR alpha-subunit defined myasthenogenic sites recognized by blocking or modulating antibody; their capability of inducing an animal model suggests that synthetic antigens contain not only B-cell sites but also T-cell sites which interact with restricted MHC class II molecules. (2) In view of the conformation-dependent B-cell site expected at beta-turn and the MHC-restricted T-cell site expected at amphipathic alpha-helix, conformationally modified peptides were synthesized by linking them; some were in combination with artificially sequenced peptide to favor an enhanced beta-turn. (3) Experiments by the use of these synthetic peptides demonstrated that such antigens were enhanced in myasthenogenicity. However, the immunogenic conformation for the induction of animal model did not necessarily assume that for the detection of antibody in native AChR-immunized rats. (4) Peptides synthesized to assume specifically for binding with antibody were used as a tool for immunoadsorption via plasma perfusion in myasthenic patients, resulting in clinical improvement.     

Toward understanding the molecular pathology of Huntington's disease

Huntington's Disease (HD) is caused by expansion of a CAG trinucleotide beyond 35 repeats within the coding region of a novel gene. Recently, new insights into the relationship between CAG expansion in the HD gene and pathological mechanisms have emerged. Survival analysis of a large cohort of affected and at-risk individuals with CAG sizes between 39 and 50 repeats have yielded probability curves of developing HD symptoms and dying of HD by a certain age. Animals transgenic for the first exon of huntingtin with large CAG repeats lengths have been reported to have a complex neurological phenotype that bears interesting similarities and differences to HD. The repertoire of huntingtin-interacting proteins continues to expand with the identification of HIP1, a protein whose yeast homologues have known functions in regulating events associated with the cytoskeleton. The ability of huntingtin to interact with two of its four known protein partners appears to be influenced by CAG length. Caspase 3 (apopain), a key cysteine protease known to play a seminal role in neural apoptosis, has also been demonstrated to specifically cleave huntingtin in a CAG length-dependent manner. Many of these features are combined in a model suggesting mechanisms by which the pathogenesis of HD may be initiated. The development of appropriate in vitro and animal models for HD will allow the validity of these models to be tested.     

Community-based care in cystic fibrosis: role of the cystic fibrosis nurse specialist and implications for patients and families

Improved survival for cystic fibrosis has rapidly increased over the past four decades, with patients now living well into adult life. With changes in the structure of the National Health Service and the formation of provider units and general practitioner (GP) fund-holding practices, it is important to strengthen links between the hospital and community teams to ensure that the CF patient receives adequate care. Increasingly, treatment is being carried out at home, and this emphasis on home-based therapy demands that parents/carers and patients must acquire the skills and knowledge of complex therapies in order to optimize health. It is the role of the CF nurse specialist (NS) to educate those who will deliver the care, co-ordinate the provision of services at home, liaise with the CF team and community health-care professionals and to support the patient and their carers.     

Mutant cystic fibrosis transmembrane conductance regulator inhibits acidification and apoptosis in C127 cells: possible relevance to cystic fibrosis

We have shown elsewhere that acidification is an early event in apoptosis, preceding DNA cleavage. Cells expressing the most common mutation (delF508) of the cystic fibrosis transmembrane regulator (CFTR) exhibit a higher resting intracellular pH and are unable to secrete chloride and bicarbonate in response to cAMP. We hypothesized that defective acidification in cells expressing delF508 CFTR would interfere with the acidification that accompanies apoptosis, which in turn, would prevent endonuclease activation and cleavage of DNA. We therefore determined whether the function of the CFTR would affect the process of apoptosis in mouse mammary epithelial C127 cells stably transfected with the wild-type CFTR (C127/wt) or the delF508 mutation of the CFTR (C127/508). C127 cells possessed an acid endonuclease capable of DNA degradation at low pH. Sixteen hours after treatment with cycloheximide, C127/wt cells underwent cytoplasmic acidification. In contrast, C127/508 cells failed to demonstrate acidification. Furthermore, the C127/508 cells did not show nuclear condensation or DNA fragmentation detected by in situ nick-end labeling after treatment with cycloheximide or etoposide, in contrast to the characteristic features of apoptosis demonstrated by the C127/wt cells. Measurement of cell viability indicated a preservation of cell viability in C127/508 cells but not in C127/wt cells. That this resistance to the induction of apoptosis depended upon the loss of CFTR activity is shown by the finding that inhibition of the CFTR with diphenylamine carboxylate in C127/wt cells conferred similar protection. These findings suggest a role for the CFTR in acidification during the initiation of apoptosis in epithelial cells and imply that a failure to undergo programmed cell death could contribute to the pathogenesis of cystic fibrosis.     

Identification of common cystic fibrosis mutations in African-Americans with cystic fibrosis increases the detection rate to 75%

Cystic fibrosis (CF)--an autosomal recessive disorder caused by mutations in CF transmembrane conductance regulator (CFTR) and characterized by abnormal chloride conduction across epithelial membranes, leading to chronic lung and exocrine pancreatic disease--is less common in African-Americans than in Caucasians. No large-scale studies of mutation identification and screening in African-American CF patients have been reported, to date. In this study, the entire coding and flanking intronic sequence of the CFTR gene was analyzed by denaturing gradient-gel electrophoresis and sequencing in an index group of 82 African-American CF chromosomes to identify mutations. One novel mutation, 3120+1G-->A, occurred with a frequency of 12.3% and was also detected in a native African patient. To establish frequencies, an additional group of 66 African-American CF chromosomes were screened for mutations identified in two or more African-American patients. Screening for 16 "common Caucasian" mutations identified 52% of CF alleles in African-Americans, while screening for 8 "common African" mutations accounted for an additional 23%. The combined detection rate of 75% was comparable to the sensitivity of mutation analysis in Caucasian CF patients. These results indicate that African-Americans have their own set of "common" CF mutations that originate from the native African population. Inclusion of these "common" mutations substantially improves CF mutation detection rates in African-Americans.     

Assessment of fitness in patients with cystic fibrosis and mild lung disease

The purpose of this investigation was to examine if exercise-induced arterial oxyhemoglobin desaturation selectively observed in highly trained endurance athletes could be related to differences in the pulmonary diffusing capacity (DL) measured during exercise. The DL of 24 male endurance athletes was measured using a 3-s breath-hold carbon monoxide procedure (to give DLCO) at rest as well as during cycling at 60% and 90% of these previously determined VO2max. Oxyhemoglobin saturation (SaO2%) was monitored throughout both exercise protocols using an Ohmeda Biox II oximeter. Exercise-induced oxyhemoglobin desaturation (DS) (SaO2% < 91% at VO2max) was observed in 13 subjects [88.2 (0.6)%] but not in the other 11 nondesaturation subjects [NDS: 92.9 (0.4)%] (P < or = 0.05), although VO2max was not significantly different between the groups [DS: 4.34 (0.65) l/min vs NDS: 4.1 (0.49) l/min]. At rest, no differences in either DLCO [ml CO.mmHg-1.min-1: 41.7 (1.7) (DS) vs 41.1 (1.8) (NDS)], DLCO/VA [8.2 (0.4) (DS) vs 7.3 (0.9) (NDS)], MVV [l/min: 196.0 (10.4) (DS) vs 182.0 (9.9) (NDS)] or FEV1/FVC [86.3 (2.2) (DS) vs 82.9 (4.7) (NDS)] were found between groups (P > or = 0.05). However, VE/VO2 at VO2max was lower in the DS group [33.0 (1.1)] compared to the NDS group [36.8 (1.5)] (P < or = 0.05). Exercise DLCO (ml CO.mmHg-1.min-1) was not different between groups at either 60% VO2max [DS: 55.1 (1.4) vs NDS: 57.2 (2.1)] or at 90% VO2max [DS: 61.0 (1.8) vs NDS: 61.4 (2.9)]. A significant relationship (r = 0.698) was calculated to occur between SaO2% and VE/VO2 during maximal exercise. The present findings indicate that the exercise-induced oxyhemoglobin desaturation seen during submaximal and near-maximal exercise is not related to differences in DL, although during maximal exercise SaO2 may be limited by a relatively lower exercise ventilation.     

Severe small airways disease resistant to medical treatment in a child with cystic fibrosis

At the age of 12, a child with cystic fibrosis developed severe small airways obstruction of unknown aetiology, in the absence of significant bronchiectasis. He remained resistant to medical treatment until, following an exacerbation of allergic bronchopulmonary aspergillosis 18 months later, he responded to high dose oral steroids. He now remains steroid-dependent, and suffering from multiple side-effects. Possible aetiology and further therapeutic strategies are discussed.     

Allergen-induced cytokine production in atopic disease and its relationship to disease severity

The Th2 cytokines, interleukin (IL)-4 and IL-5, have an important role in atopic disease. CD30 is a transmembrane molecule that may be expressed on a proportion of activated T-lymphocytes and has been reported to be a marker for Th2 phenotype. Our objective was to compare the in vitro cytokine responses and CD30 expression of peripheral blood mononuclear cells (PBMCs) to stimulation with house dust mite antigen (Dermatophagoides pteronyssinus) in atopic asthmatics, atopic nonasthmatics, and normal subjects, and to see if atopic asthmatic cytokine production correlated with symptomatic disease activity and whether cytokine production was allergen-specific. Eighteen atopic asthmatics (all were allocated a symptomatic disease score), 6 atopic nonasthmatics, and 7 healthy nonatopic individuals were studied. Resting serum IL-4 levels were measured, then PBMCs were separated using Lymphoprep density centrifugation and cultured in modified RPMI 1640 medium. PBMCs were stimulated with IL-2 alone or with D. pteronyssinus (1,000 subcutaneous units/ml) with IL-2 and harvested after 5 and 10 d. Using monoclonal antibodies and flow cytometry we obtained the percentage of CD4+ T cells expressing CD30 and the intensity of CD30 staining. Culture supernatants were analyzed for IL-4 and interferon gamma (IFN-gamma) using an enzyme-linked immunosorbent assay. In 9 atopic asthmatics PBMCs were also stimulated nonspecifically using phytohemagglutinin (PHA). IL-4 was detectable in the serum of atopic subjects but not in normal subjects. Stimulation of PBMCs with D. pteronyssinus produced significant amounts of IL-4 in atopic asthmatics and atopic nonasthmatics, but minimal quantities in normal subjects. Much lower levels of IFN-gamma were produced by atopic asthmatics in response to D. pteronyssinus compared to atopic nonasthmatics. IFN-gamma levels had an inverse correlation with asthmatic symptom score. CD4+ T-cell expression of CD30 also correlated inversely with IFN-gamma production and IFN-gamma:IL-4 ratio. PHA produced minimal levels of IL-4 compared to specific allergen stimulation. It is concluded that different groups of atopic patients exhibit different patterns of allergen-induced cytokine production. In vitro allergen-induced cytokine production in atopic asthmatics correlated with symptomatic disease activity, and is allergen-specific.