Prostanoid biosynthesis in patients with cystic fibrosis

The urinary excretion rate (ng/h/1.73 m²) of prostanoids was determined with a capillary gas-liquid chromatographic mass spectrometric method in 19 patients with cystic fibrosis (CF) aged 1–29 years. Patients with CF showed an increased excretion of prostaglandin E₂ metabolites (PGE-M) and thromboxane B₂ and its metabolites at all ages. An imbalance in the excretion pattern of thromboxane B₂ metabolites also suggested a relative impairment of β-oxidation. There was no increased excretion of dinor-6-keto-PGF_1α, indicating normal prostacyclin biosynthesis. No correlation was found to genotype, clinical score, lung function or bacterial colonization but a significant negative relation was found between the main prostanoids in the urine and serum phospholipid levels of essential fatty acids. The results show that, contrary to the generally accepted decrease of prostanoid excretion in essential fatty acid deficiency, patients with CF increase their production parallel to the development of the deficiency. Since prostanoid synthesis is rate limited by arachidonic acid release, our data support a previously presented hypothesis about a pathological regulation of the release of arachidonic acid in CF.     

The cystic fibrosis locus

The identification of the cystic fibrosis locus (CF) provides a model for the study of single gene defects where the biochemical lesion is not known. Using families each of which has several affected siblings, it was possible to exclude a number of 'candidate genes' which had previously been proposed as possible sites of the CF mutation. Exclusion mapping of the genome using polymorphic protein and DNA markers showed that CF is on the long arm of human chromosome 7. The most closely linked flanking markers were identified, and human chromosome fragments containing them (and therefore the CF locus) were isolated in rodent cell lines by chromosome-mediated gene transfer. The transgenome was then analysed using cosmid contig mapping, pulse-field gel electrophoresis, HTF island identification and linkage disequilibrium. In this way, a candidate coding sequence has been identified which always segregates with CF.     

Cystic fibrosis mutations and associated haplotypes in Turkish cystic fibrosis patients.

Identification of mutations causing cystic fibrosis (CF) in the Turkish population is essential for assessment of the molecular basis of CF in Turkey and the development of strategies for prenatal diagnosis and genetic counseling. Here, we present an updated report of mutations found in the Turkish CF population from an extensive screening study of the entire coding region, including exon-intron boundaries and the promoter region. Cases for which mutations could not be identified were also screened for previously defined large alterations and (TG)mTn-M470V loci. This study revealed a total of 27 different mutations accounting for almost 60% of disease genes in the Turkish population. In this study, we also identified the haplotypes associated with 17 mutations and those associated with unknown mutations. The mutation spectrum of CF in Turkey and its associated haplotypes indicated the presence of a major Mediterranean component in the contemporary Turkish population.     

The spectrum of CFTR mutations in south-west German cystic fibrosis patients

The cystic fibrosis transmembrane conductance regulator (CFTR) gene of 110 cystic fibrosis (CF) patients from the south-west of Germany was screened for 12 different mutations. This analysis resulted in an identification of 79% of all CF mutations and a complete genotype in 66% of the families. The most common mutation found was F508 (67%). Another 5 mutations accounted for a further 12.5% (4% G542X; 3% R553X; 3% N1303K; 2% 1717-1 GA; 0.5% G551D) whereas 6 mutations (R117H, A455E, I507, S549I, S549N, and R1162X) were not found. Fifty-four (49%) patients were AF508 homozygotes and 18 (16.5%) were compound heterozygotes for F508 and one of the rarer mutations. These frequencies differ slightly from those found in the north of Germany and considerably from those reported from the south of Europe, which seems to be consistent with a north to south decline of the relative abundance of F508. Two patients, age 6 and 25 years, were compound heterozygotes for G542X and N1303K. The clinical features of the 6 year old were characterised by severe gastrointestinal and as yet only mild pulmonary complications whereas the 25 year old manifested severe pulmonary and gastrointestinal symptoms indicating that the N1303K mutation of the C-terminal CFTR nucleotide binding fold significantly impairs protein function in both the pancreas and the lungs.     

The activity of cathepsin D in saliva of cystic fibrosis patients

Cystic fibrosis (CF) is genetically determined illness, which is caused by the mutation in the CFTR gene. CFTR protein is also expressed in epithelial cells of parotid glands, therefore parotid glands are also affected in CF patients. Cathepsin D is one of the proteolitic cascade enzymes. Physiological wearing out result in occurrence of trace quantities of this enzyme in serum and body fluids, including saliva. Among different enzymes, saliva contains cathepsin D (CTSD, EC 3.4.23.5). The aim of this study was to determine cathepsin D activity in mixed saliva in cystic fibrosis patients and healthy controls. The study was performed in a group of 26 CF patients (10F, 16M). The results obtained in CF group was compared with the results of thirty healthy subjects (12F, 14M). From each subject 8 ml of mixed saliva was obtained: before and after the stimulation of saliva excretion using paraffin pledgets. Protein and glycoprotein content was assessed using Winzler's method. Protein concentration in controls and CF group before stimulation of excretion was 1.15+/-0.714 mg/mL and 1.54+/-0.925 mg/mL. After stimulation protein concentration in saliva has lowered to 0.88+/-0.77 mg/mL in CF group and 1.24+/-1.213 mg/mL in controls. Glycoprotein concentration in controls and in CF group was respectively: before stimulation 1.08+/-0.271 mg/mL and 1.05+/-0.344 mg/mL; after stimulation 0.92+/-0.292 mg/mL and 0.86+/-0.283 mg/mL. The activity of CTSD in controls was 45.9+/-24.98 Tyr nmol/mL/4h before stimulation and 109.3+/-56.94 Tyr nmol/mL/4h after stimulation of excretion. In CF group CTSD activity before stimulation was 134.5+/-81.80 Tyr nmol/mL/4h and after stimulation 134.4+/-62.18 Tyr nmol/mL/4h. Comparing the CTSD activity in both groups statistically significant difference has been revealed in samples collected before stimulation of excretion (p=0.013). The activity of cathepsin D in saliva of cystic fibrosis patient is significantly higher than in healthy controls before the stimulation of excretion with paraffin pledgets.     

Serum ribonuclease levels in patients with cystic fibrosis

The levels of serum pancreatic-type ribonuclease (RNase) activity in normal individuals and in individuals homozygous and heterozygous for the cystic fibrosis (CF) gene have been studied. In 21 CF patients, ages 3–24 years, RNase levels of 459 ± 95 units/ml did not differ significantly from the RNase levels of 467 ± 105 units/ml in 17 normal individuals of the same age group nor from those of 490 ± 52 units/ml in 5 individuals heterozygous for the CF gene.     

Thiocyanate concentration in saliva of cystic fibrosis patients

Thiocyanates (SCN-) are ubiquitous in nature. There are indispensable part of host defense system that act as a substrate for lactoperoxidase (LPO). In our study we present initial data on SCN- concentration in saliva of CF patients in comparison to healthy non-smokers and healthy smokers. 5 ml of saliva was collected from each subject to a sterile tube and thiocyanate concentration was measured in each sample. The results of the measurements are presented on Fig. 1. Mean concentration of SCN- in saliva of CF patients was 0.031 +/- 0.0052 g/l, in healthy non-smokers 0.039 +/- 0.0048 g/l and in healthy smokers 0.048 +/- 0.0161 g/l. The differences between each group were statistically significant. Studies on larger group of patients and probably on different material (BALF or induced sputum) should present interesting data complementing the in vitro studies.     

Cell-mediated immunity in patients with cystic fibrosis.

Leucocytes from 26 patients with cystic fibrosis (CF) and 18 healthy controls were investigated by migration inhibition induced by a variety of antigens. In patients with CF cell-mediated immunity was found to human lung and pancreatic tissue extracts as well as to Aspergillus fumigatus, Pseudomonas aeruginosa, and food antigens but not to brain, heart, or kidney. Those patients with the severest form of the disease had the greatest impairment of cell-mediated immunity, but this impairment could be reversed by steroid treatment. Cell-mediated cytotoxicity may also be concerned in the pathogenesis of CF.     

Plasma chromium concentrations in normal infants and cystic fibrosis patients

Plasma Cr concentrations have been studied in normal children aged 0–14 yr. Levels ranged from 0.65 to 0.88 μg/1 and did not change with age. Plasma concentrations of CF patients given 0.5–0.75 μg Cr/kg/d in addition to their diet were similar to normal values. There was no correlation between these plasma values and growth retardation.     

The ΔF508 mutation in cystic fibrosis patients of Southern Italy

Summary Fifty one independent cystic fibrosis (CF) families originating from a restricted area of Southern Italy (Campania) have been analyzed for KM19 and XV2c haplotypes and the ΔF508 mutation: 54% of the total CF chromosomes show the ΔF508 mutation. No significative correlations were obtained when clinical score, radiological score,Pseudomonas colonization, or clinical symptoms at presentation were matched with the presence or absence of the ΔF508 mutation.     

The structure of tracheobronchial mucins from cystic fibrosis and control patients.

Tracheobronchial mucin samples from control and cystic fibrosis patients were purified by gel filtration chromatography on Sephacryl S-1000 and by density gradient centrifugation. Normal secretions contained high molecular weight (approximately 10(7] mucins, whereas the cystic fibrosis secretions contained relatively small amounts of high molecular weight mucin together with larger quantities of lower molecular weight mucin fragments. These probably represent products of protease digestion. Reducing the disulfide bonds in either the control or cystic fibrosis high molecular weight mucin fractions released subunits of approximately 2000 kDa. Treating these subunits with trypsin released glycopeptides of 300 kDa. Trypsin treatment of unreduced mucin also released fragments of 2000 kDa that could be converted into 300-kDa glycopeptides upon disulfide bond reduction. Thus, protease-susceptible linkages within these mucins must be cross-linked by disulfide bonds so that the full effects of proteolytic degradation of mucins remain cryptic until disulfide bonds are reduced. Since various combinations of protease treatment and disulfide bond reduction release either 2000- or 300-kDa fragments, these fragments must represent important elements of mucin structure. The high molecular weight fractions of cystic fibrosis mucins appear to be indistinguishable from control mucins. Their amino acid compositions are the same, and various combinations of disulfide bond reduction and protease treatment release products of identical size and amino acid composition. Sulfate and carbohydrate compositions did vary considerably from sample to sample, but the limited number of samples tested did not demonstrate a cystic fibrosis-specific pattern. Thus, tracheobronchial mucins from cystic fibrosis and control patients are very similar, and both share the same generalized structure previously determined for salivary, cervical, and intestinal mucins.     

Characterization of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> isolated from chronically infected children with cystic fibrosis in India

Background  

Pseudomonas aeruginosa is the leading cause of morbidity and mortality in patients with cystic fibrosis (CF). With chronicity of infection, the organism resides as a biofilm, shows multi-drug resistance, diversifies its colony morphology and becomes auxotrophic. The patients have been found to be colonized with multiple genotypes. The present work was carried out to characterize P. aeruginosa isolated from children with cystic fibrosis using phenotypic and genotypic methods.     

Quantitative variation in cystic fibrosis-associated proteins in cystic fibrosis patients,carriers, and controls

Summary Serum samples from patients with cystic fibrosis (CF), obligate heterozygotes, and normal controls have been examined by isoelectric focusing (IEF). Our results suggest that cystic fibrosis protein (CFP) is a normal serum protein exhibiting quantitative variation primarily dependent on possession of the CF allele. It is concluded that detection of CFP by IEF is an inappropriate screening test for the CF gene due to lack of specificity.     

The basic defect in cystic fibrosis.

Recent evidence strongly suggests that the cystic fibrosis gene product (CFTR) is a Cl- channel. Its properties, however, differ from those of a 30-50 pS outwardly rectifying channel previously implicated as defective in cystic fibrosis. It is still uncertain whether the pleiotropic effects of the CF defect, such as increased airway Na+ absorption and mucus sulfation, are secondary to reduced Cl- conductance, or reflect additional functions of CFTR.     

Pseudomonas aeruginosa antibody detection in cystic fibrosis patients.

Chronic respiratory infection due to Pseudomonas aeruginosa remains the most important prognostic factor in cystic fibrosis patients. One method to lengthen the patient's life is to extend the initial state of the illness with an early diagnosis, before Ps. aeruginosa infection becomes chronic. Often this is difficult because of the young age of the patients. This study tested an immunoenzymatic system to evaluate antibody response against three Ps. aeruginosa purified antigens, alkaline protease, elastase and exotoxin A. We studied 40 patients with cystic fibrosis, 20 affected and 20 unaffected by apparent Ps. aeruginosa infection, also from the bacteriological point of view. Serological and bacteriological results were compared for each patient and showed that serological screening can be useful in young subjects, who often have no bacteriological evidence of Ps. aeruginosa colonization.     

The genus Prevotella in cystic fibrosis airways

Airway disease resulting from chronic bacterial colonization and consequential inflammation is the leading cause of morbidity and mortality in patients with Cystic Fibrosis (CF). Although traditionally considered to be due to only a few pathogens, recent re-examination of CF airway microbiology has revealed that polymicrobial communities that include many obligate anaerobes colonize lower airways. The purpose of this study was to examine Prevotella species in CF airways by quantitative culture and phenotypic characterization. Expectorated sputum was transferred to an anaerobic environment immediately following collection and examined by quantitative microbiology using a variety of culture media. Isolates were identified as facultative or obligate anaerobes and the later group was identified by 16S rRNA sequencing. Prevotella spp. represented the majority of isolates. Twelve different species of Prevotella were recovered from 16 patients with three species representing 65% of isolates. Multiple Prevotella species were often isolated from the same sputum sample. These isolates were biochemically characterized using Rapid ID 32A kits (BioMérieux), and for their ability to produce autoinducer-2 and β-lactamases. Considerable phenotypic variability between isolates of the same species was observed. The quantity and composition of Prevotella species within a patients’ airway microbiome varied over time. Our results suggest that the diversity and dynamics of Prevotella in CF airways may contribute to airway disease.     

The cystic fibrosis and is product CFTR

Cystic fibrosis is the commonest, fatal, inherited disease of caucasian populations occurring with a frequency of 1 in 2000 live births. The CF gene spans about 230 kb of genomic DNA and encodes a protein of 1480 amino acids named the cystic fibrosis transmembrane conductance regulator (CFTR). The primary sequence predicts that CFTR is an ABC type protein with twelve transmembrane spans, two nucleotide binding domains and a cytoplasmic regulatory domain. CFTR functions as a cyclic AMP-regulated, low conductance, chloride channel in epithelial cells, but other roles are possible. Failure of the CFTR channel in CF reduces epithelial salt and water secretion, leading to a dehydration of epithelial surfaces which initiates the pathology of the disease.     

Reversibility of cardiac fibrosis in mice chronically infected with Trypanosoma cruzi, under specific chemotherapy.

This investigation was performed to verify the effect of specific chemotherapy (Benznidazole or MK-436) on the inflammatory and fibrotic cardiac alterations in mice chronically infected with the strains 21 SF (Type II) and Colombian (Type III) of Trypanosoma cruzi. To obtain chronically infected mice, two groups of 100 Swiss mice each, were infected with either the 21 SF or the Colombian strain (2 x 10(4) and 5 x 10(4) blood forms respectively). The rate of mortality in the acute phase was of 80% for both groups. Twenty surviving mice chronically infected with the 21 SF strain and 20 with the Colombian strain were then divided in treated and untreated groups. Excluding those that died during the course of treatment, 14 mice chronically infected with the 21 SF strain and 15 with the Colombian strain were finally evaluated in the present study. Chemotherapy was performed with Benznidazole (N-benzil-2-nitro-1-imidazolacetamide) in the dose of 100 mg/k.b.w/day, for 60 days, or with the MK-436 (3(1-methyl-5 nitroimidazol-2-yl) in two daily doses of 250 mg/k.b.w, for 20 days. Parasitological cure tests were performed (xenodiagnosis, haemoculture, subinoculation of the blood into newborn mice), and serological indirect immunofluorescence test. The treated and untreated mice as well as intact controls were killed at different periods after treatment and the heart were submitted to histopathological study with hematoxilineosin and picrosirius staining; ultrastructural study; collagen immunotyping, fibronectin and laminin identification by immunofluorescence tests. Results: the untreated controls either infected with 21 SF or Colombian strain, showed inflammatory and fibrotic alterations that were mild to moderate with the 21 SF strain and intense with the Colombian strain. Redpicrosirius staining showed bundles of collagen in the interstitial space and around cardiac fibers. Increased deposits of matritial components and collagen fibers, macrophages and fibroblasts appeared at the ultrastructural examination. Deposits of fibronectin, laminin, pro-III and IV collagens were seen, most intense in those infected with the Colombian strain. Treated mice, parasitologically cured, presented clear-cut regression of the inflammatory lesions and of the interstitial matrix thickening. Mice infected with the Colombian strain and treated with MK-436, was parasitologically cured in 5/6 cases and showed mild inflammatory infiltration and fibrosis. The mice treated with Benznidazole (Colombian strain) did not cure and showed moderate fibrosis and inflammation. Treatment of the mice infected with the 21 SF with Benznidazole determined parasitological cure of all animals, that showed mild inflammation and fibrosis of the myocardium.(ABSTRACT TRUNCATED AT 400 WORDS)