The Reserpinized Rat in the Study of Cystic Fibrosis: X-ray Microanalysis of Submandibular Gland and Pancreas

The chronically reserpinized rat has been suggested as an animal model for cystic fibrosis. X-ray microanalysis of thick and thin cryosections was carried out to assess elemental redistribution in the submandibular glands and the pancreas of reserpinized rats at the cellular and subcellular level. In the submandibular gland of reserpinized rats, calcium and magnesium concentrations were significantly elevated. Mucus globules, secretory granules, and endoplasmic reticulum were the primary sites of the localization of excess calcium and magnesium. A significant potassium loss from the gland had occurred, particularly from the serous cells. Electron microscopy of conventionally prepared tissue showed marked swelling of the endoplasmic reticulum, especially in mucous cells.

The elemental changes in the pancreatic acinar cells of reserpinized rats were reminiscent of elemental redistribution connected with cell death: increased levels of sodium, chlorine, and calcium and decreased levels of magnesium and potassium. Ultrastructural changes included swelling of the endoplasmic reticulum and obstruction of the acinar lumen. It is concluded that elemental redistribution in chronically reserpinized rats presents interesting parallels with cystic fibrosis.     

Effects of Cystic Fibrosis Serum and Fibroblast Culture Medium on Ion Distribution in Rat Submandibular Gland

The effects of cystic fibrosis (CF) serum and culture medium from CF fibroblasts on ion distribution in rat submandibular gland cells were investigated by X-ray microanalysis. These effects were compared to the effects of normal serum and culture medium from normal fibroblasts, of cholinergic and adrenergic agonists, and of the uncoupler 2,4-dinitrophenol.

Incubation of gland tissue with CF serum or normal serum caused a significant decrease in potassium and calcium concentrations and an increase in sodium in mucous acinar and serous granular duct cells. CF serum gave a significantly larger decrease of the potassium level than normal serum.

Culture medium from CF fibroblasts altered the cellular ion content in a way similar to CF serum. Exposure to medium from cultured normal fibroblasts did not affect the elemental composition of the gland cells significantly, compared to incubation with fresh medium or buffer. Hence, fibroblast culture medium is more suitable than serum to test specific effects of CF-associated factors.

The changes in elemental composition of gland eelIs caused by CF serum or CF fibroblast culture medium mimic some of the effects of the agonist car-bachol. They could, however, also in part result from nonspecific changes in membrane permeability.     

Effects of Culture Medium on Cystic Fibrosis and Normal Fibroblasts Studied by X-Ray Microanalysis

The effect of culture medium from fibroblast cultures of cystic fibrosis (CF) patients and healthy controls on the elemental composition of fibroblasts was investigated by X-ray microanalysis. Exposure of normal fibroblasts to culture medium from CF fibroblasts caused an increase in calcium level. Exposure of CF fibroblasts to culture medium from normal cells caused an increase of the sodium content of CF cells to approximately normal levels; the calcium level of the CF fibroblasts, however, remained abnormally high. The results may indicate that CF fibroblasts lack a factor needed for the regulation of sodium transport. CF fibroblast medium apparently contains a factor that interferes with the regulation of calcium transport.     

Effects of Culture Medium on Cystic Fibrosis and Normal Fibroblasts Studied by X-Ray Microanalysis

The effect of culture medium from fibroblast cultures of cystic fibrosis (CF) patients and healthy controls on the elemental composition of fibroblasts was investigated by X-ray microanalysis. Exposure of normal fibroblasts to culture medium from CF fibroblasts caused an increase in calcium level. Exposure of CF fibroblasts to culture medium from normal cells caused an increase of the sodium content of CF cells to approximately normal levels; the calcium level of the CF fibroblasts, however, remained abnormally high. The results may indicate that CF fibroblasts lack a factor needed for the regulation of sodium transport. CF fibroblast medium apparently contains a factor that interferes with the regulation of calcium transport.     

Multicenter Surveillance of Cystic Fibrosis in Korean Children

PurposeCystic fibrosis (CF), caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, is rare among non-Caucasians. We aimed to identify the clinical features and CFTR mutations in Korean children.MethodsWe included 18 pediatric patients with CF diagnosed using sweat chloride test or genetic analysis for 30 years. HEK293 cells were transfected with wild-type CFTR, ΔF508-CFTR, and L441P-CFTR mutant plasmids for 24 hours and treated with CFTR correctors (VX809 and VX661).ResultsThe median age at diagnosis was 9.2 years. Eleven patients had growth retardation, and 6 had a respiratory failure at diagnosis. Genetic analysis was used for all patients, while sweat testing was for 8 patients. At diagnosis, the median z scores of forced expiratory volume in one second (FEV1), FEV1/forced vital capacity, and forced expiratory flow at 25%–75% of forced vital capacity were −3.61 (−5.78, 1.78), −3.38 (−4.40, −0.60), and −4.45 (−5.78, 0.54), respectively. Two patients were treated with dornase alfa and only one with CFTR modulator. Patients were followed up for 3.7 years as a median. Four patients died at 10.6 years, with 4.2 years of post-diagnosis survival. The most common mutation was exon 16-17b deletion (19.4%). Among 11 single nucleotide variants, c.1322T>C (p.Leu441Pro, L441P) was detected in 4 patients. In the functional assay, L441P-CFTR correction was well restored by CFTR correctors compared with ΔF508.ConclusionsCF is extremely rare in Korean children and is caused by different mutations from those commonly observed in Caucasians. Early diagnosis and treatment availability may improve outcomes. CFTR modulators may be effective for Asian patients with rare CFTR mutations, c.1322T>C (p.Leu441Pro).     

Analysis of Ejaculates from Patients with Cystic Fibrosis

Ejaculates from two men with cystic fibrosis (CF) were examined. Both had azoospermia. A considerable decrease in volume and fructose content was noted. The absolute amounts of calcium, magnesium, and zinc per ejaculate showed normal values in one of the patients but were two to three times increased in the other compared to mean values of a control group. Thus the concentrations of these cations were increased at least fivefold in both patients.

The amount of Mg²⁺- and Ca²⁺dependent ATPase was comparable to that of controls, but values were higher than in men with oligospermia. Both the divalent cations and the Mg²⁺- and Ca²⁺ -dependent ATPase curve profiles of split ejaculate fractions were atypical. Secretory granules and vesicles were plentiful in the seminal plasma of both patients while amorphous substance was practically absent.

The present findings agree with a less affected function of the prostate gland and a dysfunction of the seminal vesicles in these patients.     

Spectrum of Mutations in the CFTR Gene in Cystic Fibrosis Patients of Spanish Ancestry

We analyzed 1,954 Spanish cystic fibrosis (CF) alleles in order to define the molecular spectrum of mutations in the CFTR gene in Spanish CF patients. Commercial panels showed a limited detection power, leading to the identification of only 76% of alleles. Two scanning techniques, denaturing gradient gel electrophoresis (DGGE) and single strand conformation polymorphism/hetroduplex (SSCP/HD), were carried out to detect CFTR sequence changes. In addition, intragenic markers IVS8CA, IVS8-6(T)n and IVS17bTA were also analyzed. Twelve mutations showed frequencies above 1%, p.F508del being the most frequent mutation (51%). We found that eighteen mutations need to be studied to achieve a detection level of 80%. Fifty-one mutations (42%) were observed once. In total, 121 disease-causing mutations were identified, accounting for 96% (1,877 out of 1,954) of CF alleles. Specific geographic distributions for the most common mutations, p.F508del, p.G542X, c.1811 + 1.6kbA > G and c.1609delCA, were confirmed. Furthermore, two other relatively common mutations (p.V232D and c.2789 + 5G > A) showed uneven geographic distributions. This updated information on the spectrum of CF mutations in Spain will be useful for improving genetic testing, as well as to facilitate counselling in people of Spanish ancestry. In addition, this study contributes to defining the molecular spectrum of CF in Europe, and corroborates the high molecular mutation heterogeneity of Mediterranean populations.     

Pseudomonas Aeruginosa Allergy in Cystic Fibrosis

Basophil histamine release by P. aeruginosa standard antigen was examined in cystic fibrosis patients chronically infected with mucoid P. aeruginosa (CF +P) and with pronounced antibody response against these bacteria, and in patients without P. aeruginosa infection (CF +P). All the patients showed eosinophil counts and total IgE, which did not differ significantly from that of normal persons. In the absence of patient's sera, histamine release was only found in two patients in the CF +P group, indicating that type I allergy to P. aeruginosa is not predominating in cystic fibrosis. In the presence of patients' sere significantly more of the CF+P patients responded to P. aeruginosa with histamine release compared with the CF-P patients. The response was lost by complement inactivation and regained by reconstitution of the complement activity. The involvement of a type III-mediated complement-dependent histamine release is therefore suggested in the pathogenesis of lung damage in cystic fibrosis.     

Controlled evaluation of the STARBRIGHT CD-ROM program for children and adolescents with Cystic Fibrosis

OBJECTIVE: To evaluate the effectiveness of the STARBRIGHT Fitting Cystic Fibrosis Into Your Life Everyday CD-ROM. Data were analyzed to evaluate the effectiveness of the program as an educational tool for children and adolescents with cystic fibrosis (CF). METHODS: Forty-seven children and adolescents with CF between the ages of 7 and 17 years were enrolled in the study. Participants completed an initial evaluation of CF-related knowledge and coping skills and were then randomly assigned to one of two groups: the treatment group or the wait-list control group. Participants then viewed the CD-ROM, and researchers completed posttest measures. RESULTS: Analyses indicated that both disease-related knowledge and coping strategies generated by children and adolescents with CF improved as a result of the intervention and that this effect was replicated in the wait-list group. CONCLUSIONS: In sum, the results of the current study indicate that the STARBRIGHT CD-ROM program is a promising intervention for increasing CF-related knowledge and the competence of children's and adolescents' coping strategies. These positive results are enhanced by the brief, inexpensive, and portable nature of this educational program.     

Illness Perceptions of Cystic Fibrosis: A Comparison of Young Adults with CF and Same-Aged Peers

In the last two decades, the life expectancy for individuals with Cystic Fibrosis (CF) has increased significantly. The limited research examining the psychosocial experiences of young adults with CF indicates that other young adults lack awareness and understanding of CF. Using the Illness Perception Questionnaire, perceptions of CF were examined in individuals with CF aged 16 to 25 and two groups of same-aged peers: those who did, or did not, know someone with CF. ANOVA with pairwise comparisons revealed that individuals with CF perceived significantly fewer physical symptoms of illness and fewer emotional and practical consequences of CF than both groups of peers. Individuals with CF also perceived significantly more personal control and greater understanding of CF than peers without experience of CF. Implications for enhancing opportunities for social engagement and for the provision of social support for young people with CF are identified.     

Review of a 7-Year Record of the Bacteriological Profile of Airway Secretions of Children with Cystic Fibrosis in North India

Background: Cystic fibrosis (CF) is now a recognised entity in India, with prevalence rates between 1/10,000 and 1/50,000. However, no data were available with regard to the profile of respiratory pathogens in the Indian setting. Materials and Methods: The records of respiratory secretion bacterial cultures of children with CF in a tertiary care hospital in North India from January 2010 to December 2016 were reviewed. Culture data were evaluated; the organisms were noted and their antimicrobial susceptibilities were analysed. The microbiological profile and antimicrobial susceptibility pattern of CF patients were evaluated. Results: A total of 445 samples from 146 children were processed, of which 246 (55%) samples showed bacterial growth. Mixed infections 48 (19.5%) were common in older children. Children aged 3–6 months (62.5%) showed the highest culture positivity. The most commonly isolated organisms were Pseudomonas aeruginosa (52.6%) and Staphylococcus aureus. Children with initial cultures positive for P. aeruginosa had 55% of their subsequent cultures showing polymicrobial infections. P. aeruginosa was most susceptible to ciprofloxacin (89%) and piperacillin-tazobactum (88%). Among the staphylococcal isolates, 38% were methicillin-resistant S. aureus (MRSA). The percentage of MRSA increased from 66% in 2010 to 75% in 2012, followed by a decline to 24% in 2016. Conclusions: The pattern of airway colonisation in the Indian setting is different from the Caucasian population, and P. aeruginosa and Burkholderia cepacia complex appear early. Colonisation with P. aeruginosa benefits from therapy. In case of infection, care must be taken while initiating empiric therapy. It should be based on local antibiograms to prevent the emergence of resistant microbes.     

Localization of Hsp27 in the Rat Submandibular Gland Following the Application of Various Surgical Treatments

Salivary glands repair and regenerate following various types of injuries and surgical procedures. However, the tissue responses induced in the contralateral glands have yet to be elucidated in detail. Hsp27, a member of the heat-shock protein (Hsp) family, is strongly expressed in physiological environments, particularly during development. Hsp27 was previously shown to play a role in the regulation of acinar cell proliferation and differentiation in the rat submandibular gland.The present study performed the following surgical treatments on the right submandibular glands of adult rats: 1) duct ligation followed by unligation after one week; 2) partial sialoadenectomy; and 3) total sialoadenectomy. Immunohistochemistry for Hsp27 and Ki67 was performed in the experimental and normal contralateral glands, and localization was histologically and morphometrically analyzed.The results obtained revealed the localization of Hsp27 to the intercalated duct in the submandibular glands of non-treated rats. The expression of Hsp27 was strongly induced in both the uninjured contralateral control glands as well as treated glands of experimental rats regardless of the surgical procedure performed. The number of Hsp27-immunopositive cells increased rapidly following surgery, and subsequently returned to the same level as that in non-treated rats after 4 weeks. However, no marked changes were observed in the number of Ki67-immunopositive proliferating cells. Therefore, the change in the number of Hsp27-immunopositive cells may have contributed to compensatory hypertrophy. The results of the present study indicate that the expression of Hsp27 in the intercalated duct in the submandibular gland may play a role in the differentiation of acinar cells.     

Carrier screening for cystic fibrosis in primary care: evaluation of a project in South Wales The South Wales Cystic Fibrosis Carrier Screening Research Team

Population carrier screening for cystic fibrosis (CF) was offered to all patients aged 16–45 in one general practice in South Wales, excluding those in couples with a current pregnancy. Out of 1553 patients in this group, 481 subjects were tested, giving an overall uptake rate of more than 30%. The rate of uptake varied with the mode of invitation. Twenty-six carriers were identified, giving a prevalence of identified carriers of 5.4% (1 in 18.5) for those with no family history of CF. A further 18 carriers were identified by cascade testing of these 26. We describe the practical difficulties encountered in setting up this programme in primary care in South Wales. Questionnaires were administered or distributed to all subjects before and after testing. The response rate for the pre-test questionnaire was 95%, and 40–50% for the post-test questionnaires. These showed that, at 3 months post-test, 1 in 4 screen-negative subjects did not appreciate that they had a residual risk of being a carrier. At the same time, 15% of this group thought that there was a 1 in 4 chance of a child being affected if one parent was screen-positive (carried an identified mutation) and the other was screen-negative, and 40% thought there was no risk. Anxiety in relation to testing did not appear to be a major problem, although individual patterns of response to carrier status varied widely and more sensitive indicators of psychosocial impact of genetic tests are required. A pilot study of couple screening showed that this approach is unlikely to be useful in primary care, although we did not assess couple testing during pregnancy. For any programme of CF carrier screening to be established in primary care, it will be necessary to involve the primary care team from the earliest planning stage, so that the opportunity costs, training needs and other costs of the programme can be fully resourced.     

Perceived Body Image and Eating Behavior in Young Adults with Cystic Fibrosis and Their Healthy Peers

Treatment aimed at achieving an ideal nutritional status is an integral part of the management of patients with cystic fibrosis (CF). Emphasis is continually placed upon dietary intake and weight. The effects of this on eating behavior and self-perceptions are unclear. This work compared male and female CF adults with a healthy male and female control population with regard to (a) clinical variables, (b) actual, perceived, and desired body shape/body mass index (BMI), and (c) body satisfaction, eating behaviors and attitudes, and self-esteem. Clinical data were recorded for 221 adults with CF and 148 healthy controls. All subjects completed BMI Charts (perception of body weight/BMI), the Eating Attitudes Test, and scales of body satisfaction and self-esteem. CF patients had poorer lung function and nutritional status than controls. Control males accurately perceived their body shape/BMI and were content with it, whereas CF males viewed their BMI as greater than it actually was and desired to be much heavier. Control females viewed their body shape/BMI as less than it actually was and desired to be even slimmer, in comparison with CF females, who perceived their BMI as less than it actually was but were happy with their perceived shape/weight. Control subjects, especially females, dieted to a greater extent and were more preoccupied with food (with binge eating and intended vomiting) than CF patients. Conversely, those with CF reported greater pressure from others to eat than did controls. More problems with food/eating behavior were associated with less body satisfaction and reduced self-esteem. In comparison with a healthy control population, the perceptions and behaviors of CF adults relating to eating, weight, and body image are not abnormal. Indeed, females with CF have fewer problems than their healthy peers.     

X-ray micro-analysis of cultured respiratory epithelial cells from patients with cystic fibrosis

X-ray micro-analysis was carried out on cultured respiratory cells from polyps removed from individuals with and without cystic fibrosis (CF). In a first set of experiments, proper experimental conditions were established. Washing the cells with 300 mmol 1^-1 mannitol in distilled water was found to give the best removal of the culture medium. The elemental concentrations stabilized in about 10 min after the start of the preincubation. Intracellular [Na] and [Cl] increased slightly with increasing passage number, whereas intracellular [K] decreased. Under resting conditions there were no significant differences in elemental content between CF and control cells, and there were no indications for abnormally high total [Ca] in CF cells. In normal cells, stimulation with a cAMP-analogue resulted in a decrease of cellular [CI], whereas in CF cells an increase was measured. Exposure of both normal and CF cells to ouabain resulted in decreased [K] and increased [Na] and [CI] level. The calcium ionophore A23187 had a similar effect on normal cells but did not affect CF cells markedly. Application of amiloride to the apical side of the cells resulted in a decrease of cellular [Na] in CF cells, whereas [Na] in control cells was not affected. The results correspond with what is known about the defective cAMP-regulated transepithelial Cl-transport in CF cells. The effect of the calcium ionophore on cellular electrolyte content is more complicated and may be the result of two separate effects: efflux of Cl^- via a Ca²⁺-dependent mechanism and inhibition of the Na⁺-K⁺-ATPase by intracellular Ca²⁺ ions causing an influx of Na⁺ and Cl^- ions.     

FSH受体在大鼠下颌下腺的定位及其与FSH的共存关系

目的研究大鼠下颌下腺FSH受体的定位及其与FSH的免疫共定位关系,证明下颌下腺是FSH的一个靶器官。方法制备大鼠下颌下腺石蜡连续切片以免疫组化SABC法分别研究FSH和FSHR的免疫定位。结果大鼠下颌下腺浆液性腺泡上皮细胞、颗粒曲管、排泄管及分泌管上皮细胞内有FSHR和FSH免疫反应阳性物质共存,阳性物质分布在胞质内,胞核阴性。结论大鼠下颌下腺存在FSHR,是雌激素的靶器官,而且FSHR和FSH共存于下颌下腺相同的细胞,FSH可能通过FSHR的介导调节下颌下腺的功能。     

Analysis of Ejaculates from Patients with Cystic Fibrosis

Ejaculates from two men with cystic fibrosis (CF) were examined. Both had azoospermia. A considerable decrease in volume and fructose content was noted. The absolute amounts of calcium, magnesium, and zinc per ejaculate showed normal values in one of the patients but were two to three times increased in the other compared to mean values of a control group. Thus the concentrations of these cations were increased at least fivefold in both patients.

The amount of Mg²⁺- and Ca²⁺dependent ATPase was comparable to that of controls, but values were higher than in men with oligospermia. Both the divalent cations and the Mg²⁺- and Ca²⁺ -dependent ATPase curve profiles of split ejaculate fractions were atypical. Secretory granules and vesicles were plentiful in the seminal plasma of both patients while amorphous substance was practically absent.

The present findings agree with a less affected function of the prostate gland and a dysfunction of the seminal vesicles in these patients.     

CFTR Rearrangements in Spanish Cystic Fibrosis Patients: First New Duplication (35kb) Characterised in the Mediterranean Countries

Developments in quantitative PCR technologies have greatly improved our ability to detect large genome rearrangements. In particular oligonucleotide‐based array comparative genomic hybridisation has become a useful tool for appropriate and rapid detection of breakpoints. In this work, we have analysed 80 samples (42 unknown CF alleles) applying three quantitative technologies (MLPA, qPCR and array‐CGH) to detect recurrent as well as novel large rearrangements in the Spanish CF population. Three deletions and one duplication have been identified in five alleles (12%). Interestingly, we provide the comprehensive characterisation of the first duplication in our CF cohort. The new CFTRdupProm‐3 mutation spans 35.7 kb involving the 5′‐end of the CFTR gene. Additionally, the RNA analysis has revealed a cryptic sequence with a premature termination codon leading to a disrupted protein. This duplication has been identified in five unrelated families from Spain, France and Italy with all patients showing the same associated haplotype, which is further evidence for its likely common Mediterranean origin. Overall, considering this and other previous studies, CFTR rearrangements account for 1.3% of the Spanish CF alleles.     

Regulation of Salivary Kallikrein Secretion in the Rat Submandibular Gland

Unstimulated pairs of rat submandibular glands were compared with regard to their wet weight, total protein content and kallikrein activity quantitated by Bz-Arg-OEt-esterase and kallikrein antigenic activity. Paired glands from the same animal were found to be comparable, whereas differences from one animal to another were considerable. One of two paired glands was extirpated and used as control, and the other was subsequently subjected to stimulation. Salivary secretion was induced parasympathomimetically (intraperitoneal injections of pilocarpine; perfusion with acetylcholine and electrical stimulation of the ductal nerve plexus near the gland hilus) or sympathomimetically (cervical sympathetic nerve stimulation with or without administration of α or β-adrenergic blocker; perfusion with epinephrine, norepinephrine or isoproterenol). The effect was studied by measuring the change in total gland kallikrein content and by quantitation of kallikrein in saliva. A small secretion of kallikrein was always observed. However, oc-adrenergic stimulation was 40 and 1500 fold more effective in releasing kallikrein than β-adrenergic and parasympathomimetic stimulation, respectively. Also, significantly more kallikrein was released by β-adrenergic than parasympathomimetic stimulation. Immunohistochemistry confirmed the observed depletion of kallikrein following α-adrenergic stimulation. No alteration in kallikrein localization was observed in stimulated glands.