Pregnancy Research: Cystic Fibrosis and Beyond.

High-fat vs low-fat diets for patients with cystic fibrosis: Authors' reply

The last 25 years have shown that it has been comparatively slow and difficult to develop cystic fibrosis (CF) gene therapy; the lung is a complex target organ. However, research has steadily progressed and recently it was shown that non-viral gene therapy can stabilise CF lung disease. These data, in addition to the development of potent lentiviral vectors, have renewed interest in CF gene therapy within academia and industry.

Clinical, chemical and pathologic data are presented on 9 patients with the generalized disease, "fibrocytic disease of the pancreas," but with normal or partial pancreatic exocrine function. Pancreatic function was evaluated not only by duodenal assay, but by studies of intestinal absorption of fat and nitrogen. Pancreatic function was entirely normal in 3 patients, partially present in 3 others, and in the remaining 3, normal at first, but decreasing to low levels at a later date. Pathologic findings in the pancreas at autopsy in 1 case are presented and the histologic appearance of a biopsy specimen of this organ is described in another. It is emphasized that the nutritional symptoms of the disease are secondary to pancreatic deficiency and may be absent if this gland is functioning normally. No evidence is found for a close relation between pancreatic function and pulmonary involvement. The data reported in this paper strongly support the view that partial or even normal pancreatic function may occur in "fibrocystic disease of the pancreas." The diagnostic implications are discussed. It is concluded that finding of tryptic activity in normal concentration in duodenal juice does not negate the diagnosis. Evidence is presented supporting the reliability of the "sweat test," since it may be positive at a time when other findings are not present. In the light of recent knowledge, criteria for the diagnosis of "fibrocystic disease of the pancreas" are restated. The diagnostic importance of the demonstration of an abnormality of sweat electrolytes in association with the chronic pulmonary pathology is stressed, especially if pancreatic function is normal. The use of the stool trypsin test, increased viscosity of duodenal contents and microscopic examination of stool for excess fat as screening tests is discussed. The latter is preferred. In patients having the disease without pancreatic deficiency, excess fecal fat is not present. Nevertheless if the diagnosis is suspected on the basis of the respiratory involvement, a "sweat test" should be done. The conclusion is reached that "fibrocystic disease of the pancreas," is not primarily a disease of the pancreas, but a generalized condition in which this organ is usually, but not necessarily affected. Varying degrees of involvement of the pancreas, lungs, liver and sweat glands are inherent in this disease. Complete and incomplete forms of the condition exist. The basic nature of the disturbance is discussed. There is an accumulating body of evidence that the secretory activity of many and perhaps all exocrine glands, mucus-producing and others, is affected in this disorder. Possible mechanisms of hereditary transmission of the basic defect are considered. A plea is made for retention of the name "cystic fibrosis of the pancreas" or "fibrocystic disease of the pancreas" until a better one is proposed, perhaps after the etiology and pathogenesis of the disease are further clarified. The term "mucoviscidosis," by implying that only mucus-secreting structures are affected, tends to prolong an erroneous concept and introduce another improper term to further confuse an already muddled field.

Thrombophilia Screening in Adults Is Not Predictive of a Thrombotic Event after Bipulmonary Lung Transplantation

Cystic fibrosis (CF) cells exhibit an increase in the protein expression of β-arrestin-2 (βarr2) coincident with perinuclear accumulation of free cholesterol. Arrestins are proteins that both serve as broad signaling regulators and contribute to G-protein coupled receptor internalization after agonist stimulation. The hypothesis of this study is that βarr2 is an important component in the mechanisms leading to cholesterol accumulation characteristic of CF cells. To test this hypothesis, epithelial cells stably expressing GFP-tagged βarr2 (βarr2-GFP) and respective GFP-expressing control cells (cont-GFP) were analyzed by filipin staining. The βarr2-GFP cells show a late endosomal/lysosomal cholesterol accumulation that is identical to that seen in CF cells. This βarr2-mediated accumulation is sensitive to Rp-cAMPS treatment, and depleting βarr2 expression in CF-model cells by shRNA alleviates cholesterol accumulation compared with controls. Cftr/βarr2 double knockout mice also exhibit wild-type (WT) levels of cholesterol synthesis, and WT profiles of signaling protein expression have previously been shown to be altered in CF due to cholesterol-related pathways. These data indicate a significant regulatory role for βarr2 in the development of CF-like cholesterol accumulation and give further insight into cholesterol processing mechanisms. An impact of βarr2 expression on Niemann-Pick type C-1 (NPC1)-containing organelle movement is proposed as the mechanism of βarr2-mediated alterations on cholesterol processing. It is concluded that βarr2 expression contributes to altered cholesterol trafficking observed in CF cells.

Regulation of Chemokine Expression by NaCl Occurs Independently of Cystic Fibrosis Transmembrane Conductance Regulator in Macrophages

Toward the understanding and improvement of cystic fibrosis (CF) airway health, our laboratories have studied the tracheobronchial mucous glycoproteins (mGP) of individuals with and without CF. With the increased mucus viscosity and charged character of the mGP oligosaccharide species, obstructions in the CF gastrointestinal and respiratory tracts readily occur and lead to malnutrition, frequent and persistent pulmonary infections with opportunistic pathogens, and COPD. With new medications (“modulators”) targeting the basic defects, we anticipate marked improvements in CF airway health. With respect for the current airway clearance challenges faced by many with CF, we reflect on our experiences in handling sputum specimens over the years as we processed them toward mGP oligosaccharide structural elucidation. These studies provided opportunities for microscopic, chemical and biochemical observations about CF sputum relative to non‐CF sputum and insights about sputa heterogeneity of an individual. Toward improving therapeutic strategies, here we briefly report these observations. Microscopically, dense CF sputum typically depicted a neutrophil inflammatory response with large numbers of clumped neutrophils, many with degenerating nuclei, and much cellular debris, whereas non‐CF purulent sputa commonly displayed abundant mucous material and scattered inflammatory cells. Average chemical compositions of >1000 sputa (213 non‐purulent, NP; 322 purulent, P; and 468 CF), showed for CF less water content per specimen and greater dry weight (NP, P, CF respectively: water mg/mL 959, 939, 862; macromolecular dry weight [MDW] mg/mL 43, 61, 98 respectively). CF sputa possessed a higher % of MDW as lipid and DNA than non‐CF sputa and surprisingly, decreased total carbohydrate (CHO), (NP, P, CF respectively: lipid 25, 31, 34 % MDW; DNA 0.01, 1.4, 2.7 % MDW, with CF DNA ranging 0.1 – 9.1% MDW; CHO 21, 20, 14 % MDW.) Sputa of 4 CF patients collected over 5 days of hospitalization, showed >35% reduction in sputa DNA content regardless of initial DNA % MDW at admission. Sputa solubilization with guanidinium HCl showed no correlation of lipid content to hours to complete solubilization (hS); a trend to longer times with increased glycoprotein content (GP); and a marked impact of DNA content on solubilization. With CF focus, GP, DNA content, and hS were determined for 77 CF sputa. Rather than linear concentration trends, there were scattered relationships of % MDW of DNA and of GP. 3D graphical analysis revealed longer solubilization times (i.e. 10–20 h) when both mucins and DNA were in significant quantity, than specimens of high levels of DNA with little GP (i.e. 4–8h). Compiling these types of observations, we see CF sputa, over the population, and for a given individual, reflect a heterogenous spectrum of DNA‐mucin relative concentrations. As therapeutic strategies continue to evolve for the mobilization of the viscous obstructive materials, these data suggest that approaches which address both the DNA and mucous glycoprotein components will be the most beneficial in the ever‐changing airway dynamic. As COPDs share many features with CF, these CF sputum reflections may also guide airway clearance strategies in general.Support or Funding InformationUniversity of Missouri Agricultural Experiment Station Chemical Laboratories; Cystic Fibrosis Associations of Missouri and West Plains.

The Registry of the International Society for Heart and Lung Transplantation: Thirty-third Adult Lung and Heart–Lung Transplant Report—2016; Focus Theme: Primary Diagnostic Indications for Transplant

McDonagh, Ellen M.; Clancy, John P.; Altman, Russ B.; Klein, Teri E. Author Information

Editorial for CF pregnancy guidelines

Background and Aims: The bacterium Pseudomonas aeruginosa is a versatile environmental organism which is capable of persisting within a diverse range of habitats. For people with cystic fibrosis (CF), P. aeruginosa is the most common bacterial species isolated from respiratory secretions and infection is associated with poor health outcomes and decreased life expectancy. Molecular-based epidemiological studies have shown that patients with CF can harbour indistinguishable genotypes (shared strains) of P. aeruginosa that have not been isolated from natural or commonly encountered environments sources. It is speculated that these shared strains are acquired by person-to-person transmission and have become highly niche-adapted to the CF airway. However, the specific mechanisms relating to the transmission, host adaptation and persistence of these strains remain uncertain. By thoroughly characterising P. aeruginosa isolates from a defined cohort of patients this thesis aims to determine the prevalence and diversity of strains among adults with CF, and using phenotypic and genomic methods, to explore the key differences pertaining to adaptation between shared and unique strains of P. aeruginosa strains sourced from a range of ecological settings. Methods: Longitudinal analysis of respiratory pathogens from adults with CF was undertaken to determine the prevalence of P. aeruginosa and other major CF pathogens. Univariate analysis was conducted to determine change in prevalence over time. P. aeruginosa isolates at two time points from adults with CF underwent single nucleotide polymorphism (SNP) based genotyping to assess the incidence and prevalence of shared strains. Statistical analyses was undertaken to find associations between acquisition of shared strains and health outcomes. Bacterial motility and adhesion was determined for clinical (CF, non-CF and animal) and environmental P. aeruginosa isolates. Each assay was performed under varied atmospheric conditions. Regression analysis was performed to assess an association between genotype and phenotype. CF and environmental P. aeruginosa isolates were grown a dynamic flow cell system to allow for the development and visualisation of biofilm structures. Following incubation and staining, three dimensional images were captured and resulting data analysed using Comstat software. Mixed effects models were used to compare relevant biofilm parameters. Bioinformatic analysis was performed on CF and environmental P. aeruginosa isolates. Genetic variants associated with phenotypic characteristics were identified. Results: The prevalence of significant airway pathogens declined over the study period. For paediatric patients at the time of transition, this decline was correlated with an improvement in lung function. Interestingly, the prevalence of chronic P. aeruginosa infection remained stable. While the proportion of patients harbouring the shared strain AUST-02 declined over time, there was a marked increase in those infected with the AUST-06 strain. In contrast, the prevalence of AUST-01 and other non-shared strains remained stable. Patients who acquired AUST-06 reported the largest decline in lung function over time and patients harbouring AUST-02 showed an increased risk of death or lung transplantation. Environmental, animal and non-CF isolates displayed enhanced motility and adherence to a plastic surface in the microtitre assay; while those collected from CF patients, particularly shared strains displayed a reduction in these phenotypic characteristics. Incubation under anaerobic conditions further reduced phenotypes for all isolates. Unlike results from the microtitre assay, all isolates assessed using the flow cell system demonstrated adherence to the glass substrate and the ability to form biofilms. Morphologically all isolates were similar, although CF isolates produced significantly thicker biofilms with a greater biomass when compared to the environmental isolates. Bioinformatic analysis identified a range of genetic variants associated with the assessed phenotypic characteristics. Non-synonymous mutations in genes associated with recognised virulence genes, in particular those which regulate motility and biofilm formation, were identified in most strains. Conclusions: This thesis demonstrated that P. aeruginosa is still the most common pathogen isolated from adults with CF; although the prevalence of this organism has declined in recent years. Furthermore, despite few changes in patient management there has been an overall shift in the epidemiology of shared strain infections within this patient cohort. P. aeruginosa has developed a range of mechanisms in order to avoid attack by the host immune system and killing via antimicrobial substances. One of these mechanisms is growth within a protective biofilm structure. Although motility and adhesion, two traits required for biofilm development, were reduced amongst the CF isolates, they were still capable of forming a biofilm. Discordance of the bacterial adherence and flow cell assays provide evidence that the static microtitre assay may not be suitable for assessing biofilm forming capacity of P. aeruginosa isolates derived from the CF airway. Finally, a bacterial GWAS approach identified genetic variants suitable for use as targets in future studies aimed at determining the genetic mechanisms of these phenotypic characteristics. Overall, the results of this thesis demonstrate that adaptation and virulence of P. aeruginosa to the airways is multifactorial, complex and influences from the environment, host and the bacterium itself all affect its ability to infect and persist.

Background: Patients with cystic fibrosis with the same mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene differ widely in survival suggesting other factors have a substantial role in...

To determine whether the activity of cytochrome P450 isoforms involved in the metabolism of (R)-warfarin is enhanced in cystic fibrosis. Six adult subjects with cystic fibrosis and six healthy control subjects, matched by age and sex, were administered (R)-warfarin as a single intravenous bolus dose (0.375 mg/kg), and urine and plasma samples were collected for 192 hours. The concentration of (R)-warfarin in plasma and the concentration of (R)-warfarin and its metabolites in urine were determined by HPLC and GC/MS, respectively. Plasma protein binding of (R)-warfarin was measured by ultrafiltration. The unbound plasma clearance of (R)-warfarin was not significantly (p > 0.05) different between the cystic fibrosis and the control groups (cystic fibrosis, 997 +/- 483 ml/hr/kg; control, 788 +/- 219 ml/hr/kg). The unbound metabolic clearances of (R)-warfarin to its oxidative metabolites--6-hydroxywarfarin, 7-hydroxywarfarin, 8-hydroxywarfarin, and 10-hydroxywarfarin (mediated by P450 3A4)--were also similar (p > 0.05) in the two groups (6-hydroxywarfarin: cystic fibrosis: 124.2 +/- 72.8 ml/hr/kg, control: 99.4 +/- 37.3 ml/hr/kg; 7-hydroxywarfarin: cystic fibrosis: 43.8 +/- 32.2 ml/hr/kg, control: 34.5 +/- 10.6 ml/hr/kg; 8-hydroxywarfarin: cystic fibrosis: 80.4 +/- 85.4 ml/hr/kg, control: 69.5 +/- 39.5 ml/hr/kg; 10-hydroxywarfarin: cystic fibrosis: 4.38 +/- 2.72 ml/hr/kg, control: 16.28 +/- 13.71 ml/hr/kg). The in vivo activity of cytochrome P450 isoforms involved in the metabolism of (R)-warfarin, including P450 3A4, is not enhanced in cystic fibrosis.

“Typical” cystic fibrosis in an elderly woman

Cystic fibrosis (CF) is a frequent autosomal recessive disorder caused by mutation of a gene encoding a multifunctional transmembrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), located in the apical membrane of epithelial cells lining exocrine glands. In an attempt to get a more complete picture of the pleiotropic effects of the CFTR defect on epithelial cells and particularly on the membrane compartment, a bidimensional blue native (BN)/SDS-PAGE-based proteomic approach was used on colonic crypt samples from control and CFTR knock-out mice (cftr-/-). This approach overcomes the difficulties of membrane protein analysis by conventional two-dimensional PAGE and is able to resolve multiprotein complexes. Used here for the first time on crude membrane proteins that were extracted from murine colonic crypts, BN/SDS-PAGE allows effective separation of protein species and complexes of various origins, including mitochondria, plasma membrane, and intracellular compartments. The major statistically significant difference in protein maps obtained with samples from control and cftr-/- mice was unambiguously identified as mClCA3, a member of a family of calcium-activated chloride channels considered to be key molecules in mucus secretion by goblet cells. On the basis of this finding, we evaluated the overall expression and localization of mClCA3 in the colonic epithelium and in the lung of mice by immunoblot analysis and immunohistochemistry. We found that mClCA3 expression was significantly decreased in the colon and lung of the cftr-/- mice. In an ex vivo assay, we found that the Ca2+-dependent (carbachol-stimulated) glycoprotein secretion strongly inhibited by the calcium-activated chloride channel blocker niflumic acid (100 microm) was impaired in the distal colon of cftr-/- mice. These results support the conclusion that a ClCA-related function in the CF colon depends on CFTR expression and may be correlated with the impaired expression of mClCA3.