CF Tissues Research Articles

Immunohistochemical detection of MUC5AC and MUC5B mucins in ferrets

ObjectiveCystic fibrosis (CF) is a genetic condition that causes abnormal mucus secretions in affected organs. MUC5AC and MUC5B are gel-forming mucins and frequent targets for investigations in CF tissues. Our objective was to qualify MUC5AC and MUC5B immunohistochemical techniques to provide a useful tool to identify, localize and interpret mucin expression in ferret tissues.ResultsMUC5AC and MUC5B mucins were detected most commonly in large airways and least in small airways, consistent with reported goblet cell density in airway surface epithelia. We evaluated whether staining method affected the detection of goblet cell mucins in serial sections of bronchial surface epithelia. Significant differences between stains were not observed suggesting common co-expression MUC5AC and MUC5B proteins in goblet cells of airway surface epithelia. Gallbladder and stomach tissues are reported to have differential mucin enrichment, so we tested these tissues in wildtype ferrets. Stomach tissues were enriched in MUC5AC and gallbladder tissues enriched in MUC5B, mucin enrichment similar to human tissues. Mucin immunostaining techniques were further qualified for specificity using lung tissue from recently generated MUC5AC−/− and MUC5B−/− ferrets. Qualified techniques for MUC5AC and MUC5B immunohistochemistry will be useful tools for mucin tissue studies in CF and other ferret models.

A Comprehensive and Updated Review of MicroRNA Pathogenesis and Gene Expression in Cystic Fibrosis

MicroRNAs (miRNAs) are short (~22 nucleotides) non-coding sequences that regulate gene expression. It has been recently identified that miR-145 regulates cystic fibrosis transmembrane regulator (CFTR) expression, and response to first-generation miRNAs is recognized as major biomedical research in CF. Abnormal miRNA activity causes numerous diseases, thus the critical role of miRNA is to focus on miRNA bindings to target proteins. miRNA classifications depend on their genomic location and gene structure, and they include intergenic and intronic regions. Intronic miRNA location is in the introns of annotated genes. Intergenic and intronic miRNA genes are controlled with their promoters. Some miRNAs negatively regulate the cystic fibrosis transmembrane conductance regulator (CFTR) gene expression (e.g., miR-101) in adult lung cells and have no effect on fetal epithelial cells. MiR-145, miR-223, and miR-494 directly regulate the CFTR gene expression. MiR-509-3p and miR-494 regulate CFTR more strongly than the other miRNAs. MiR-138 enhances CFTR channels on the cell surface of epithelial cells. Polymorphisms in the 3′-UTR region of CFTR decrease the expression of CFTR. Mutations in the CF (CFTR) gene, located on chromosome 7, diminish ion transport. The consequent loss of chloride and bicarbonate secretion and absorption lead to a multi-organ disease called CF marked by mucus inspissation, chronic inflammation, and tissue destruction. The most affected organs are the lung and pancreas, but the liver, bowel, sinuses, and vas deferens, among others, are prominently involved as well. The loss of CFTR also results in an altered miRNA profile that regulates the response to therapy and disease pathogenesis. This review summarizes miRNA alterations in the CF tissue, the role of miRNA in disease pathogenesis, and the therapeutic opportunity for oligotherapeutic manipulation to improve CF outcomes.

Microbial Community Composition in Explanted Cystic Fibrosis and Control Donor Lungs

To date, investigations of the microbiota in the lungs of people with Cystic Fibrosis (PWCF) have primarily focused on microbial community composition in luminal mucus, with fewer studies observing the microbiota in tissue samples from explanted lung tissue. Here, we analysed both tissue and airway luminal mucus samples extracted from whole explanted lungs of PWCF and unused donor lungs. We determined if the lung microbiota in end-stage CF varied within and between patients, was spatially heterogeneous and related to localized structural damage. Microbial community composition was determined by Illumina MiSeq sequencing and related to the CF-Computed Tomography (CT) score and features of end-stage lung disease on micro-CT. Ninety-eight CF tissue (n=11 patients), 20 CF luminal mucus (n=8 patients) and 33 donor tissue (n=4 patients) samples were analysed. Additionally, we compared 20 paired CF tissue and luminal mucus samples that enabled a direct “geographical” comparison of the microbiota in these two niches. Significant differences in microbial communities were apparent between the 3 groups. However, overlap between the three groups, particularly between CF and donor tissue and CF tissue and CF luminal mucus was also observed. Microbial diversity was lower in CF luminal mucus compared to CF tissue, with dominance higher in luminal mucus. For both CF and donor tissue, intra- and inter-patient variability in ecological parameters was observed. No relationships were observed between ecological parameters and CF-CT score, or features of end-stage lung disease. The end-stage CF lung is characterised by a low diversity microbiota, differing within and between individuals. No clear relationship was observed between regional microbiota variation and structural lung damage.

405: Calcium activated chloride channel regulator 1 VWA domain can potentiate TMEM16A anion channel in primary CF airway cells and tissues to enhance airway defense properties

405: Calcium activated chloride channel regulator 1 VWA domain can potentiate TMEM16A anion channel in primary CF airway cells and tissues to enhance airway defense properties

Diagnosis and Exercise Rehabilitation of Knee Joint Anterior Cruciate Ligament Injury Based on 3D-CT Reconstruction

The joint capsule of the knee joint is attached to the edges of various articular surfaces and is thin and loose. Therefore, ligament reinforcement is needed to protect the knee joint and increase the stability of the joint. It plays a vital role in human activities. In this paper, a 3D-CT three-dimensional reconstruction method is used to reconstruct the ACL natural femoral imprint and double-bone tract. The relative positional relationship between the two center points is compared, and the law is summarized to guide the improvement of ACL anatomic double-beam reconstruction under arthroscopy. The 3D reconstruction results suggest that the bone layer in the anterior medial portion is the thickest, forming a peak, and the thickness of the bone layer in the posterior medial portion gradually decreases in a stepwise manner. The entire bone tissue in the anterior medial portion and posterior medial portion is integrated into one body. The tissues are connected as a whole, and the thickness is relatively uniform. The two parts of the bone tissues are not connected. The CF tissue was inserted into the bone tissue in a zigzag pattern. The changes of CF tissues in the anterior medial and posteromedial CF tissues were similar, and they were distributed stepwise from the inside to the outside. According to the bone and CF spatial structure and changing rules, ACL is divided into medial and lateral beams. According to this study, it can be summarized that (1) 3D reconstruction can clearly reconstruct the natural footprint of ACL femoral stops and postoperative osseous position and (2) 3D reconstruction can be used to evaluate the position of osseous postoperative ACL anatomic double-beam reconstruction. Arthroscopy double-beam reconstruction of ACL is instructive.

Disrupted local innervation results in less VIP expression in CF mice tissues

Vasoactive Intestinal Peptide (VIP) is the major physiological agonist of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) chloride channel activity. VIP functions as a neuromodulator and neurotransmitter secreted by neurons innervating all exocrine glands. VIP is also a potent vasodilator and bronchodilator that regulates exocrine gland secretions, contributing to local innate defense by stimulating the movement of water and chloride transport across intestinal and tracheobronchial epithelia. Previous human studies have shown that the rich intrinsic neuronal networks for VIP secretion around exocrine glands could be lost in tissues from patients with cystic fibrosis. Our research has since confirmed, in vitro and in vivo, the need for chronic VIP exposure to maintain functional CFTR chloride channels at the cell surface of airways and intestinal epithelium, as well as normal exocrine tissues morphology [1]. The goal of the present study was to examine changes in VIP in the lung, duodenum and sweat glands of 8- and 17-weeks old F508del/F508del mice and to investigate VIPergic innervation in the small intestine of CF mice, before important signs of the disease development. Our data show that a low amount of VIP is found in CF tissues prior to tissue damage. Moreover, we found a specific reduction in VIPergic and cholinergic innervation of the small intestine. The general innervation of the primary and secondary myenteric plexus was lost in CF tissues, with the presence of enlarged ganglionic cells in the tertiary layer. We propose that low amount of VIP in CF tissues is due to a reduction in VIPergic and cholinergic innervation and represents an early defect that constitutes an aggravating factor for CF disease progression.

Stressors and Stress Responses in Cystic Fibrosis

Abstract Cystic fibrosis (CF) is a life-shortening, genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). The primary cause of CF is reduced CFTR-mediated chloride and bicarbonate transport, due to mutations in CFTR. However, inflammation and persistent infections influence clinical outcome. Cellular stress response pathways, such as the unfolded protein response (UPR) and the integrated stress response (ISR), referred to here as cellular stress response pathways (SRPs), contribute to the pathology of human disorders. Multiple studies have indicated activation of SRPs in CF tissues. We review our present understanding of how SRPs are activated in CF and their contribution to pathology. We conclude that reduced CFTR function in CF organs establishes a tissue environment in which internal or external insults activate SRPs. SRPs contribute to CF pathogenesis by reducing CFTR expression, enhancing inflammation with consequent tissue remodeling. Understanding the contribution of SRPs to CF pathogenesis is crucial even in the era of CFTR “modulators” that are designed to potentiate, correct or amplify CFTR function, since there is an urgent need for supportive treatments. Importantly, CF patients with established pathology could benefit from the targeted use of drugs that modulate SRPs to reduce the symptoms.

WS1.2 Vardenafil promotes relocalization of F508del-CFTR in human and mouse airways

Introduction Vardenafil, a clinically approved cGMP-dependent phosphodiesterase type 5 inhibitor (PDE5i), normalizes defective F508del-CFTR chloride transport across the nasal mucosa of mice carrying the F508del mutation (CF). This work aimed at evaluating the influence of vardenafil on CFTR expression and localization in mouse lungs and in human bronchial epithelial cells in culture. Methods CFTR localization was studied by immunohistostaining and evaluated by a normalized ratio of specific fluorescence (CFTR antibody clone 24-1) between the apical and the subapical compartments of cells. Incubation of slides with Pontamine Sky Blue was performed 10 minutes before immunostaining to overcome lung tissue autofluorescence. Results Immunohistostaining studies showed reduced CFTR expression in apical compartments of CF airways: in untreated conditions, the apical/subapical fluorescence ratio was lower in CF (1.48 +/- 0.09) than in WT tissues (2.24 +/- 0.12; p < 0.0001). In CF tissues, vardenafil treatment increased both the ratio of fluorescence and the peak intensity of the CFTR signal to WT values. Conclusion These data show that vardenafil acts as a CFTR corrector by promoting CFTR relocalization of the protein towards and into the apical compartment where the wild-type protein is mainly expressed. This work provides compelling support for studying cGMP pathway as therapeutic strategies in CF pharmacotherapy.

Myofibroblast Differentiation and Enhanced Tgf-B Signaling in Cystic Fibrosis Lung Disease

RationaleTGF-β, a mediator of pulmonary fibrosis, is a genetic modifier of CF respiratory deterioration. The mechanistic relationship between TGF-β signaling and CF lung disease has not been determined.ObjectiveTo investigate myofibroblast differentiation in CF lung tissue as a novel pathway by which TGF-β signaling may contribute to pulmonary decline, airway remodeling and tissue fibrosis.MethodsLung samples from CF and non-CF subjects were analyzed morphometrically for total TGF-β1, TGF-β signaling (Smad2 phosphorylation), myofibroblast differentiation (α-smooth muscle actin), and collagen deposition (Masson trichrome stain).ResultsTGF-β signaling and fibrosis are markedly increased in CF (p<0.01), and the presence of myofibroblasts is four-fold higher in CF vs. normal lung tissue (p<0.005). In lung tissue with prominent TGF-β signaling, both myofibroblast differentiation and tissue fibrosis are significantly augmented (p<0.005).ConclusionsThese studies establish for the first time that a pathogenic mechanism described previously in pulmonary fibrosis is also prominent in cystic fibrosis lung disease. The presence of TGF-β dependent signaling in areas of prominent myofibroblast proliferation and fibrosis in CF suggests that strategies under development for other pro-fibrotic lung conditions may also be evaluated for use in CF.

COMPARATIVE GROWTH ANALYSIS OF TISSUE CULTURED CLONES OF CORSICAN PINE WITH NATURAL SEEDLINGS

The maturity or the physiological state of the mother plant in which the explants were collectedhas a greater effect on the growth of in vitro plants. Younger explants respond better in theculture medium. The physiological state of the mother plant has a direct effect on the growth invitro. Seasonal changes, also has a greater effect on the growth. Morphological andphysiological disorders in the growth of the tissue cultured plants also reported by manyresearchers. Therefore, this experiment was designed in order to determine: Any deviation of thegrowth comparing to the growth of the natural seedliugsThe effect of the nature Istatus of the mother plant of which the explants were collected on thegrowth of the plants produced in vitro.Five clones of Corsican pine (Clone 1 - originated from embryo culture; Clone 2 - Originatedfrom meristems of 21-day -old seedlings; Clone 3 - Originated from axillary buds of 5-year-oldplants Clone 4 - Originated from mature shoot tips of lO-year-old plants: Clone 5 - Originatedfrom resting buds from 5-year-old plants) wen: produced by tissue culture. They wereacclimatized and were established in the field for a period of one year. A set of natural seedlingswas also cultivated in the same field in order to compare the growth of in vitro clones with thenatural seedlings.Natural seedlings were from a same seed lot and the initial height of all the plants were aroundI5cm at the time of planting in the nursery. They were maintained over a period of one year andthe height of plants (em), mean shoot diameter at the soil level (cm) and number of branches pershoot was observed. Plants were fertilized regularly. There were 20 plants in each clone. Theresults were analysed using ANOVA and the pairwise comparisons were done with Turkey's testin Minitab Statistical packageThere was no significant difference between clones 1-4 and natural seedlings on height; but theheight of the clone 5 - which the original explants from resting buds, was significantly lowerthan all other plants. There was a slight (non-sign'ficant) differences within height betweenclones 1 and 4-in which the original material from ru.ture (10 years-old) plants. The diameter atthe soil level also shows the similar pattern of growth. Branching patterns were almost the samein all four clones and also in plants from natural seedlingsIt can be suggested that there is no significant difference in the growth pattern of the tissueculturedclones when comparing them with natural »lants. There is an effect ofthe physiologicalstate (dormancy) of the mother plant on the growth cf tissue cultured plants in the field but theage of the mother plant does not show any effect on the growth of the in vitro plants in the field.Therefore, the use of in vitro techniques for mass clonal propagation of Corsican pine forcommercial purposes is recommended.

Status of Fluid and Electrolyte Absorption in Cystic Fibrosis

Salt and fluid absorption is a shared function of many of the body's epithelia, but its use is highly adapted to the varied physiological roles of epithelia-lined organs. These functions vary from control of hydration of outward-facing epithelial surfaces to conservation and regulation of total body volume. In the most general context, salt and fluid absorption is driven by active Na(+) absorption. Cl(-) is absorbed passively through various available paths in response to the electrical driving force that results from active Na(+) absorption. Absorption of salt creates a concentration gradient that causes water to be absorbed passively, provided the epithelium is water permeable. Key differences notwithstanding, the transport elements used for salt and fluid absorption are broadly similar in diverse epithelia, but the regulation of these elements enables salt absorption to be tailored to very different physiological needs. Here we focus on salt absorption by exocrine glands and airway epithelia. In cystic fibrosis, salt and fluid absorption by gland duct epithelia is effectively prevented by the loss of cystic fibrosis transmembrane conductance regulator (CFTR). In airway epithelia, salt and fluid absorption persists, in the absence of CFTR-mediated Cl(-) secretion. The contrast of these tissue-specific changes in CF tissues is illustrative of how salt and fluid absorption is differentially regulated to accomplish tissue-specific physiological objectives.

Lubiprostone targets prostanoid EP4 receptors in ovine airways

Lubiprostone, a prostaglandin E₁ derivative, is reported to activate ClC-2 chloride channels located in the apical membranes of a number of transporting epithelia. Lack of functioning CFTR chloride channels in epithelia is responsible for the genetic disease cystic fibrosis, therefore, surrogate channels that can operate independently of CFTR are of interest. This study explores the target receptor(s) for lubiprostone in airway epithelium. All experiments were performed on the ventral tracheal epithelium of sheep. Epithelia were used to measure anion secretion from the apical surface as short circuit current or as fluid secretion from individual airway submucosal glands, using an optical method. The EP₄ antagonists L-161982 and GW627368 inhibited short circuit current responses to lubiprostone, while EP₁(,)₂(&)₃ receptor antagonists were without effect. Similarly, lubiprostone induced secretion in airway submucosal glands was inhibited by L-161982. L-161982 effectively competed with lubiprostone with a K(d) value of 0.058 µM, close to its value for binding to human EP₄ receptors (0.024 µM). The selective EP₄ agonist L-902688 and lubiprostone behaved similarly with respect to EP₄ receptor antagonists. Results of experiments with H89, a protein kinase A inhibitor, were consistent with lubiprostone acting through a G(s) -protein coupled EP₄ receptor/cAMP cascade. Lubiprostone-induced short-circuit currents and submucosal gland secretions were inhibited by selective EP₄ receptor antagonists. The results suggest EP₄ receptor activation by lubiprostone triggers cAMP production necessary for CFTR activation and the secretory responses, a possibility precluded in CF tissues.

Tissue Transglutaminase Activation Modulates Inflammation in Cystic Fibrosis via PPARγ Down-Regulation

Cystic fibrosis (CF), the most common life-threatening inherited disease in Caucasians, is due to mutations in the CF transmembrane conductance regulator (CFTR) gene and is characterized by airways chronic inflammation and pulmonary infections. The inflammatory response is not secondary to the pulmonary infections. Indeed, several studies have shown an increased proinflammatory activity in the CF tissues, regardless of bacterial infections, because inflammation is similarly observed in CFTR-defective cell lines kept in sterile conditions. Despite recent studies that have indicated that CF airway epithelial cells can spontaneously initiate the inflammatory cascade, we still do not have a clear insight of the molecular mechanisms involved in this increased inflammatory response. In this study, to understand these mechanisms, we investigated ex vivo cultures of nasal polyp mucosal explants of CF patients and controls, CFTR-defective IB3-1 bronchial epithelial cells, C38 isogenic CFTR corrected, and 16HBE normal bronchial epithelial cell lines. We have shown that a defective CFTR induces a remarkable up-regulation of tissue transglutaminase (TG2) in both tissues and cell lines. The increased TG2 activity leads to functional sequestration of the anti-inflammatory peroxisome proliferator-activated receptor gamma and increase of the classic parameters of inflammation, such as TNF-alpha, tyrosine phosphorylation, and MAPKs. Specific inhibition of TG2 was able to reinstate normal levels of peroxisome proliferator-activated receptor-gamma and dampen down inflammation both in CF tissues and CFTR-defective cells. Our results highlight an unpredicted central role of TG2 in the mechanistic pathway of CF inflammation, also opening a possible new wave of therapies for sufferers of chronic inflammatory diseases.

Responses of well-differentiated airway epithelial cell cultures from healthy donors and patients with cystic fibrosis to Burkholderia cenocepacia infection.

Well-differentiated cultures established from airway epithelia of patients with cystic fibrosis (CF cultures) exhibited goblet cell hyperplasia, increased secretion of mucus, and higher basal levels of interleukin-8 than similarly cultured cells from healthy donors. Upon apical infection with low doses (10(4) to 10(5) CFU) of Burkholderia cenocepacia isolate BC7, the two cultures gave different responses. While normal cultures trapped the added bacteria in the mucus layer, killed and/or inhibited bacterial replication, and prevented bacterial invasion of the cells, CF cultures failed to kill and/or supported the growth of bacteria, leading to invasion of underlying epithelial cells, compromised transepithelial permeability, and cell damage. Depletion of the surface mucus layer prior to bacterial infection rendered the normal cultures susceptible to bacterial invasion, but the invading bacteria were mainly confined to vacuoles within the cells and appeared to be nonviable. In contrast, bacteria that invaded cells in CF cultures were found free in the cytoplasm surrounded by intermediate filaments and also between cells. Cultured CF airway epithelium was therefore more susceptible to infection than normal epithelium. This mimics CF tissue in vivo and illustrates differences in the way epithelia in CF patients and normal subjects handle bacterial infection. In addition, we found that the CF and normal cell cultures responded differently not only to isolate BC7 but also to isolates of other B. cepacia complex species. We therefore conclude that this cell culture model is suitable for investigation of B. cepacia complex pathogenesis in CF patients.

Effect of genistein on native epithelial tissue from normal individuals and CF patients and on ion channels expressed in Xenopus oocytes.

The flavonoid genistein has been shown to activate a Cl(-) conductance in various cell types expressing CFTR. We examined if similar effects can be observed when genistein is applied to native ex vivo tissues from human respiratory tract and rectum. We further compared the effects when genistein was applied to oocytes of Xenopus laevis expressing CFTR. In oocytes, both wtCFTR and DeltaF508-CFTR were activated by genistein while both cyclic AMP (K(v)LQT1) and Ca(2+) (SK4) activated K(+) channels were inhibited at high concentrations of genistein. Biopsies from nasal polyps and rectal mucosa were obtained from normal individuals (non-CF) and CF patients and in the presence of amiloride (10 micromol l(-1); mucosal side) the effects of genistein were assessed using a perfused Ussing chamber. In non-CF airway epithelia, genistein (50 micromol l(-1); mucosal side) increased lumen negative I(sc) but had no additional effects on tissues pre-stimulated with IBMX and forskolin (100 micromol l(-1) and 1 micromol l(-1); both sides). In non-CF rectal biopsies, in the presence of amiloride (10 micromol l(-1); mucosal side) and indomethacin (10 micromol l(-1); basolateral side), genistein increased lumen negative I(sc) and enabled cholinergic (carbachol; CCH, 100 micromol l(-1); basolateral side) stimulation of Cl(-) secretion indicating activation of luminal CFTR Cl(-) channels. However, after stimulation with IBMX/forskolin, genistein induced opposite effects and significantly inhibited CCH activated I(sc). In CF airway and intestinal tissues genistein failed to induce Cl(-) secretion. Thus, genistein is able to activate luminal CFTR Cl(-) conductance in non-CF tissues and mutant CFTR in oocytes. However, additional inhibitory effects on basolateral K(+) conductance and missing effects in native CF tissues do not support the use for pharmacological intervention in CF.

&lt;b&gt;CATION CHANNELS IN NORMAL AND CYSTIC EIBROSIS HUMAN AIRWAY EPITHELIAL CELLS &lt;/b&gt;

The single channel patch clamp technique was used to characterize cation channels in the apical membranes of both normal and cystic fibrosis human nasal airway epithelial cells maintained in primary culture. Single channels were studied in the cell-attached and the excised inside-out configurations. Channels were characterized as cation or anion permeable using ion substitutions. Two groups of cation channels were found with mean conductances of about 8 and 24 pS, both groups of channels being completely impermeable to choline chloride. Both types of cation channels behaved similarly in normal and cystic fibrosis tissues. However, the number of active 24 pS cation channels in CF tissues was significantly higher than in normal cells

Altered sensitivity to amiloride in cystic fibrosis. Observations using cultured sweat glands.

1. Using cultured epithelia from sweat glands, derived from both cystic fibrosis and normal subjects, the relationship between amiloride concentration and the inhibition of electrogenic sodium transport was measured, under short circuit conditions. 2. The Kd for amiloride in cultures from normal subjects was 0.64 microM (n = 6) while in cultures derived from CF patients the value was 1.07 microM (n = 4). The values were significantly different (P less than 0.02, ANOVA). 3. In cultures from normal sweat glands, bathed in solutions free of permeable anions (chloride/bicarbonate), the Kd for amiloride rose to a value greater than found in CF tissues (2.3 microM). In CF epithelia subject to the same conditions the abnormally high value increased further, so that in solutions without permeable anions normal and CF cultures behaved similarly. 4. In cultures derived from normal and CF tissues lowering the sodium concentration to 10 mM also lowered the Kd for amiloride, however the shift was greater for CF cultures. 5. Several possible explanations for the results are discussed. The most probable is that the relatively more positive apical membrane potential in CF epithelia opposes the interaction of amiloride with the sodium channel, implying that complex formation is potential sensitive.

A novel method for culturing sweat gland epithelia: comparison of normal and cystic fibrosis tissues.

A new method is described to produce epithelial sheets by direct explantation of human sweat glands onto matrigel-coated millipore filters. The method is applicable to whole glands, separated coils or ducts and to normal and CF tissues. Electrogenic transport studies show that epithelia develop sodium transporting capability, even when explants are derived from secretory coils.