Reducing Lung Disease in Cystic Fibrosis: Model Systems and Anti-Inflammatory Treatment

Cystic fibrosis (CF), the most common lethal autosomal recessive disease in Caucasians resulting in a median life span of approximately 35 years, is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). The primary cause of morbidity and mortality is respiratory failure caused by progressive obstructive lung disease. CF airway disease is characterized by the abundance of pro inflammatory cells, chemokines and cytokines with a deficiency of regulatory/anti-inflammatory cytokine interleukin 10 (lL10). We hypothesized that CF airway obstruction originates from excessive inflammation and gene transfer of IL-I0 would decrease inflammation in the airways of mice infected with Pseudomonas aeruginosa, a common pathogen in CF. IL-10 gene transfer was accomplished using an adeno-associated viral vector (AAV) selected for the inherent benefits of long-term gene expression, a proven safety profile, and the ability to elicit a minimal inflammatory response in comparison to other gene transfer agents. Using intratracheal injection, we administered AAV5.β-mIL10 to IL-10 deficient mice, chronically infected the mice with P. aeruginosa, and observed high levels of IL-10, decreased lung pathology, and IL-β and IL-8 (MIP-1α and KC in mice), cytokines frequently seen at increased levels in patients with CF. To characterize IL-10 gene transfer in CFTR knockout mice, we modified a noninvasive procedure, aspiration challenge, to include surfactant phospholipids as a vehicle for transfer of P. aeruginosa and AAV-based vectors. This was important because CFTR knockout mice are sensitive to manipulations such as intratracheal injection, resulting in mortality from the surgical procedure. We demonstrated aspiration challenge with surfactant phospholipids was similar to intratracheal injection for P. aeruginosa and AAV delivery, as measured by percentage weight loss, proinflammatory cytokine production, inflammation associated histopathology, and bacterial burden for P. aeruginosa and vector distribution and transgene expression for AAV. Then, we characterized AAV5.Cβ-mIL-10 in CFTR knockout mice chronically infected with P. aeruginosa, observing decreased percentage weight loss, lung pathology, pro-inflammatory cytokines (IL-lβ, IL-6, TNF-α, KC, MIP-1α), histopathology, and neutrophil migration with no change in bacterial burden. These studies support that inflammation is excessive to bacterial infection in CF and demonstrate AAV -based IL-10 gene transfer is a promising anti-inflammatory therapeutic for CF lung disease

Sequence-Specific Gene Correction of Cystic Fibrosis Airway Basal Cells

Cystic fibrosis (CF) is a lethal monogenic disease resulting from mutations in the CFTR gene which encodes a protein involved in regulating anion trans-epithelial transport. A three-base deletion in CFTR (termed as ΔF508 mutation), wherein CFTR protein is misfolded leading to its pre-mature degradation in the endoplasmic reticulum (ER), is the most common cause of this debilitating disease. Since CFTR is expressed in multiple body systems, CF affects different organs, but lung pathology is the greatest cause of death in affected patients. We achieved site-specific gene correction with an efficiency of ~10 % in CF airway basal cells homozygous for the ΔF508 mutation. Basal cells are a multipotent stem cell population of the respiratory epithelium and therefore, their gene correction could provide a long-term, permanent remedy for CF. Delivery of engineered sequence-specific zinc finger nucleases (ZFNs) and single-stranded oligo DNA (ssODN) carrying the correcting sequence via electroporation facilitated the correction. The gene-corrected cells upon in vitro differentiation using air-liquid interface showed presence of fully-glycosylated mature CFTR protein as opposed to differentiated mutant cells which synthesized only the core-glycosylated immature form. Most importantly, we demonstrated CFTR ion channel activity in the gene-corrected cells by Ussing chamber electrophysiology

Survey of CF mutations in the clinical laboratory

BACKGROUND: Since it is impossible to sequence the complete CFTR gene routinely, clinical laboratories must rely on test systems that screen for a panel of the most frequent mutations causing disease in a high percentage of patients. Thus, in a cohort of 257 persons that were referred to our laboratory for analysis of CF gene mutations, reverse line probe assays for the most common CF mutations were performed. These techniques were evaluated as routine first-line analyses of the CFTR gene status. METHODS: DNA from whole blood specimens was extracted and subjected to PCR amplification of 9 exons and 6 introns of the CFTR gene. The resulting amplicons were hybridised to probes for CF mutations and polymorphisms, immobilised on membranes supplied by Roche Molecular Systems, Inc. and Innogenetics, Inc.. Denaturing gradient gel electrophoresis and sequencing of suspicious fragments indicating mutations were done with CF exon and intron specific primers. RESULTS: Of the 257 persons tested over the last three years (referrals based on 1) clinical symptoms typical for/indicative of CF, 2) indication for in vitro fertilisation, and 3) gene status determination because of anticipated parenthood and partners or relatives affected by CF), the reverse line blots detected heterozygote or homozygote mutations in the CFTR gene in 68 persons (26%). Eighty-three percent of those affected were heterozygous (47 persons) or homozygous (10 persons) for the ΔF508 allele. The only other CF-alleles that we found with these tests were the G542X allele (3 persons), the G551D allele (3 persons), the 3849+10kb C-T allele (2 persons) the R117H allele (2 persons) and the 621+1G-T allele (1 person). Of the fifteen IVS8-5T-polymorphisms detected in intron 8, seven (47%) were found in males referred to us from IVF clinics. These seven 5T-alleles were all coupled with a heterozygous ΔF508 allele, they make up 35% of the males with fertility problems (20 men) referred to us. CONCLUSIONS: In summary, the frequency of CF chromosomes in the cohort examined with these tests was 26%, with the ΔF508 allele affecting 83% of the CF chromosomes. It is a substantial improvement for routine CF diagnostics to have available a test system for 30 mutations plus the polypyrimidine length variants in intron 8. Our results show that this test system allows a routine first-line analyses of the CFTR gene status

Chronic cough with normal sweat chloride: Phenotypic descriptions of two rare cystic fibrosis genotypes

AbstractWhile our understanding of cystic fibrosis genetics has expanded in recent decades, the genetics and clinical manifestations of the disease remains highly heterogeneous. Diagnosis of CF in non-classical mutations remains a clinical challenge. We describe the clinical presentation of two patients with chronic cough found to have normal sweat chlorides. We discuss the subsequent evaluation that lead to the diagnosis of two rare CF mutations. We briefly discuss the use of the expanded 106-panel of CF mutations (homozygous 3849 + 10  kb C > T), and the role of whole CFTR gene sequencing (heterozygous c.2752-26 A > G/5T)

Integrating omics reveals that miRNA-guided genetic regulation on plant hormone level and defense response pathways shape resistance to Cladosporium fulvum in the tomato Cf-10-gene-carrying line

Invasion of C. fulvum causes the most serious diseases affecting the reproduction of tomatoes. Cf-10-gene-carrying line showed remarkable resistance to Cladosporium fulvum. To exploit its defense response mechanism, we performed a multiple-omics profiling of Cf-10-gene-carrying line and a susceptible line without carrying any resistance genes at non-inoculation and 3 days post-inoculation (dpi) of C. fulvum. We detected 54 differentially expressed miRNAs (DE-miRNAs) between the non-inoculation and 3 dpi in the Cf-10-gene-carrying line, which potentially regulated plant-pathogen interaction pathways and hormone signaling pathways. We also revealed 3,016 differentially expressed genes (DEGs) between the non-inoculated and 3 dpi in the Cf-10-gene-carrying line whose functions enriched in pathways that were potentially regulated by the DE-miRNAs. Integrating DE-miRNAs, gene expression and plant-hormone metabolites indicated a regulation network where the downregulation of miRNAs at 3 dpi activated crucial resistance genes to trigger host hypersensitive cell death, improved hormone levels and upregulated the receptors/critical responsive transcription factors (TFs) of plant hormones, to shape immunity to the pathogen. Notably, our transcriptome, miRNA and hormone metabolites profiling and qPCR analysis suggested that that the downregulation of miR9472 potentially upregulated the expression of SAR Deficient 1 (SARD1), a key regulator for ICS1 (Isochorismate Synthase 1) induction and salicylic acid (SA) synthesis, to improve the level of SA in the Cf-10-gene-carrying line. Our results exploited potential regulatory network and new pathways underlying the resistance to C. fulvum in Cf-10-gene-carrying line, providing a more comprehensive genetic circuit and valuable gene targets for modulating resistance to the virus

Atypical cystic fibrosis: from the genetic causes to current and future treatments

Cystic Fibrosis (CF) is a life threatening autosomal recessive disorder caused by a mutation in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, leading to irregular secretions and inflammation in tubular organs. Disease manifestations of CF are heterogeneous in severity and can be present in the sinopulmonary, hepatic, gastrointestinal, and genitourinary tract. Since the 1960’s, physicians and scientists have described a less severe form of CF known as atypical CF, usually seen in adults. Patients with atypical CF tend to have one severe CF mutation on one chromosome, and one less common, mild CF mutation on their other chromosome; or have one severe mutation on one chromosome and an abnormal number of trinucleotide repeats in the CFTR gene on their other chromosome. Today, of the approximately 1000 patients diagnosed with CF per year in the United States, roughly 10% are diagnosed with the atypical presentation of the disease as adults. Patients suffering from atypical CF typically have only one organ system that is dysfunctional, and their clinical symptoms may be less severe than those of a classical case where there are two severe CF mutations. Common symptoms include idiopathic bronchiectasis, chronic sinusitis, congenital bilateral absence of the vas deferens (CBAVD), and idiopathic pancreatitis. Unlike patients suffering from the classical presentation of the disease, most are pancreatic sufficient – however the possibility of pancreatic insufficiency still exists. Patients with atypical CF represent a diagnostic challenge for physicians due to the mild, slowly progressing array of clinical symptoms, the general lack of knowledge about atypical CF, and the general association of CF as a childhood disease. Increasing physician awareness of the adult population with CF is a paramount in improving the diagnosis, care and treatment of patients with atypical CF. Missed diagnoses can result in hospital admissions and morbidity that may have been avoidable. The goal of this thesis is to describe the causes of CF, the common symptoms seen in both CF and atypical CF, the proper diagnosis of atypical CF, and to identify the therapies, both current and in development, used to treat atypical CF

Lubiprostone ameliorates the cystic fibrosis mouse intestinal phenotype

<p>Abstract</p> <p>Background</p> <p>Cystic fibrosis (CF) is caused by mutations in the <it>CFTR </it>gene that impair the function of CFTR, a cAMP-regulated anion channel. In the small intestine loss of CFTR function creates a dehydrated, acidic luminal environment which is believed to cause an accumulation of mucus, a phenotype characteristic of CF. CF mice have small intestinal bacterial overgrowth, an altered innate immune response, and impaired intestinal transit. We investigated whether lubiprostone, which can activate the CLC2 Cl^-channel, would improve the intestinal phenotype in CF mice.</p> <p>Methods</p> <p><it>Cftr^tm1UNC</it>(CF) and wildtype (WT) littermate mice on the C57BL/6J background were used. Lubiprostone (10 μg/kg-day) was administered by gavage for two weeks. Mucus accumulation was estimated from crypt lumen widths in periodic acid-Schiff base, Alcian blue stained sections. Luminal bacterial load was measured by qPCR for the bacterial 16<it>S </it>gene. Gastric emptying and small intestinal transit in fasted mice were assessed using gavaged rhodamine dextran. Gene expression was evaluated by Affymetrix Mouse430 2.0 microarray and qRT-PCR.</p> <p>Results</p> <p>Crypt width in control CF mice was 700% that of WT mice (<it>P </it>< 0.001). Lubiprostone did not affect WT crypt width but, unexpectedly, increased CF crypt width 22% (<it>P </it>= 0.001). Lubiprostone increased bacterial load in WT mice to 490% of WT control levels (<it>P </it>= 0.008). Conversely, lubiprostone decreased bacterial overgrowth in CF mice by 60% (<it>P </it>= 0.005). Lubiprostone increased gastric emptying at 20 min postgavage in both WT (<it>P </it>< 0.001) and CF mice (<it>P </it>< 0.001). Lubiprostone enhanced small intestinal transit in WT mice (<it>P </it>= 0.024) but not in CF mice (<it>P </it>= 0.377). Among other innate immune markers, expression of mast cell genes was elevated 4-to 40-fold in the CF intestine as compared to WT, and lubiprostone treatment of CF mice decreased expression to WT control levels.</p> <p>Conclusions</p> <p>These results indicate that lubiprostone has some benefits for the CF intestinal phenotype, especially on bacterial overgrowth and the innate immune response. The unexpected observation of increased mucus accumulation in the crypts of lubiprostone-treated CF mice suggests the possibility that lubiprostone increases mucus secretion.</p

Variation in MSRA Modifies Risk of Neonatal Intestinal Obstruction in Cystic Fibrosis

Meconium ileus (MI), a life-threatening intestinal obstruction due to meconium with abnormal protein content, occurs in approximately 15 percent of neonates with cystic fibrosis (CF). Analysis of twins with CF demonstrates that MI is a highly heritable trait, indicating that genetic modifiers are largely responsible for this complication. Here, we performed regional family-based association analysis of a locus that had previously been linked to MI and found that SNP haplotypes 5′ to and within the MSRA gene were associated with MI (P = 1.99×10−5 to 1.08×10−6; Bonferroni P = 0.057 to 3.1×10−3). The haplotype with the lowest P value showed association with MI in an independent sample of 1,335 unrelated CF patients (OR = 0.72, 95% CI [0.53–0.98], P = 0.04). Intestinal obstruction at the time of weaning was decreased in CF mice with Msra null alleles compared to those with wild-type Msra resulting in significant improvement in survival (P = 1.2×10−4). Similar levels of goblet cell hyperplasia were observed in the ilea of the Cftr−/− and Cftr−/−Msra−/− mice. Modulation of MSRA, an antioxidant shown to preserve the activity of enzymes, may influence proteolysis in the developing intestine of the CF fetus, thereby altering the incidence of obstruction in the newborn period. Identification of MSRA as a modifier of MI provides new insight into the biologic mechanism of neonatal intestinal obstruction caused by loss of CFTR function

Proposal of a quantitative PCR-based protocol for an optimal Pseudomonas aeruginosa detection in patients with cystic fibrosis

BACKGROUND: The lung of patients with cystic fibrosis (CF) is particularly sensitive to Pseudomonas aeruginosa. This bacterium plays an important role in the poor outcome of CF patients. During the disease progress, first acquisition of P. aeruginosa is the key-step in the management of CF patients. Quantitative PCR (qPCR) offers an opportunity to detect earlier the first acquisition of P. aeruginosa by CF patients. Given the lack of a validated protocol, our goal was to find an optimal molecular protocol for detection of P. aeruginosa in CF patients. METHODS: We compared two formerly described qPCR formats in early detection of P. aeruginosa in CF sputum samples: a qPCR targeting oprL gene, and a multiplex PCR targeting gyrB and ecfX genes. RESULTS: Tested in vitro on a large panel of P. aeruginosa isolates and others gram-negative bacilli, oprL qPCR exhibited a better sensitivity (threshold of 10 CFU/mL versus 730 CFU/mL), whereas the gyrB/ecfX qPCR exhibited a better specificity (90% versus 73%). These results were validated ex vivo on 46 CF sputum samples positive for P. aeruginosa in culture. Ex vivo assays revealed that qPCR detected 100 times more bacterial cells than culture-based method did. CONCLUSION: Based on these results, we proposed a reference molecular protocol combining the two qPCRs, which offers a sensitivity of 100% with a threshold of 10 CFU/mL and a specificity of 100%. This combined qPCR-based protocol can be adapted and used for other future prospective studies

Diagnosing cystic fibrosis in South Africa

Cystic fibrosis (CF) occurs in all South Africa’s population groups. While well described in the white and coloured populations, its presence in black African populations is less well known. Recent evidence from the group of CF patients in the Western Cape suggests an incidence of 1 in 3 000 and 1 in 10 300 live births in the white and coloured populations respectively.1 In black South African populations, carrier frequency estimates have been used to project an incidence of 1 in 4 624 live births.2 Further evidence of the presence of CF in these populations is presented in this issue of the Journal. 3 While considering or being aware of the diagnosis is the first step in identifying CF, diagnosing the disease presents challenges at clinical and laboratory levels in South Africa. In simple terms, the diagnosis of CF requires a patient to have suggestive clinical features as well as 2 positive sweat tests and/or 2 identified disease-causing CF transmembrane conductance regulator (CFTR) gene mutations