A model for stimulation of enzyme activity by a competitive inhibitor based on the interaction of terazosin and phosphoglycerate kinase 1.

The drug terazosin (TZ) binds to and can enhance the activity of the glycolytic enzyme phosphoglycerate kinase 1 (PGK1) and can increase ATP levels. That finding prompted studies of TZ in Parkinson's disease (PD) in which decreased neuronal energy metabolism is a hallmark feature. TZ was neuroprotective in cell-based and animal PD models and in large epidemiological studies of humans. However, how TZ might increase PGK1 activity has remained a perplexing question because structural data revealed that the site of TZ binding to PGK1 overlaps with the site of substrate binding, predicting that TZ would competitively inhibit activity. Functional data also indicate that TZ is a competitive inhibitor. To explore the paradoxical observation of a competitive inhibitor increasing enzyme activity under some conditions, we developed a mass action model of TZ and PGK1 interactions using published data on PGK1 kinetics and the effect of varying TZ concentrations. The model indicated that TZ-binding introduces a bypass pathway that accelerates product release. At low concentrations, TZ binding circumvents slow product release and increases the rate of enzymatic phosphotransfer. However, at high concentrations, TZ inhibits PGK1 activity. The model explains stimulation of enzyme activity by a competitive inhibitor and the biphasic dose-response relationship for TZ and PGK1 activity. By providing a plausible mechanism for interactions between TZ and PGK1, these findings may aid development of TZ or other agents as potential therapeutics for neurodegenerative diseases. The results may also have implications for agents that interact with the active site of other enzymes.

Injectable long-acting ivacaftor-loaded poly (lactide-co-glycolide) microparticle formulations for the treatment of cystic fibrosis: In vitro characterization and in vivo pharmacokinetics in mice.

Optimizing a sustained-release drug delivery system for the treatment of cystic fibrosis (CF) is crucial for decreasing the dosing frequency and improving patients' compliance with the treatment regimen. In the current work, we developed an injectable poly(D,L-lactide-co-glycolide) (PLGA) microparticle formulation loaded with ivacaftor, a cystic fibrosis transmembrane conductance regulator (CFTR) potentiator that increases the open probability of the CFTR anion channel, using a single emulsion solvent evaporation technique. We aimed to study the effect of different parameters on the characteristics of the prepared formulations to select an optimized microparticle formulation to be used in an in vivo pharmacokinetic study in mice. First, a suite of ivacaftor-loaded microparticles were prepared using different formulation parameters in order to study the effect of varying these parameters on microparticle size, morphology, drug loading, encapsulation efficiency, and in vitro release profiles. Prepared microparticles were spherical with diameters ranging from 1.91-6.93 µm, percent drug loading (% DL) of 3.91-10.3%, percent encapsulation efficiencies (% EE) of 26.6-100%, and an overall slow cumulative release profile. We selected the formulation that demonstrated optimal combined % DL and % EE values (8.25 and 90.7%, respectively) for further studies. These microparticles had an average particle size of 6.83 µm and a slow tri-phasic in vitro release profile (up to 6 weeks). In vivo pharmacokinetic studies in mice showed that the subcutaneously injected microparticles resulted in steady plasma levels of ivacaftor over a period of 28 days, and a 6-fold increase in AUC 0 - t (71.6 µg/mL*h) compared to the intravenously injected soluble ivacaftor (12.3 µg/mL*h). Our results suggest that this novel ivacaftor-loaded microparticle formulation could potentially eliminate the need for the frequent daily administration of ivacaftor to people with CF thus improving their compliance and ensuring successful treatment outcomes.

Evaluating the psychometric properties of the Fear of Food Measure in adolescents across three independent samples.

The Fear of Food Measure (FOFM) was developed to assess eating-related anxiety and evaluate outcomes of food exposure treatment. The FOFM scores in adult community and clinical samples have demonstrated good factor structure, reliability, and validity, but the FOFM has yet to be evaluated in adolescents, despite eating disorders (EDs) being extremely prevalent during adolescence. The current research evaluated the psychometric properties of the FOFM in three independent child and adolescent samples ages 11-18: patients at two separate intensive treatment programs for EDs (N = 688, N = 151) and students in an all-girl high school (N = 310). The revised adolescent version of FOFM (FOFM-A) consists of 10 items and three subscales: Anxiety About Eating, Food Anxiety Rules, and Social Eating Anxiety. We also found support for the use of a global FOFM-A score in an adolescent population. The FOFM-A scores evidenced good internal consistency as well as convergent, discriminant, and incremental validity across all samples. FOFM-A subscales strongly correlated with other measures of ED symptoms and moderately to strongly correlated with measures of anxiety and depression. Adolescents diagnosed with EDs scored significantly higher on all subscales of FOFM-A compared to a community high school sample without ED diagnoses. We identified that a total FOFM-A cutoff score of 1.93 best differentiates between those with and without ED diagnoses. The FOFM-A may be useful in the assessment and treatment of eating-related anxiety and avoidance in adolescents. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Composition of gastrointestinal microbiota in association with treatment response in individuals with metastatic castrate resistant prostate cancer progressing on enzalutamide and initiating treatment with anti-PD-1 (pembrolizumab).

Recent studies in cancer patients and animal models demonstrate that intestinal microbiota influence the therapeutic efficacy of cancer treatments, including immune checkpoint inhibition. However, no studies to-date have investigated relationships between gastrointestinal microbiota composition and response to checkpoint inhibition in advanced metastatic castrate resistant prostate cancer (mCRPC). We performed 16S rRNA gene sequencing of fecal DNA from 23 individuals with mCRPC progressing on enzalutamide and just prior to treatment with anti-PD-1 (pembrolizumab) to determine whether certain features of the microbiome are associated with treatment response (defined as serum PSA decrease >50% at any time on treatment or radiographic response per RECIST V.1.1). Global bacterial composition was similar between responders and non-responders, as assessed by multiple alpha and beta diversity metrics. However, certain bacterial taxa identified by sequencing across multiple 16S rRNA hypervariable regions were consistently associated with response, including the archetypal oral bacterium Streptococcus salivarius. Quantitative PCR (qPCR) of DNA extracts from fecal samples confirmed increased Streptococcus salivarius fecal levels in responders, whereas qPCR of oral swish DNA extracts showed no relationship between oral Streptococcus salivarius levels and response status. Contrary to previous reports in other cancer types, Akkermansia muciniphila levels were reduced in responder samples as assessed by both 16S rRNA sequencing and qPCR. We further analyzed our data in the context of a previously published "integrated index" describing bacteria associated with response and non-response to checkpoint inhibition. We found that the index was not reflective of response status in our cohort. Lastly, we demonstrate little change in the microbiome over time, and with pembrolizumab treatment. Our results suggest that the association between fecal microbiota and treatment response to immunotherapy may be unique to cancer type and/or previous treatment history.

Incorporation of Data From Multiple Hypervariable Regions when Analyzing Bacterial 16S rRNA Gene Sequencing Data.

Short read 16 S rRNA amplicon sequencing is a common technique used in microbiome research. However, inaccuracies in estimated bacterial community composition can occur due to amplification bias of the targeted hypervariable region. A potential solution is to sequence and assess multiple hypervariable regions in tandem, yet there is currently no consensus as to the appropriate method for analyzing this data. Additionally, there are many sequence analysis resources for data produced from the Illumina platform, but fewer open-source options available for data from the Ion Torrent platform. Herein, we present an analysis pipeline using open-source analysis platforms that integrates data from multiple hypervariable regions and is compatible with data produced from the Ion Torrent platform. We used the ThermoFisher Ion 16 S Metagenomics Kit and a mock community of twenty bacterial strains to assess taxonomic classification of six amplicons from separate hypervariable regions (V2, V3, V4, V6-7, V8, V9) using our analysis pipeline. We report that different amplicons have different specificities for taxonomic classification, which also has implications for global level analyses such as alpha and beta diversity. Finally, we utilize a generalized linear modeling approach to statistically integrate the results from multiple hypervariable regions and apply this methodology to data from a representative clinical cohort. We conclude that examining sequencing results across multiple hypervariable regions provides more taxonomic information than sequencing across a single region. The data across multiple hypervariable regions can be combined using generalized linear models to enhance the statistical evaluation of overall differences in community structure and relatedness among sample groups.

A pilot to assess target engagement of terazosin in Parkinson's disease.

BACKGROUND: Impaired brain energy metabolism is a key feature of Parkinson's disease (PD). Terazosin (TZ) binds phosphoglycerate kinase 1 and stimulates its activity, which enhances glycolysis and increases ATP levels. Preclinical and epidemiologic data suggest that TZ may be neuroprotective in PD. We aimed to assess target engagement and safety of TZ in people with PD. METHODS: We performed a 12-week pilot study in people with PD. Participants were randomized to receive 5 mg TZ or placebo. Participants and study personnel were blinded. We assessed TZ target engagement by measuring brain ATP with 31P-magnetic resonance spectroscopy (MRS) and whole blood ATP with a luminescence assay. Robust linear regression models compared changes between groups controlling for baseline brain and blood ATP levels, respectively. We also assessed clinical measures of PD and adverse events. RESULTS: Thirteen participants were randomized. Mild dizziness/lightheadedness was more common in the TZ group, and three participants taking TZ dropped out because of dizziness and/or orthostatic hypotension. Compared to the placebo group, the TZ group had a significant increase in the ratio of ßATP to inorganic phosphate in the brain. The TZ group also had a significant increase in blood ATP levels compared to the placebo group (p < 0.01). CONCLUSIONS: This pilot study suggests that TZ may engage its target and change ATP levels in the brain and blood of people with PD. Further studies may be warranted to test the disease-modifying potential of TZ.

Selenium and the 15kDa Selenoprotein Impact Colorectal Tumorigenesis by Modulating Intestinal Barrier Integrity.

Selenoproteins play important roles in many cellular functions and biochemical pathways in mammals. Our previous study showed that the deficiency of the 15 kDa selenoprotein (Selenof) significantly reduced the formation of aberrant crypt foci (ACF) in a mouse model of azoxymethane (AOM)-induced colon carcinogenesis. The objective of this study was to examine the effects of Selenof on inflammatory tumorigenesis, and whether dietary selenium modified these effects. For 20 weeks post-weaning, Selenof-knockout (KO) mice and littermate controls were fed diets that were either deficient, adequate or high in sodium selenite. Colon tumors were induced with AOM and dextran sulfate sodium. Surprisingly, KO mice had drastically fewer ACF but developed a similar number of tumors as their littermate controls. Expression of genes important in inflammatory colorectal cancer and those relevant to epithelial barrier function was assessed, in addition to structural differences via tissue histology. Our findings point to Selenof's potential role in intestinal barrier integrity and structural changes in glandular and mucin-producing goblet cells in the mucosa and submucosa, which may determine the type of tumor developing.

Oncogenic gene fusions in nonneoplastic precursors as evidence that bacterial infection can initiate prostate cancer.

Prostate adenocarcinoma is the second most commonly diagnosed cancer in men worldwide, and the initiating factors are unknown. Oncogenic TMPRSS2:ERG (ERG+) gene fusions are facilitated by DNA breaks and occur in up to 50% of prostate cancers. Infection-driven inflammation is implicated in the formation of ERG+ fusions, and we hypothesized that these fusions initiate in early inflammation-associated prostate cancer precursor lesions, such as proliferative inflammatory atrophy (PIA), prior to cancer development. We investigated whether bacterial prostatitis is associated with ERG+ precancerous lesions in unique cases with active bacterial infections at the time of radical prostatectomy. We identified a high frequency of ERG+ non-neoplastic-appearing glands in these cases, including ERG+ PIA transitioning to early invasive cancer. These lesions were positive for ERG protein by immunohistochemistry and ERG messenger RNA by in situ hybridization. We additionally verified TMPRSS2:ERG genomic rearrangements in precursor lesions using tricolor fluorescence in situ hybridization. Identification of rearrangement patterns combined with whole-prostate mapping in three dimensions confirmed multiple (up to eight) distinct ERG+ precancerous lesions in infected cases. We further identified the pathogen-derived genotoxin colibactin as a potential source of DNA breaks in clinical cases as well as cultured prostate cells. Overall, we provide evidence that bacterial infections can initiate driver gene alterations in prostate cancer. In addition, our observations indicate that infection-induced ERG+ fusions are an early alteration in the carcinogenic process and that PIA may serve as a direct precursor to prostate cancer.

GSTP1 positive prostatic adenocarcinomas are more common in Black than White men in the United States.

GSTP1 is a member of the Glutathione-S-transferase (GST) family silenced by CpG island DNA hypermethylation in 90-95% of prostate cancers. However, prostate cancers expressing GSTP1 have not been well characterized. We used immunohistochemistry against GSTP1 to examine 1673 primary prostatic adenocarcinomas on tissue microarrays (TMAs) with redundant sampling from the index tumor from prostatectomies. GSTP1 protein was positive in at least one TMA core in 7.7% of cases and in all TMA cores in 4.4% of cases. The percentage of adenocarcinomas from Black patients who had any GSTP1 positive TMA cores was 14.9%, which was 2.5 times higher than the percentage from White patients (5.9%; P < 0.001). Further, the percentages of tumors from Black patients who had all TMA spots positive for GSTP1 (9.5%) was 3-fold higher than the percentage from White patients (3.2%; P<0.001). In terms of association with other molecular alterations, GSTP1 positivity was enriched in ERG positive cancers among Black men. By in situ hybridization, GSTP1 mRNA expression was concordant with protein staining, supporting the lack of silencing of at least some GSTP1 alleles in GSTP1-positive tumor cells. This is the first report revealing that GSTP1-positive prostate cancers are substantially over-represented among prostate cancers from Black compared to White men. This observation should prompt additional studies to determine whether GSTP1 positive cases represent a distinct molecular subtype of prostate cancer and whether GSTP1 expression could provide a biological underpinning for the observed disparate outcomes for Black men.

A network investigation of core symptoms and pathways across duration of illness using a comprehensive cognitive-behavioral model of eating-disorder symptoms.

BACKGROUND: In the past decade, network analysis (NA) has been applied to psychopathology to quantify complex symptom relationships. This statistical technique has demonstrated much promise, as it provides researchers the ability to identify relationships across many symptoms in one model and can identify central symptoms that may predict important clinical outcomes. However, network models are highly influenced by node selection, which could limit the generalizability of findings. The current study (N = 6850) tests a comprehensive, cognitive-behavioral model of eating-disorder symptoms using items from two, widely used measures (Eating Disorder Examination Questionnaire and Eating Pathology Symptoms Inventory). METHODS: We used NA to identify central symptoms and compared networks across the duration of illness (DOI), as chronicity is one of the only known predictors of poor outcome in eating disorders (EDs). RESULTS: Our results suggest that eating when not hungry and feeling fat were the most central symptoms across groups. There were no significant differences in network structure across DOI, meaning the connections between symptoms remained relatively consistent. However, differences emerged in central symptoms, such that cognitive symptoms related to overvaluation of weight/shape were central in individuals with shorter DOI, and behavioral central symptoms emerged more in medium and long DOI. CONCLUSIONS: Our results have important implications for the treatment of individuals with enduring EDs, as they may have a different core, maintaining symptoms. Additionally, our findings highlight the importance of using comprehensive, theoretically- or empirically-derived models for NA.

Distinct Genomic Alterations in Prostate Tumors Derived from African American Men.

We aim to understand, from acquired genetic alterations in tumors, why African American (AA) men are more likely to develop aggressive prostate cancer. By analyzing somatic mutations in 39 genes using deeper next-generation sequencing with an average depth of 2,522 reads for tumor DNA and genome-wide DNA copy-number alterations (CNA) in prostate cancer in a total of 171 AA/black men and comparing with those in 860 European American (EA)/white men, we here present several novel findings. First, >35% of AA men harbor damaging mutations in APC, ATM, BRCA2, KDM6A, KMT2C, KMT2D, MED12, ZFHX3, and ZMYM3, each with >1% of mutated copies. Second, among genes with >10% of mutated copies in tumor cells, ZMYM3 is the most frequently mutated gene in AA prostate cancer. In a patient's tumor with >96% frameshift mutations of ZMYM3, we find allelic imbalances in 10 chromosomes, including losses of five and gains of another four chromosomes, suggesting its role in maintaining genomic integrity. Third, when compared to prostate cancer in EA/white men, a higher frequency of CNAs of MYC, THADA, NEIL3, LRP1B, BUB1B, MAP3K7, BNIP3L and RB1, and a lower frequency of deletions of RYBP, TP53, and TMPRSS2-ERG are observed in AA/black men. Finally, for the above genes with higher frequency of CNAs in AA than in EA, deletion of MAP3K7, BNIP3L, NEIL3 or RB1, or gain of MYC significantly associates with both higher Gleason grade and advanced pathologic stage in AA/black men. Deletion of THADA associates with advanced pathologic stage only. IMPLICATIONS: A higher frequency of damaging mutation in ZMYM3 causing genomic instability along with higher frequency of altered genomic regions including deletions of MAP3K7, BNIP3L, RB1, and NEIL3, and gain of MYC appear to be distinct somatically acquired genetic alterations that may contribute to more aggressive prostate cancer in AA/black men.

Early pathogenesis of cystic fibrosis gallbladder disease in a porcine model.

Hepatobiliary disease causes significant morbidity in people with cystic fibrosis (CF), yet this problem remains understudied. We previously found that newborn CF pigs have microgallbladders with significant luminal obstruction in the absence of infection and consistent inflammation. In this study, we sought to better understand the early pathogenesis of CF pig gallbladder disease. We hypothesized that loss of CFTR would impair gallbladder epithelium anion/liquid secretion and increase mucin production. CFTR was expressed apically in non-CF pig gallbladder epithelium but was absent in CF. CF pig gallbladders lacked cAMP-stimulated anion transport. Using a novel gallbladder epithelial organoid model, we found that Cl- or HCO3- was sufficient for non-CF organoid swelling. This response was absent for non-CF organoids in Cl-/HCO3--free conditions and in CF. Single-cell RNA-sequencing revealed a single epithelial cell type in non-CF gallbladders that coexpressed CFTR, MUC5AC, and MUC5B. Despite CF gallbladders having increased luminal MUC5AC and MUC5B accumulation, there was no significant difference in the epithelial expression of gel-forming mucins between non-CF and CF pig gallbladders. In conclusion, these data suggest that loss of CFTR-mediated anion transport and fluid secretion contribute to microgallbladder development and luminal mucus accumulation in CF.

Avoidance coping during mealtimes predicts higher eating disorder symptoms.

OBJECTIVE: Eating disorders (EDs) are characterized by significant anxiety during mealtime that contributes to food avoidance and weight loss. Individuals with EDs commonly use avoidance coping (e.g., distraction) to tolerate meals and comply with meal plans. Although this strategy may be effective short term, a large body of anxiety literature suggests that avoidance can lead to worsening of psychological symptoms long term. METHOD: The current study (N = 66 individuals diagnosed with ED) used ecological momentary assessment (EMA) to examine the short-term and long-term associations of avoidance coping on ED symptoms. RESULTS: Distraction during meals predicted a reduction in anxiety in the short term, and both distraction and avoidance of emotions predicted increases in excessive exercise in the short term. Distraction and avoidance of emotions predicted increases in bulimic symptoms 1 month after completion of EMA. DISCUSSION: These results are consistent with prior literature on avoidance and suggest that avoidance coping during meals may contribute to the increase of ED behaviors in the long term. Coping strategies that encourage approach and tolerance of difficult thoughts and emotions (e.g., acceptance-based strategies) rather that avoidance coping may promote longer-term symptom reduction.

Inflammation-associated pathologies in a case of prostate schistosomiasis: Implications for a causal role in prostate carcinogenesis.

BACKGROUND: Urogenital infection with Schistosoma haematobium is a risk factor for the development of squamous cell carcinoma of the urinary bladder. The pathophysiology is thought to be mediated in part by inflammation, cellular damage, and bladder regeneration induced by the parasitic infection. Herein, we report an unusual case of schistosomiasis of the prostate that was found concurrent with prostate adenocarcinoma in a radical prostatectomy specimen from a man in the United States. METHODS: The infecting Schistosoma species was characterized via histomorphology and acid-fast stain. The concurrent Gleason score 6 prostate cancer was assessed for ETS transcription factor ERG (ERG), phosphatase and tensin homolog (PTEN), p27, and p53 status using immunohistochemistry (IHC). Cellular proliferation and the presence of intermediate cells in prostatic atrophy were assessed via immunostaining for Ki67 and CK903, respectively. RESULTS: Histomorphology and acid-fast stain of the infecting species were consistent with S. haematobium. We classified the Gleason score 6 prostate adenocarcinoma via IHC as ERG positive, PTEN intact, p27 intact, and without p53 nuclear accumulation. The prostatic epithelium immediately adjacent to the schistosomiasis-related granulomatous inflammation was atrophic and accompanied by increased cellular proliferation and the presence of intermediate cells. Upon literature review, we determined that prostate schistosomiasis is associated with a young age of prostate cancer diagnosis and highly aggressive prostate cancer. CONCLUSIONS: This is a rare case of prostate schistosomiasis in the United States; however, prostate schistosomiasis occurs frequently in endemic areas. The patient had traveled to a Schistosoma-endemic region, which was the likely location of exposure to the parasite. To our knowledge, this is the first report of the association of proliferative inflammatory atrophy and intermediate cells with schistosomiasis of the prostate. We propose that prostate schistosomiasis may be considered as a risk factor for the development of prostate cancer in geographic regions where Schistosoma species are endemic.

Compositional differences in gastrointestinal microbiota in prostate cancer patients treated with androgen axis-targeted therapies.

BACKGROUND: It is well known that the gastrointestinal (GI) microbiota can influence the metabolism, pharmacokinetics, and toxicity of cancer therapies. Conversely, the effect of cancer treatments on the composition of the GI microbiota is poorly understood. We hypothesized that oral androgen receptor axis-targeted therapies (ATT), including bicalutamide, enzalutamide, and abiraterone acetate, may be associated with compositional differences in the GI microbiota. METHODS: We profiled the fecal microbiota in a cross-sectional study of 30 patients that included healthy male volunteers and men with different clinical states of prostate cancer (i.e., localized, biochemically recurrent, and metastatic disease) using 16S rDNA amplicon sequencing. Functional inference of identified taxa was performed using PICRUSt. RESULTS: We report a significant difference in alpha diversity in GI microbiota among men with versus without a prostate cancer diagnosis. Further analysis identified significant compositional differences in the GI microbiota of men taking ATT, including a greater abundance of species previously linked to response to anti-PD-1 immunotherapy such as Akkermansia muciniphila and Ruminococcaceae spp. In functional analyses, we found an enriched representation of bacterial gene pathways involved in steroid biosynthesis and steroid hormone biosynthesis in the fecal microbiota of men taking oral ATT. CONCLUSIONS: There are measurable differences in the GI microbiota of men receiving oral ATT. We speculate that oral hormonal therapies for prostate cancer may alter the GI microbiota, influence clinical responses to ATT, and/or potentially modulate the antitumor effects of future therapies including immunotherapy. Given our findings, larger, longitudinal studies are warranted.

Identifying New Small Proteins in Escherichia coli.

The number of small proteins (SPs) encoded in the Escherichia coli genome is unknown, as current bioinformatics and biochemical techniques make short gene and small protein identification challenging. One method of small protein identification involves adding an epitope tag to the 3' end of a short open reading frame (sORF) on the chromosome, with synthesis confirmed by immunoblot assays. In this study, this strategy was used to identify new E. coli small proteins, tagging 80 sORFs in the E. coli genome, and assayed for protein synthesis. The selected sORFs represent diverse sequence characteristics, including degrees of sORF conservation, predicted transmembrane domains, sORF direction with respect to flanking genes, ribosome binding site (RBS) prediction, and ribosome profiling results. Of 80 sORFs, 36 resulted in encoded synthesized proteins-a 45% success rate. Modeling of detected versus non-detected small proteins analysis showed predictions based on RBS prediction, transcription data, and ribosome profiling had statistically-significant correlation with protein synthesis; however, there was no correlation between current sORF annotation and protein synthesis. These results suggest substantial numbers of small proteins remain undiscovered in E. coli, and existing bioinformatics techniques must continue to improve to facilitate identification.

Lack of cystic fibrosis transmembrane conductance regulator disrupts fetal airway development in pigs.

Loss of cystic fibrosis transmembrane conductance regulator (CFTR) function causes cystic fibrosis (CF), predisposing the lungs to chronic infection and inflammation. In young infants with CF, structural airway defects are increasingly recognized before the onset of significant lung disease, which suggests a developmental origin and a possible role in lung disease pathogenesis. The role(s) of CFTR in lung development is unclear and developmental studies in humans with CF are not feasible. Young CF pigs have structural airway changes and develop spontaneous postnatal lung disease similar to humans; therefore, we studied lung development in the pig model (non-CF and CF). CF trachea and proximal airways had structural lesions detectable as early as pseudoglandular development. At this early developmental stage, budding CF airways had smaller, hypo-distended lumens compared to non-CF airways. Non-CF lung explants exhibited airway lumen distension in response to forskolin/IBMX as well as to fibroblast growth factor (FGF)-10, consistent with CFTR-dependent anion transport/secretion, but this was lacking in CF airways. We studied primary pig airway epithelial cell cultures and found that FGF10 increased cellular proliferation (non-CF and CF) and CFTR expression/function (in non-CF only). In pseudoglandular stage lung tissue, CFTR protein was exclusively localized to the leading edges of budding airways in non-CF (but not CF) lungs. This discreet microanatomic localization of CFTR is consistent with the site, during branching morphogenesis, where airway epithelia are responsive to FGF10 regulation. In summary, our results suggest that the CF proximal airway defects originate during branching morphogenesis and that the lack of CFTR-dependent anion transport/liquid secretion likely contributes to these hypo-distended airways.

Motile cilia of human airway epithelia contain hedgehog signaling components that mediate noncanonical hedgehog signaling.

Differentiated airway epithelia produce sonic hedgehog (SHH), which is found in the thin layer of liquid covering the airway surface. Although previous studies showed that vertebrate HH signaling requires primary cilia, as airway epithelia mature, the cells lose primary cilia and produce hundreds of motile cilia. Thus, whether airway epithelia have apical receptors for SHH has remained unknown. We discovered that motile cilia on airway epithelial cells have HH signaling proteins, including patched and smoothened. These cilia also have proteins affecting cAMP-dependent signaling, including Gαi and adenylyl cyclase 5/6. Apical SHH decreases intracellular levels of cAMP, which reduces ciliary beat frequency and pH in airway surface liquid. These results suggest that apical SHH may mediate noncanonical HH signaling through motile cilia to dampen respiratory defenses at the contact point between the environment and the lung, perhaps counterbalancing processes that stimulate airway defenses.

Relationships among CFTR expression, HCO3- secretion, and host defense may inform gene- and cell-based cystic fibrosis therapies.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. Airway disease is the major source of morbidity and mortality. Successful implementation of gene- and cell-based therapies for CF airway disease requires knowledge of relationships among percentages of targeted cells, levels of CFTR expression, correction of electrolyte transport, and rescue of host defense defects. Previous studies suggested that, when ∼10-50% of airway epithelial cells expressed CFTR, they generated nearly wild-type levels of Cl(-) secretion; overexpressing CFTR offered no advantage compared with endogenous expression levels. However, recent discoveries focused attention on CFTR-mediated HCO3 (-) secretion and airway surface liquid (ASL) pH as critical for host defense and CF pathogenesis. Therefore, we generated porcine airway epithelia with varying ratios of CF and wild-type cells. Epithelia with a 50:50 mix secreted HCO3 (-) at half the rate of wild-type epithelia. Likewise, heterozygous epithelia (CFTR(+/-) or CFTR(+/∆F508)) expressed CFTR and secreted HCO3 (-) at ∼50% of wild-type values. ASL pH, antimicrobial activity, and viscosity showed similar relationships to the amount of CFTR. Overexpressing CFTR increased HCO3 (-) secretion to rates greater than wild type, but ASL pH did not exceed wild-type values. Thus, in contrast to Cl(-) secretion, the amount of CFTR is rate-limiting for HCO3 (-) secretion and for correcting host defense abnormalities. In addition, overexpressing CFTR might produce a greater benefit than expressing CFTR at wild-type levels when targeting small fractions of cells. These findings may also explain the risk of airway disease in CF carriers.

Airway acidification initiates host defense abnormalities in cystic fibrosis mice.

Cystic fibrosis (CF) is caused by mutations in the gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. In humans and pigs, the loss of CFTR impairs respiratory host defenses, causing airway infection. But CF mice are spared. We found that in all three species, CFTR secreted bicarbonate into airway surface liquid. In humans and pigs lacking CFTR, unchecked H(+) secretion by the nongastric H(+)/K(+) adenosine triphosphatase (ATP12A) acidified airway surface liquid, which impaired airway host defenses. In contrast, mouse airways expressed little ATP12A and secreted minimal H(+); consequently, airway surface liquid in CF and non-CF mice had similar pH. Inhibiting ATP12A reversed host defense abnormalities in human and pig airways. Conversely, expressing ATP12A in CF mouse airways acidified airway surface liquid, impaired defenses, and increased airway bacteria. These findings help explain why CF mice are protected from infection and nominate ATP12A as a potential therapeutic target for CF.