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Cystic fibrosis (CF) patients and CF mouse models have increased risk for gastrointestinal tumors. CF mice show augmented intestinal proliferation of unknown etiology and an altered intestinal environment. We examined the role of the cystic fibrosis transmembrane conductance regulator (Cftr) in Wnt/β-catenin signaling, stem cell proliferation, and its functional expression in the active intestinal stem cell (ISC) population. Dysregulation of intracellular pH (pHi) in CF ISCs was investigated for facilitation of Wnt/β-catenin signaling. Methods: Crypt epithelia from wild-type (WT) and CF mice were compared ex vivo and in intestinal organoids (enteroids) for proliferation and Wnt/β-catenin signaling by standard assays. Cftr in ISCs was assessed by immunoblot of sorted Sox9enhanced green fluorescent protein(EGFP) intestinal epithelia and pHi regulation by confocal microfluorimetry of leucine-rich G-protein–coupled receptor 5 ISCs. Plasma membrane association of the Wnt transducer Dishevelled 2 (Dvl2) was assessed by fluorescence imaging of live enteroids from WT and CF mice crossed with Dvl2-EGFP/ACTB-tdTomato,-EGFP)Luo/J (RosamT/mG) mice. Results: Relative to WT, CF intestinal crypts showed an ∼30% increase in epithelial and Lgr5+ ISC proliferation and increased Wnt/β-catenin signaling. Cftr was expressed in Sox9EGFPLo ISCs and loss of Cftr induced an alkaline pHi in ISCs. CF crypt-base columnar cells showed a generalized increase in plasma membrane Dvl2-EGFP association as compared with WT. Dvl2-EGFP membrane association was charge- and pH-dependent and increased in WT crypt-base columnar cells by Cftr inhibition. Conclusions: CF intestine shows increased ISC proliferation and Wnt/β-catenin signaling. Loss of Cftr increases pHi in ISCs, which stabilizes the plasma membrane association of the Wnt transducer Dvl, likely facilitating Wnt/β-catenin signaling. Absence of Cftr-dependent suppression of ISC proliferation in the CF intestine may contribute to increased risk for intestinal tumors.

Increased intestinal permeability is a manifestation of cystic fibrosis (CF) in people with CF (pwCF) and in CF mouse models. CF transmembrane conductance regulator knockout (Cftr KO) mouse intestine exhibits increased proliferation and Wnt/β-catenin signaling relative to wild-type mice (WT). Since the Rho GTPase Cdc42 plays a central role in intestinal epithelial proliferation and tight junction remodeling, we hypothesized that Cdc42 may be altered in the Cftr KO crypts. Immunofluorescence showed distinct tight junction localization of Cdc42 in Cftr KO fresh crypts and enteroids, the latter indicating an epithelial-autonomous feature. Quantitative PCR and immunoblots revealed similar expression of Cdc42 in the Cftr KO crypts/enteroids relative to WT, whereas pulldown assays showed increased GTP-bound (active) Cdc42 in proportion to total Cdc42 in Cftr KO enteroids. Cdc42 activity in the Cftr KO and WT enteroids could be reduced by inhibition of the Wnt transducer Disheveled. With the use of a dye permeability assay, Cftr KO enteroids exhibited increased paracellular permeability to 3 kDa dextran relative to WT. Leak permeability and Cdc42 tight junction localization were reduced to a greater extent by inhibition of Wnt/β-catenin signaling with endo-IWR1 in Cftr KO relative to WT enteroids. Increased proliferation or inhibition of Cdc42 activity with ML141 in WT enteroids had no effect on permeability. In contrast, inhibition of Cdc42 with ML141 increased permeability to both 3 kDa dextran and tight junction impermeant 500 kDa dextran in Cftr KO enteroids. These data suggest that increased constitutive Cdc42 activity may alter the stability of paracellular permeability in Cftr KO crypt epithelium. NEW & NOTEWORTHY: Increased tight junction localization and GTP-bound activity of the Rho GTPase Cdc42 was identified in small intestinal crypts and enteroids of cystic fibrosis (CF) transmembrane conductance regulator knockout (Cftr KO) mice. The increase in epithelial Cdc42 activity was associated with increased Wnt signaling. Paracellular flux of an uncharged solute (3 kDa dextran) in Cftr KO enteroids indicated a moderate leak permeability under basal conditions that was strongly exacerbated by Cdc42 inhibition. These findings suggest increased activity of Cdc42 in the Cftr KO intestine underlies alterations in intestinal permeability. [ABSTRACT FROM AUTHOR]

Mucocele formation in dogs is a unique and enigmatic muco-obstructive disease of the gallbladder caused by the amassment of abnormal mucus that bears striking pathological similarity to cystic fibrosis. We investigated the role of cystic fibrosis transmembrane conductance regulatory protein (CFTR) in the pathogenesis of this disease. The location and frequency of disease-associated variants in the coding region of CFTR were compared using whole genome sequence data from 2,642 dogs representing breeds at low-risk, high-risk, or with confirmed disease. Expression, localization, and ion transport activity of CFTR were quantified in control and mucocele gallbladders by NanoString, Western blotting, immunofluorescence imaging, and studies in Ussing chambers. Our results establish a significant loss of CFTR-dependent anion secretion by mucocele gallbladder mucosa. A significantly lower quantity of CFTR protein was demonstrated relative to E-cadherin in mucocele compared with control gallbladder mucosa. Immunofluorescence identified CFTR along the apical membrane of epithelial cells in control gallbladders but not in mucocele gallbladder epithelium. Decreases in mRNA copy number for CFTR were accompanied by decreases in mRNA for the Cl−/ HCO 3 − exchanger SLC26A3, K+ channels (KCNQ1, KCNN4), and vasoactive intestinal polypeptide receptor (VIPR1), which suggest a driving force for change in secretory function of gallbladder epithelial cells in the pathogenesis of mucocele formation. There were no significant differences in CFTR gene variant frequency, type, or predicted impact comparing low-risk, high-risk, and definitively diagnosed groups of dogs. This study describes a unique, naturally occurring muco-obstructive disease of the canine gallbladder, with uncanny similarity to cystic fibrosis, and driven by the underlying failure of CFTR function. NEW & NOTEWORTHY: Cystic fibrosis transmembrane conductance regulatory protein (CFTR) genomic variants and expression of mRNA, protein, and electrogenic anion secretory activity of CFTR were characterized in dog gallbladder. Acquired inhibition of CFTR expression by gallbladder epithelium was identified as underpinning a naturally occurring muco-obstructive disease of the dog gallbladder that bears striking pathological similarity to animal models of cystic fibrosis. [ABSTRACT FROM AUTHOR]

Chronic intestinal inflammation is a poorly understood manifestation of cystic fibrosis (CF), which may be refractory to ion channel CF transmembrane conductance regulator (CFTR) modulator therapy. People with CF exhibit intestinal dysbiosis, which has the potential for stimulating intestinal and systemic inflammation. CFTR is expressed in organ epithelia, leukocytes, and other tissues. Here, we investigate the contribution of intestinal epithelium-specific loss of Cftr [iCftr knockout (KO)] to dysbiosis and inflammation in mice treated with either of two antiobstructive dietary regimens necessary to maintain CF mouse models [polyethylene glycol (PEG) laxative or a liquid diet (LiqD)]. Feces collected from iCftr KO mice and their wild-type (WT) sex-matched littermates were used to measure fecal calprotectin to evaluate inflammation and to perform 16S rRNA sequencing to characterize the gut microbiome. Fecal calprotectin was elevated in iCftr KO relative to WT mice that consumed either PEG or LiqD. PEG iCftr KO mice did not show a change in α diversity versus WT mice but demonstrated a significant difference in microbial composition (b diversity) with included increases in the phylum Proteobacteria, the family Peptostreptococcaceae, four genera of Clostridia including C. innocuum, and the mucolytic genus Akkermansia. Fecal microbiome analysis of LiqD-fed iCftr KO mice showed both decreased α diversity and differences in microbial composition with increases in the Proteobacteria family Enterobacteriaceae, Firmicutes families Clostridiaceae and Peptostreptococcaceae, and enrichment of Clostridium perfringens, C. innocuum, C. difficile, mucolytic Ruminococcus gnavus, and reduction of Akkermansia. It was concluded that epithelium-specific loss of Cftr is a major driver of CF intestinal dysbiosis and inflammation with significant similarities to previous studies of pan Cftr KO mice. NEW & NOTEWORTHY Chronic intestinal inflammation is a manifestation of cystic fibrosis (CF), a disease caused by loss of the anion channel CF transmembrane conductance regulator (CFTR) that is expressed in many tissues. This study shows that intestinal epithelial cell-specific loss of CFTR [inducible Cftr knockout (KO)] in mice is sufficient to induce intestinal dysbiosis and inflammation. Experiments were performed on mice consuming two dietary regimens routinely used to prevent obstruction in CF mice. [ABSTRACT FROM AUTHOR]

Objective: We posit that evaluators of relationship education interventions can explore a policy relevant outcome by assessing relationship stability and conflict in a single index that is based on the literature on the effects of divorce and marital distress on children. We provide an empirical example from a randomized trial. Background: The U.S. Administration for Children and Families funds community‐based projects using relationship education with a foundational goal of fostering stable and healthy relationships. Assessing this outcome requires an approach different from separately analyzing stability and relationship quality. Method: We used data (N = 1,156 couples) from a randomized trial of Family Expectations to test the Stable Low‐Conflict Index, comparing couples assigned to the intervention to couples assigned to an untreated control group at a follow‐up 8 to 9 months postintervention. Results: Intervention couples were more likely to be in a stable, low‐conflict relationship at the follow up than control couples (b =.36, SE =.15, odds ratio = 1.44, p =.014). Conclusion: An index based on empirical precedence showed evidence of an intervention impact in a community‐based program. Implications: Evaluators of family policy linked interventions may advance the field by exploring outcomes that encapsulate aspects of both relationship stability and quality. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) Intestinal disease Is associated with the pathological manifestation mucoviscidosis, which is the secretion of tenacious, viscid mucus that plugs ducts and glands of epithelial-lined organs. Goblet cells are the principal cell type involved in exocytosis of mucin granules; however, little is known about the exocytotic process of goblet cells in the CF intestine. Using intestinal organoids from a CF mouse model, we determined that CF goblet cells have altered exocytotic dynamics, which involved intrathecal granule swelling that was abruptly followed by incomplete release of partially decondensated mucus. Some CF goblet cells exhibited an ectopic granule location and distorted cellular morphology, a phenotype that is consistent with retrograde intracellular granule movement during exocytosis. Increasing the luminal concentration of bicarbonate, which mimics CF transmembrane conductance regulator-mediated anion secretion, increased spontaneous degranulation in WT goblet cells and improved exocytotic dynamics in CF goblet cells; however, there was still an apparent incoordination between granule decondensation and exocytosis in the CF goblet cells. Compared with those within WT goblet cells, mucin granules within CF goblet cells had an alkaline pH, which may adversely affect the polyionic composition of the mucins. Together, these findings indicate that goblet cell dysfunction is an epithelial-autonomous defect in the CF intestine that likely contributes to the pathology of mucoviscidosis and the intestinal manifestations of obstruction and inflammation., Introduction Cystic fibrosis (CF), an autosomal recessive disease, is caused by loss-of-activity mutations in CF transmembrane conductance regulator (CFTR) (1), an epithelial anion channel largely responsible for transepithelial secretion of [...]

The federal government, through the Administration for Children and Families (ACF), has funded community‐based relationship education programs for couples, individuals, and families, with a strong focus on serving economically disadvantaged and racially diverse families. This study evaluated the impact of a 36‐hour, workshop‐based couple relationship education program that was funded by ACF using a randomized controlled trial (RCT) design and intent‐to‐treat (ITT) analyses. Participants were 1320 couples who were either expecting a baby or had a baby within the past 3 months, at the time of enrollment. Follow‐up surveys were administered 12 months later. Analyses evaluated program impacts on relationship stability, constructive communication, and destructive conflict compared to a no‐treatment control group. Analyses showed a statistically significant impact of the program on destructive conflict (d = 0.10) but not on constructive communication (d = 0.06) or stability (dCox = 0.10). Based on findings from previous evaluations, we also examined whether participants' levels of sociodemographic disadvantage moderated these effects. There was significant moderation by sociodemographic disadvantage on constructive communication and destructive conflict, but not on stability. Effects were observed for those at higher levels of sociodemographic disadvantage. [ABSTRACT FROM AUTHOR]

The majority of dietary amino acids are absorbed via the [H.sup.+]-di-/tripeptide transporter Pept1 of the small intestine. Proton influx via Pept1 requires maintenance of intracellular pH ([pH.sub.i]) to sustain the driving force for peptide absorption. The apical membrane [Na.sup.+]/[H.sup.+] exchanger Nhe3 plays a major role in minimizing epithelial acidification during [H.sup.+]-di-/tripeptide absorption. However, the contributions of HC[O.sup.-.sub.3]-dependent transporters to this process have not been elucidated. In this study, we investigate the role of putative anion transporter-1 (Pat-1), an apical membrane anion exchanger, in epithelial [pH.sub.i] regulation during [H.sup.+]-peptide absorption. Using wild-type (WT) and Pat-1(-) mice, Ussing chambers were employed to measure the short-circuit current ([I.sub.SC]) associated with Pept1-mediated glycyl-sarcosine (Gly-Sar) absorption. Microfluorometry was used to measure [pH.sub.i] and [Cl.sup.-]/HC[O.sup.-.sub.3] exchange in the upper villous epithelium. In C[O.sub.2]/HC[O.sup.-.sub.3]-buffered Ringers, WT small intestine showed significant Gly-Sar-induced [I.sub.SC] and efficient [pH.sub.i] regulation during pharmacological inhibition of Nhe3 activity. In contrast, epithelial acidification and reduced [I.sub.SC] response to Gly-Sar exposure occurred during pharmacological inhibition of [Cl.sup.-]/HC[O.sup.-.sub.3] exchange and in the Pat-1(-) intestine. Pat-1 interacts with carbonic anhydrase II (CAII), and studies using CAII(-) intestine or the pharmacological inhibitor methazolamide on WT intestine resulted in increased epithelial acidification during Gly-Sar exposure. Increased epithelial acidification during Gly-Sar exposure also occurred in WT intestine during inhibition of luminal extracellular CA activity. Measurement of [Cl.sup.-]/HC[O.sup.-.sub.3] exchange in the presence of Gly-Sar revealed an increased rate of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] exchange that was both Pat-1 dependent and CA dependent. In conclusion, Pat-1 [Cl.sup.-]/HC[O.sup.-.sub.3] exchange contributes to [pH.sub.i] regulation in the villous epithelium during [H.sup.+]-dipeptide absorption, possibly by providing a HC[O.sup.-.sub.3] import pathway. acid-base transport; anion exchange; Pept1; carbonic anhydrase II; mouse doi:10.1152/ajpgi.00293.2009.

The Ussing chamber provides a physiological system to measure the transport of ions, nutrients, and drugs across various epithelial tissues. One of the most studied epithelia is the intestine, which has provided several landmark discoveries regarding the mechanisms of ion transport processes. Adaptation of this method to mouse intestine adds the dimension of investigating genetic loss or gain of function as a means to identify proteins or processes affecting transepithelial transport. In this review, the principles underlying the use of Ussing chambers are outlined including limitations and advantages of the technique. With an emphasis on mouse intestinal preparations, the review covers chamber design, commercial equipment sources, tissue preparation, stepby-step instruction for operation, troubleshooting, and examples of interpretation difficulties. Specialized uses of the Ussing chamber such as the pH stat technique to measure transepithelial bicarbonate secretion and isotopic flux methods to measure net secretion or absorption of substrates are discussed in detail, and examples are given for the adaptation of Ussing chamber principles to other measurement systems. The purpose of the review is to provide a practical guide for investigators who are new to the Ussing chamber method. colon; duodenum; ion transport; nutrient absorption

In cystic fibrosis, impaired secretion resulting from loss of activity of the cystic fibrosis transmembrane conductance regulator (CFTR) causes dehydration of intestinal contents and life-threatening obstructions. Conversely, impaired absorption resulting from loss of the NHE3 [Na.sup.+]/[H.sup.+] exchanger causes increased fluidity of the intestinal contents and diarrhea. To test the hypothesis that reduced NHE3-mediated absorption could increase survival and prevent some of the intestinal pathologies of cystic fibrosis, Cftr/Nhe3 double heterozygous mice were mated and their offspring analyzed. Cftr-null mice lacking one or both copies of the NHE3 gene exhibited increased fluidity of their intestinal contents, which prevented the formation of obstructions and increased survival. Goblet cell hyperplasia was eliminated, but not the accumulation of Paneth cell granules or increased cell proliferation in the crypts. Microarray analysis of small intestine RNA from Cftr-null, NHE3-null, and double-null mice all revealed downregulation of genes involved in xenobiotic metabolism, including a cohort of genes involved in glutathione metabolism. Expression of energy metabolism genes was altered, but there were no changes in genes involved in inflammation. Total intracellular glutathione was increased in the jejunum of all of the mutants and the ratio of reduced to oxidized glutathione was reduced in Cftr-null mutants, indicating that CFTR deficiency affects intestinal glutathione metabolism. The data establish a major role for NHE3 in regulating the fluidity of the intestinal contents and show that reduced NHE3-mediated absorption reverses some of the intestinal pathologies of cystic fibrosis, thus suggesting that it may serve as a potential therapeutic target. Slc9a3; distal intestinal obstructive syndrome; meconium ileus; cytochrome P-450; glutathione transferase

Ischemic injury induces breakdown of the intestinal barrier. Recent studies in porcine postischemic tissues indicate that inhibition of NHE2 results in enhanced recovery of barrier function in vitro via a process involving interepithelial tight junctions. To further study this process, recovery of barrier function was assessed in wild-type (NHE[2.sup.+/+]) and NHE[2.sup.-/-] mice in vivo and wild-type mice in vitro. Mice were subjected to complete mesenteric ischemia in vivo, after which barrier function was measured by blood-to-lumen mannitol clearance over a 3-h recovery period or measurement of transepithelial electrical resistance (TER) in Ussing chambers immediately following ischemia. Tissues were assessed for expression of select junctional proteins. Compared with NHE[2.sup.+/+] mice, NHE[2.sup.-/-] mice had greater intestinal permeability during the postischemic recovery process. In contrast to prior porcine studies, pharmacological inhibition of NHE2 in post-ischemic tissues from wild-type mice also resulted in significant reductions in TER. Mucosa from NHE[2.sup.-/-] mice displayed a shift of occludin and claudin-1 expression to the Triton-X-soluble membrane fractions and showed disruption of occludin and claudin-1 localization patterns following injury. This was qualitatively and quantitatively recovered in NHE[2.sup.+/+] mice compared with NHE[2.sup.-/-] mice by the end of the 3-h recovery period. Serine phosphorylation of occludin and claudin-1 was downregulated in NHE[2.sup.-/-] postischemia compared with wild-type mice. These data indicate an important role for NHE2 in recovery of barrier function in mice via a mechanism involving tight junctions. tight junction; intestinal permeability; ischemia; [Na.sup.+]/[H.sup.+] exchange

Basal HC[O.sup.-.sub.3] secretion across the duodenum has been shown in several species to principally involve the activity of apical membrane [Cl.sup.-]/HC[O.sup.-.sub.3] exchanger(s). To investigate the identity of relevant anion exchanger(s), experiments were performed using wild-type (WT) mice and mice with gene-targeted deletion of the following [Cl.sup.-]/HC[O.sup.-.sub.3] exchangers localized to the apical membrane of murine duodenal villi: Slc26a3 [down-regulated in adenoma (DRA)], Slc26a6 [putative anion transporter 1 (PAT-1)], and Slc4a9 [anion exchanger 4 (AEA)]. RT-PCR of the isolated villous epithelium demonstrated PAT-1, DRA, and AE4 mRNA expression. Using the pH-sensitive dye BCECF, anion exchange rates were measured across the apical membrane of epithelial cells in the upper villus of the intact duodenal mucosa. Under basal conditions, [Cl.sup.-]/HC[O.sup.-.sub.3] exchange activity was reduced by 65-80% in the PAT-l(-) duodenum, 30-40% in the DRA(-) duodenum, and bicarbonate secretion; intestine; cystic fibrosis transmembrane conductance regulator; Slc4; putative anion transporter 1; downregulated in adenoma; anion exhanger isoform 4

Genistein, a naturally occurring isoflavone, augments in vitro epithelial anion transport via activation of the cystic fibrosis transmembrane conductance regulator chloride channel. In this study, we examined whether chronic dietary exposure to 600 mg/kg genistein (600 G) for 1 mo would stimulate anion secretion across wild-type (Wt, normal) murine intestine. Anion secretion was assessed in freshly excised segments of murine jejuna by measuring short circuit current ([I.sub.sc]) and comparing with jejunal segments from mice fed 0 mg/kg genistein (0 G). Basal and forskolin-stimulated anion secretions were augmented (P < 0.05) in female but not in male mice fed 600 G, compared with their counterparts fed 0 G. Serum genistein concentrations were greater in both female and male mice fed 600 G (~3.5-6.9 [micro]mol/L) than those fed 0 G (~100 nmol/L). Anion substitution experiments and bumetanide-sensitivity demonstrated that chloride was the major anion mediating the increased secretion. A smaller bicarbonate component was not augmented by consumption of the genistein diet. These data indicate that chronic exposure to dietary genistein stimulates a sex-dependent increase in basal and forskolin-stimulated chloride secretion across murine intestine.

Villi of the proximal duodenum are situated for direct exposure to gastric acid chyme. However, little is known about active bicarbonate secretion across villi that maintains the protective alkaline mucus barrier, a process that may be compromised in cystic fibrosis (CF), i.e., in the absence of a functional CF transmembrane conductance regulator (CFTR) anion channel. We investigated [Cl.sup.-]/HC[O.sup.-.sub.3] exchange activity across the apical membrane of epithelial cells located at the midregion of villi in intact duodenal mucosa from wild-type (WT) and CF mice using the pH-sensitive dye BCECF. Under basal conditions, the [Cl.sup.-]/HC[O.sup.-.sub.3] exchange rate was reduced by ~35% in CF compared with WT villous epithelium. [Cl.sup.-]/HC[O.sup.-.sub.3] exchange in WT and CF villi responded similarly to inhibitors of anion exchange, and membrane depolarization enhanced rates of [Cl.sup.-.sub.out]/HC[O.sup.-.sub.3 in] exchange in both epithelia. In anion substitution studies, [anion.sub.in]/HC[O.sup.-.sub.3 out] exchange rates were greater in WT epithelium using [Cl.sup.-] or N[O.sup.-.sub.3], but decreased to the level of the CF epithelium using the CFTR-impermeant anion, S[O.sup.2-.sub.4] . Similarly, treatment of WT epithelium with the CFTR-selective blocker glybenclamide decreased the [Cl.sup.-]/HC[O.sup.-.sub.3] exchange rate to the level of CF epithelium. The mRNA expression of Slc26a3 (downregulated in adenoma) and Slc26a6 (putative anion exchanger-1) was similar between WT and CF duodena. From these studies of murine duodenum, we conclude 1) characteristics of [Cl.sup.-]/HC[O.sup.-.sub.3] exchange in the villous epithelium are most consistent with Slc26a6 activity, and 2) [Cl.sup.-] channel activity of CFTR facilitates apical membrane [Cl.sup.-.sub.in]/HC[O.sup.-.sub.3 out] exchange by providing a Cl 'leak' under basal conditions. bicarbonate secretion; anion exchange; cystic fibrosis; Slc26a; Slc4; mouse