Fecal dysbiosis and inflammation in intestinal-specific Cftr knockout mice on regimens preventing intestinal obstruction.

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 (ß 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.

Impact evaluation of the family expectations program and moderation by sociodemographic disadvantage.

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.

Goblet cell hyperplasia is not epithelial-autonomous in the Cftr knockout intestine.

Goblet cell hyperplasia is an important manifestation of cystic fibrosis (CF) disease in epithelial-lined organs. Explants of CF airway epithelium show normalization of goblet cell numbers; therefore, we hypothesized that small intestinal enteroids from Cftr knockout (KO) mice would not exhibit goblet cell hyperplasia. Toll-like receptors 2 and 4 (Tlr2 and Tlr4) were investigated as markers of inflammation and influence on goblet cell differentiation. Ex vivo studies found goblet cell hyperplasia in Cftr KO jejunum compared with wild-type (WT) mice. IL-13, SAM pointed domain-containing ETS transcription factor (Spdef), Tlr2, and Tlr4 protein expression were increased in Cftr KO intestine relative to WT. In contrast, WT and Cftr KO enteroids did not exhibit differences in basal or IL-13-stimulated goblet cell numbers, or differences in expression of Tlr2, Tlr4, and Spdef. Ileal goblet cell numbers in Cftr KO/Tlr4 KO and Cftr KO/Tlr2 KO mice were not different from Cftr KO mice, but enumeration was confounded by altered mucosal morphology. Treatment with Tlr4 agonist LPS did not affect goblet cell numbers in WT or Cftr KO enteroids, whereas the Tlr2 agonist Pam3Csk4 stimulated goblet cell hyperplasia in both genotypes. Pam3Csk4 stimulation of goblet cell numbers was associated with suppression of Notch1 and Neurog3 expression and upregulated determinants of goblet cell differentiation. We conclude that goblet cell hyperplasia and inflammation of the Cftr KO small intestine are not exhibited by enteroids, indicating that this manifestation of CF intestinal disease is not epithelial-automatous but secondary to the altered CF intestinal environment.NEW & NOTEWORTHY Studies of small intestinal organoids from cystic fibrosis (CF) mice show that goblet cell hyperplasia and increased Toll-like receptor 2/4 expression are not primary manifestations of the CF intestine. Intestinal goblet cell hyperplasia in the CF mice was not strongly altered by genetic ablation of Tlr2 and Tlr 4, but could be induced in both wild-type and CF intestinal organoids by a Tlr2-dependent suppression of Notch signaling.

Laparoscopic Appendectomy and Cholecystectomy Timing Predicts Hand-Offs but Not Miscounts.

INTRODUCTION: Intraoperative hand-offs are poorly coordinated and associated with risk of surgical miscount. We evaluated hand-off patterns for nursing staff during two common operations hypothesizing that hand-off patterns would be associated with increased surgical miscounts and vary during operations performed standard versus nonstandard operating hours. METHODS: We retrospectively analyzed laparoscopic cholecystectomy (N = 3888) and appendectomy (N = 1768) from 2012 to 2021 at a single institution using electronic medical records. We evaluated intraoperative hand-off patterns and the presence of miscounts for operations performed during standard versus nonstandard hours. Standard operating hours were defined as M-F 7:30 am to 5:00 pm. RESULTS: Across 5656 operations, 10 cases had surgical miscounts and were significantly longer than those without (156.5 versus 101 min P = 0.0178). More than half (51.3%) of cases had no identified hand-offs, and 42.9% of cases occurred during nonstandard hours. Cases during standard versus nonstandard hours were more likely to have hand-offs (56.0% versus 38.9%), P < 0.0001 and had shorter interval between hand-offs (64 versus 75 min), P < 0.0001. The period between patient entry to the room and intubation, which includes initial counts, had a disproportionately high percentage of hand-offs (P < 0.0001). CONCLUSIONS: Variability in hand-off occurrence and frequency in operations performed during standard and nonstandard hours suggest that hand-offs are influenced by staffing patterns. Few surgical miscounts occurred but were associated with longer cases. Hand-offs disproportionately occurred between patient entry and intubation, with a potential for disruption of initial instrument counts. Future work optimizing hand-off coordination is an opportunity to mitigate risk to patients.

Evaluation of Intraoperative Hand-Off Frequency, Duration, and Context: A Mixed Methods Analysis.

BACKGROUND: Hand-offs in the operating room contribute to poor communication, reduced team function, and may be poorly coordinated with other activities. Conversely, they may represent a missed opportunity for improved communication. We sought to better understand the coordination and impact of intraoperative hand-offs. METHODS: We prospectively audio-video (AV) recorded 10 operations and evaluated intraoperative hand-offs. Data collected included percentage of time team members were absent due to breaks, relationships between hand-offs and intraoperative events (incision, surgical counts), and occurrences of simultaneous hand-offs. We also identified announcement that a hand-off had occurred and anchoring, in which team members not involved in the hand-off participated and provided information. RESULTS: Spanning 2919 min of audio-video data, there were 74 hand-offs (range, 4-14 per case) totaling 225.2 min, representing 7.7% of time recorded. Thirty-two (45.1%) hand-offs were interrupted or delayed because of competing activities; eight hand-offs occurred during an instrument or laparotomy pad count. Six cases had simultaneous hand-offs; two cases had two episodes of simultaneous hand-offs. Eight hand-offs included an announcement. Seven included anchoring. Evaluating both temporary and permanent hand-offs, one or more original team members was absent for 40.7% of time recorded and >one team member was absent for 20.5% of time recorded. CONCLUSIONS: Intraoperative hand-offs are frequent and not well coordinated with intraoperative events including counts and other hand-offs. Anchoring and announced hand-offs occurred in a small proportion of cases. Future work must focus on optimizing timing, content, and participation in intraoperative hand-offs.

Modeling Surgical Technical Skill Using Expert Assessment for Automated Computer Rating.

OBJECTIVE: Computer vision was used to predict expert performance ratings from surgeon hand motions for tying and suturing tasks. SUMMARY BACKGROUND DATA: Existing methods, including the objective structured assessment of technical skills (OSATS), have proven reliable, but do not readily discriminate at the task level. Computer vision may be used for evaluating distinct task performance throughout an operation. METHODS: Open surgeries was videoed and surgeon hands were tracked without using sensors or markers. An expert panel of 3 attending surgeons rated tying and suturing video clips on continuous scales from 0 to 10 along 3 task measures adapted from the broader OSATS: motion economy, fluidity of motion, and tissue handling. Empirical models were developed to predict the expert consensus ratings based on the hand kinematic data records. RESULTS: The predicted versus panel ratings for suturing had slopes from 0.73 to 1, and intercepts from 0.36 to 1.54 (Average R2 = 0.81). Predicted versus panel ratings for tying had slopes from 0.39 to 0.88, and intercepts from 0.79 to 4.36 (Average R2 = 0.57). The mean square error among predicted and expert ratings was consistently less than the mean squared difference among individual expert ratings and the eventual consensus ratings. CONCLUSIONS: The computer algorithm consistently predicted the panel ratings of individual tasks, and were more objective and reliable than individual assessment by surgical experts.

Familiarity and Communication in the Operating Room.

BACKGROUND: Poor communication is implicated in many adverse events in the operating room (OR); however, many hospitals' scheduling practices permit unfamiliar operative teams. The relationship between unfamiliarity, team communication and effectiveness of communication is poorly understood. We sought to evaluate the relationship between familiarity, communication rates, and communication ineffectiveness of health care providers in the OR. MATERIALS AND METHODS: We performed purposive sampling of 10 open operations. For each case, six providers (anesthesiology attending, in-room anesthetist, circulator, scrub, surgery attending, and surgery resident) were queried about the number of mutually shared cases. We identified communication events and created dyad-specific communication rates. RESULTS: Analysis of 48 h of audio-video content identified 2570 communication events. Operations averaged 58.0 communication events per hour (range, 29.4-76.1). Familiarity was not associated with communication rate (P = 0.69) or communication ineffectiveness (P = 0.21). Cross-disciplinary dyads had lower communication rates than intradisciplinary dyads (P < 0.001). Anesthesiology-nursing, anesthesiology-surgery, and nursing-surgery dyad communication rates were 20.1%, 42.7%, and 57.3% the rate predicted from intradisciplinary dyads, respectively. In addition, cross-disciplinary dyad status was a significant predictor of having at least one ineffective communication event (P = 0.02). CONCLUSIONS: Team members do not compensate for unfamiliarity by increasing their verbal communication, and dyad familiarity is not protective against ineffective communication. Cross-disciplinary communication remains vulnerable in the OR suggesting poor crosstalk across disciplines in the operative setting. Further investigation is needed to explore these relationships and identify effective interventions, ensuring that all team members have the necessary information to optimize their performance.

A Statewide Surgical Coaching Program Provides Opportunity for Continuous Professional Development.

OBJECTIVE: We sought to develop and evaluate a video-based coaching program for board-eligible/certified surgeons. SUMMARY BACKGROUND DATA: Multiple disciplines utilize coaching for continuous professional development; however, coaching is not routinely employed for practicing surgeons. METHODS: Peer-nominated surgeons were trained as coaches then paired with participant surgeons. After setting goals, each coaching pair reviewed video-recorded operations performed by the participating surgeon. Coaching sessions were audio-recorded, transcribed, and coded to identify topics discussed. The effectiveness with which our coaches were able to utilize the core principles and activities of coaching was evaluated using 3 different approaches: self-evaluation; evaluation by the participants; and assessment by the study team. Surveys of participating surgeons and coach-targeted interviews provided general feedback on the program. All measures utilized a 5-point Likert scale format ranging from 1 (low) to 5 (high). RESULTS: Coach-participant surgeon pairs targeted technical, cognitive, and interpersonal aspects of performance. Other topics included managing intraoperative stress. Mean objective ratings of coach effectiveness was 3.1 ±â€Š0.7, ranging from 2.0 to 5.0 on specific activities of coaching. Subjective ratings by coaches and participants were consistently higher. Coaches reported that the training provided effectively prepared them to facilitate coaching sessions. Participants were similarly positive about interactions with their coaches. Identified barriers were related to audio-video technology and scheduling of sessions. Overall, participants were satisfied with their experience (mean 4.4 ±â€Š0.7) and found the coaching program valuable (mean 4.7 ±â€Š0.7). CONCLUSIONS: This is the first report of cross-institutional surgical coaching for the continuous professional development of practicing surgeons, demonstrating perceived value among participants, as well as logistical challenges for implementing this evidence-based program. Future research is necessary to evaluate the impact of coaching on practice change and patient outcomes.

Examining the influence of personal goal interference and attainability on psychological distress in non-metastatic breast cancer patients.

The present study investigated the relationship between two goal-related appraisals - perceived cancer-related interference and perceived attainability of important personal goals - and psychological distress among non-metastatic breast cancer patients across the short-term treatment and recovery period. Forty-five women completed self-report questionnaires at approximately 1 and 6 months following surgery. A mixed idiographic-nomothetic goal methodology assessed perceived cancer-related interference and attainability of self-generated important personal goals. Psychological distress symptoms were assessed with the Depression Anxiety Stress Scales short form. Correlation analyses and general linear modelling were used to evaluate the hypothesised relationships over time. Average cancer-related interference and attainability of important personal goals were significantly associated with concurrent depression, anxiety and stress symptoms at 6 months following surgery. Perceived attainability of highly important goals at 6 months post-surgery uniquely predicted change in psychological distress symptoms over time. The findings suggest that low perceived attainability of important personal goals may be an important predictor of elevated distress symptoms across the short-term following surgery. Further insight into the relationship between these negative goal appraisals and psychological functioning among different groups of cancer patients could inform the provision of targeted psychosocial support across the cancer continuum.

Cellular chloride and bicarbonate retention alters intracellular pH regulation in Cftr KO crypt epithelium.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), an anion channel providing a major pathway for Cl(-) and HCO3 (-) efflux across the apical membrane of the epithelium. In the intestine, CF manifests as obstructive syndromes, dysbiosis, inflammation, and an increased risk for gastrointestinal cancer. Cftr knockout (KO) mice recapitulate CF intestinal disease, including intestinal hyperproliferation. Previous studies using Cftr KO intestinal organoids (enteroids) indicate that crypt epithelium maintains an alkaline intracellular pH (pHi). We hypothesized that Cftr has a cell-autonomous role in downregulating pHi that is incompletely compensated by acid-base regulation in its absence. Here, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein microfluorimetry of enteroids showed that Cftr KO crypt epithelium sustains an alkaline pHi and resistance to cell acidification relative to wild-type. Quantitative real-time PCR revealed that Cftr KO enteroids exhibit downregulated transcription of base (HCO3 (-))-loading proteins and upregulation of the basolateral membrane HCO3 (-)-unloader anion exchanger 2 (Ae2). Although Cftr KO crypt epithelium had increased Ae2 expression and Ae2-mediated Cl(-)/HCO3 (-) exchange with maximized gradients, it also had increased intracellular Cl(-) concentration relative to wild-type. Pharmacological reduction of intracellular Cl(-) concentration in Cftr KO crypt epithelium normalized pHi, which was largely Ae2-dependent. We conclude that Cftr KO crypt epithelium maintains an alkaline pHi as a consequence of losing both Cl(-) and HCO3 (-) efflux, which impairs pHi regulation by Ae2. Retention of Cl(-) and an alkaline pHi in crypt epithelium may alter several cellular processes in the proliferative compartment of Cftr KO intestine.

Leveraging comparative effectiveness research to improve the quality of multidisciplinary care for breast cancer patients.

Breast cancer is the most commonly diagnosed cancer among women. To date, the use of efficacy randomized controlled trials (RCTs) in breast cancer have resulted in dramatic improvements in oncologic outcomes for this disease. However, not every question pertinent to breast cancer is amenable to such efficacy trials. This chapter will discuss some of the unique aspects of breast cancer that make efficacy RCTs challenging and/or impractical, how comparative effectiveness research can be used to address these issues, and identify several key questions which would benefit from ongoing comparative effectiveness research.

Laparoscopic versus open Ladd's procedure for intestinal malrotation in adults.

BACKGROUND: Intestinal malrotation results from errors in fetal intestinal rotation and fixation. While most patients are diagnosed in childhood, some present as adults. Laparoscopic Ladd's procedure is an accepted alternative to laparotomy in children but has not been well-studied in adults. This study was designed to investigate outcomes for adults undergoing laparoscopic Ladd's repair for malrotation. METHODS: We performed a single-institution retrospective chart review over 11 years. Data collected included patient age, details of pre-operative work-up and diagnosis, surgical management, complications, rates of re-operation, and symptom resolution. Patients were evaluated on an intent-to-treat basis based on their planned operative approach. Categorical data were analyzed using Fisher's exact test. Continuous data were analyzed using Student's t test. RESULTS: Twenty-two patients were identified (age range 18-63). Fifteen were diagnosed pre-operatively; of the remaining seven patients, four received an intra-operative malrotation diagnosis during elective surgery for another problem. Most had some type of pre-operative imaging, with computed tomography being the most common (77.3 %). Comparing patients on an intent-to-treat basis, the two groups were similar with respect to age, operative time, and estimated blood loss. Six patients underwent successful laparoscopic repair; three began laparoscopically but were converted to laparotomy. There was a statistically significant difference in hospital length of stay (LOS) (5.0 ± 2.5 days vs 11.6 ± 8.1 days, p = 0.0148) favoring the laparoscopic approach. Three patients required re-operation: two underwent side-to-side duodeno-duodenostomy and one underwent a re-do Ladd's procedure. Ultimately, three (two laparoscopic, one open) had persistent symptoms of bloating (n = 2), constipation (n = 2), and/or pain (n = 1). CONCLUSION: Laparoscopic repair appears to be safe and effective in adults. While a small sample size limits the power of this study, we found a statistically significant decrease in LOS and a trend toward decreased postoperative nasogastric decompression. There were no significant differences in complication rates, re-operation, or persistence of symptoms between groups.

Couples Relationship Education and Couples Therapy: Healthy Marriage or Strange Bedfellows?

This paper focuses on issues sparked by the Couples Relationship Education (CRE) field moving toward a more clinical model to meet the needs of an increasing number of distressed couples coming to CRE programs. We review the concerns raised and recommendations made by Bradford, Hawkins, and Acker (2015), most of which push CRE toward a more clinical model. We address these recommendations and make suggestions for best practices that preserve the prevention/education model underlying research-based CRE. The three main issues are couple screening, leader training, and service delivery models. Our suggested best practices include: conducting minimal screening including the assessment of dangerous levels of couple violence, training leaders with key skills to handle issues raised by distressed couples as well as other couples who may place additional burdens on leaders, providing referrals and choices of programs available to participants at intake and throughout the CRE program, and adding (rather than integrating) clinical services to CRE services for couples who desire additional intervention. Finally, throughout the paper, we review other key issues in the CRE field and make recommendations made for future research and practice.

Identifying and Reducing Insulin Errors in the Simulated Military Critical Care Air Transport Environment: A Human Factors Approach.

INTRODUCTION: During high-fidelity simulations in the Critical Care Air Transport (CCAT) Advanced course, we identified a high frequency of insulin medication errors and sought strategies to reduce them using a human factors approach. MATERIALS AND METHODS: Of 169 eligible CCAT simulations, 22 were randomly selected for retrospective audio-video review to establish a baseline frequency of insulin medication errors. Using the Human Factors Analysis Classification System, dosing errors, defined as a physician ordering an inappropriate dose, were categorized as decision-based; administration errors, defined as a clinician preparing and administering a dose different than ordered, were categorized as skill-based. Next, 3 a priori interventions were developed to decrease the frequency of insulin medication errors, and these were grouped into 2 study arms. Arm 1 included a didactic session reviewing a sliding-scale insulin (SSI) dosing protocol and a hands-on exercise requiring all CCAT teams to practice preparing 10 units of insulin including a 2-person check. Arm 2 contained arm 1 interventions and added an SSI cognitive aid available to students during simulation. Frequency and type of insulin medication errors were collected for both arms with 93 simulations for arm 1 (January-August 2021) and 139 for arm 2 (August 2021-July 2022). The frequency of decision-based and skill-based errors was compared across control and intervention arms. RESULTS: Baseline insulin medication error rates were as follows: decision-based error occurred in 6/22 (27.3%) simulations and skill-based error occurred in 6/22 (27.3%). Five of the 6 skill-based errors resulted in administration of a 10-fold higher dose than ordered. The post-intervention decision-based error rates were 9/93 (9.7%) and 23/139 (2.2%), respectively, for arms 1 and 2. Compared to baseline error rates, both arm 1 (P = .04) and arm 2 (P < .001) had a significantly lower rate of decision-based errors. Additionally, arm 2 had a significantly lower decision-based error rate compared to arm 1 (P = .015). For skill-based preparation errors, 1/93 (1.1%) occurred in arm 1 and 4/139 (2.9%) occurred in arm 2. Compared to baseline, this represents a significant decrease in skill-based error in both arm 1 (P < .001) and arm 2 (P < .001). There were no significant differences in skill-based error between arms 1 and 2. CONCLUSIONS: This study demonstrates the value of descriptive error analysis during high-fidelity simulation using audio-video review and effective risk mitigation using training and cognitive aids to reduce medication errors in CCAT. As demonstrated by post-intervention observations, a human factors approach successfully reduced decision-based error by using didactic training and cognitive aids and reduced skill-based error using hands-on training. We recommend the development of a Clinical Practice Guideline including an SSI protocol, guidelines for a 2-person check, and a cognitive aid for implementation with deployed CCAT teams. Furthermore, hands-on training for insulin preparation and administration should be incorporated into home station sustainment training to reduced medication errors in the operational environment.

Fecal Dysbiosis and Inflammation in Intestinal-Specific Cftr Knockout Mice on Regimens Preventing Intestinal Obstruction.

Chronic intestinal inflammation is a poorly understood manifestation of Cystic Fibrosis (CF), which may be refractory to ion channel CFTR modulator therapy. People with CF exhibit intestinal dysbiosis which has potential for stimulating intestinal and systemic inflammation. CFTR is expressed in organ epithelia and in the leukocyte population. Here, we investigate the contribution of intestinal epithelial-specific loss of Cftr (iCftr KO) to dysbiosis and inflammation in mice treated with either of two anti-obstructive dietary regimens necessary to maintain CF mouse models (PEG laxative or a liquid diet, LiqD). Feces collected from iCftr KO mice and their wildtype (WT) sex-matched littermates were used to measure fecal calprotectin and to perform 16S rRNA sequencing to characterize the gut microbiome. Fecal calprotectin was elevated in iCftr KO relative to WT samples of mice consuming either PEG or LiqD. PEG iCftr KO mice did not show a change in α-diversity versus WT but demonstrated a significant difference in microbial composition (ß-diversity) with increases in phylum Proteobacteria , family Peptostreptococcaceae , four genera of Clostridia including C. innocuum , and mucolytic genus Akkermansia . Fecal microbiome analysis of LiqD iCftr KO mice showed both decreased α-diversity and differences in microbial composition with increases in 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 epithelial-specific loss of Cftr is a major driver of CF intestinal dysbiosis and inflammation with significant similarities to previous studies of global Cftr KO mice. New and noteworthy: Chronic intestinal inflammation is a manifestation of cystic fibrosis (CF), a disease caused by loss of the anion channel CFTR that is expressed in many tissues. This study shows that intestinal epithelial cell-specific loss of CFTR (iCftr KO) in mice is sufficient to induce intestinal dysbiosis and inflammation. Studies were performed on mice consuming either dietary regimen (PEG laxative or liquid diet) routinely used to prevent obstruction in CF mice.

Functional Cftr in crypt epithelium of organotypic enteroid cultures from murine small intestine.

Physiological studies of intact crypt epithelium have been limited by problems of accessibility in vivo and dedifferentiation in standard primary culture. Investigations of murine intestinal stem cells have recently yielded a primary intestinal culture in three-dimensional gel suspension that recapitulates crypt structure and epithelial differentiation (Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, Van Es JH, Abo A, Kujala P, Peters PJ, Clevers H. Nature 459: 262-265, 2009). We investigated the utility of murine intestinal crypt cultures (termed "enteroids") for physiological studies of crypt epithelium by focusing on the transport activity of the cystic fibrosis transmembrane conductance regulator Cftr. Enteroids had multiple crypts with well-differentiated goblet and Paneth cells that degranulated on exposure to the muscarinic agonist carbachol. Modified growth medium provided a crypt proliferation rate, as measured by 5-ethynyl-2'-deoxyuridine labeling, which was similar to proliferation in vivo. Immunoblots demonstrated equivalent Cftr expression in comparisons of freshly isolated crypts with primary and passage 1 enteroids. Apparent enteroid differences in mRNA expression of other transporters were primarily associated with villous epithelial contamination of freshly isolated crypts. Microelectrode analysis revealed cAMP-stimulated membrane depolarization in enteroid epithelium from wild-type (WT) but not Cftr knockout (KO) mice. Morphological and microfluorimetric studies, respectively, demonstrated Cftr-dependent cell shrinkage and lower intracellular pH in WT enteroid epithelium in contrast to Cftr KO epithelium or WT epithelium treated with Cftr inhibitor 172. We conclude that crypt epithelium of murine enteroids exhibit Cftr expression and activity that recapitulates crypt epithelium in vivo. Enteroids provide a primary culture model that is suitable for physiological studies of regenerating crypt epithelium.

Synthesis and Evaluation of Pseudomonas aeruginosa ATP Synthase Inhibitors.

New antibiotics with unique biological targets are desperately needed to combat the growing number of resistant bacterial pathogens. ATP synthase, a critical protein found in all life, has recently become a target of interest for antibiotic development due to the success of the anti-tuberculosis drug bedaquiline, and while many groups have worked on developing drugs to target bacterial ATP synthase, few have been successful at inhibiting Pseudomonas aeruginosa (PA) ATP synthase specifically. PA is one of the leading causes of resistant nosocomial infections across the world and is extremely challenging to treat due to its various antibiotic resistance mechanisms for most commonly used antibiotics. Herein, we detail the synthesis and evaluation of a series of C1/C2 quinoline analogues for their ability to inhibit PA ATP synthase and act as antibiotics against wild-type PA. From this survey, we found six compounds capable of inhibiting PA ATP synthase in vitro showing that bulky/hydrophobic C1/C2 substitutions are preferred. The strongest inhibitor showed an IC50 of 10 µg/mL and decreased activity of PA ATP synthase to 24% relative to the control. While none of the compounds were able to inhibit wild-type PA in cell culture, two showed improved inhibition of PA growth when permeability of the outer membrane was increased or efflux was knocked out, thus demonstrating that these compounds could be further developed into efficacious antibiotics.

Effect of polarized release of CXC-chemokines from wild-type and cystic fibrosis murine airway epithelial cells.

The respiratory epithelium lining the airway relies on mucociliary clearance and a complex network of inflammatory mediators to protect the lung. Alterations in the composition and volume of the periciliary liquid layer, as occur in cystic fibrosis (CF), lead to impaired mucociliary clearance and persistent airway infection. Moreover, the respiratory epithelium releases chemoattractants after infection, inciting airway inflammation. However, characterizing the inflammatory response of primary human airway epithelial cells to infection can be challenging because of genetic heterogeneity. Using well-characterized, differentiated, primary murine tracheal cells grown at an air-liquid interface, which provides an in vitro polarized epithelial model, we compared inflammatory gene expression and secretion in wild-type and ΔF508 CF airway cells after infection with Pseudomonas aeruginosa. The expression of several CXC-chemokines, including macrophage inflammatory protein-2, small inducible cytokine subfamily member 2, lipopolysaccharide-induced chemokine, and interferon-inducible cytokine-10, was markedly increased after infection, and these proinflammatory mediators were asymmetrically released from the airway epithelium, predominantly from the basolateral surface. Equal amounts of CXC-chemokines were released from wild-type and CF cells. Secreted mediators were concentrated in the thin, periciliary fluid layer, and the dehydrated apical microenvironment of CF airway epithelial cells amplified the inflammatory signal, potentially resulting in high chemokine concentration gradients across the epithelium. Consistent with this observation, the enhanced chemotaxis of wild-type neutrophils was detected in CF airway epithelial cultures, compared with wild-type cells. These data suggest that P. aeruginosa infection of the airway epithelium induces the expression and polarized secretion of CXC-chemokines, and the increased concentration gradient across the CF airway leads to an exaggerated inflammatory response.

Native and recombinant Slc26a3 (downregulated in adenoma, Dra) do not exhibit properties of 2Cl-/1HCO3- exchange.

The recent proposal that Dra/Slc26a3 mediates electrogenic 2Cl(-)/1HCO(3)(-) exchange suggests a required revision of classical concepts of electroneutral Cl(-) transport across epithelia such as the intestine. We investigated 1) the effect of endogenous Dra Cl(-)/HCO(3)(-) activity on apical membrane potential (V(a)) of the cecal surface epithelium using wild-type (WT) and knockout (KO) mice; and 2) the electrical properties of Cl(-)/(OH(-))HCO(3)(-) exchange by mouse and human orthologs of Dra expressed in Xenopus oocytes. Ex vivo (36)Cl(-) fluxes and microfluorometry revealed that cecal Cl(-)/HCO(3)(-) exchange was abolished in the Dra KO without concordant changes in short-circuit current. In microelectrode studies, baseline V(a) of Dra KO surface epithelium was slightly hyperpolarized relative to WT but depolarized to the same extent as WT during luminal Cl(-) substitution. Subsequent studies indicated that Cl(-)-dependent V(a) depolarization requires the anion channel Cftr. Oocyte studies demonstrated that Dra-mediated exchange of intracellular Cl(-) for extracellular HCO(3)(-) is accompanied by slow hyperpolarization and a modest outward current, but that the steady-state current-voltage relationship is unaffected by Cl(-) removal or pharmacological blockade. Further, Dra-dependent (36)Cl(-) efflux was voltage-insensitive in oocytes coexpressing the cation channels ENaC or ROMK. We conclude that 1) endogenous Dra and recombinant human/mouse Dra orthologs do not exhibit electrogenic 2Cl(-)/1HCO(3)(-) exchange; and 2) acute induction of Dra Cl(-)/HCO(3)(-) exchange is associated with secondary membrane potential changes representing homeostatic responses. Thus, participation of Dra in coupled NaCl absorption and in uncoupled HCO(3)(-) secretion remains compatible with electroneutrality of these processes, and with the utility of electroneutral transport models for predicting epithelial responses in health and disease.