FOXO inhibition rescues α-defensin expression in human intestinal organoids.

To mediate critical host-microbe interactions in the human small intestine, Paneth cells constitutively produce abundant levels of α-defensins and other antimicrobials. We report that the expression profile of these antimicrobials is dramatically askew in human small intestinal organoids (enteroids) as compared to that in paired tissue from which they are derived, with a reduction of α-defensins to nearly undetectable levels. Murine enteroids, however, recapitulate the expression profile of Paneth cell α-defensins seen in tissue. WNT/TCF signaling has been found to be instrumental in the regulation of α-defensins, yet in human enteroids exogenous stimulation of WNT signaling appears insufficient to rescue α-defensin expression. By stark contrast, forkhead box O (FOXO) inhibitor AS1842856 induced the expression of α-defensin mRNA in enteroids by >100,000-fold, restoring DEFA5 and DEFA6 to levels comparable to those found in primary human tissue. These results newly identify FOXO signaling as a pathway of biological and potentially therapeutic relevance for the regulation of human Paneth cell α-defensins in health and disease.

Early investigations into improving bowel and bladder function in fetal ovine myelomeningocele repair.

INTRODUCTION: Fetal myelomeningocele (MMC) repair improves lower extremity motor function. We have previously demonstrated that augmentation of fetal MMC repair with placental mesenchymal stromal cells (PMSCs) seeded on extracellular matrix (PMSC-ECM) further improves motor function in the ovine model. However, little progress has been made in improving bowel and bladder function, with many patients suffering from neurogenic bowel and bladder. We hypothesized that fetal MMC repair with PMSC-ECM would also improve bowel and bladder function. METHODS: MMC defects were surgically created in twelve ovine fetuses at median gestational age (GA) 73 days, followed by defect repair at GA101 with PMSC-ECM. Fetuses were delivered at GA141. Primary bladder function outcomes were voiding posture and void volumes. Primary bowel function outcome was anorectal manometry findings including resting anal pressure and presence of rectoanal inhibitory reflex (RAIR). Secondary outcomes were anorectal and bladder detrusor muscle thickness. PMSC-ECM lambs were compared to normal lambs (n = 3). RESULTS: Eighty percent of PMSC-ECM lambs displayed normal voiding posture compared to 100% of normal lambs (p = 1). Void volumes were similar (PMSC-ECM 6.1 ml/kg vs. normal 8.8 ml/kg, p = 0.4). Resting mean anal pressures were similar between cohorts (27.0 mmHg PMSC-ECM vs. normal 23.5 mmHg, p = 0.57). RAIR was present in 3/5 PMSC-ECM lambs that underwent anorectal manometry and all normal lambs (p = 0.46). Thicknesses of anal sphincter complex, rectal wall muscles, and bladder detrusor muscles were similar between cohorts. CONCLUSION: Ovine fetal MMC repair augmented with PMSC-ECM results in near-normal bowel and bladder function. Further work is needed to evaluate these outcomes in human patients.

Risk Stratification and Personal Protective Equipment Use in Pediatric Endoscopy During the Coronavirus Disease 2019 Outbreak: A Single-center Protocol.

SARS-CoV-2, the novel coronavirus causing coronavirus disease 2019 (COVID-19), is now a global pandemic. Human-to-human transmission has been documented to occur through respiratory secretions, feces, aerosols, and contaminated environmental surfaces. Pediatric patients present a unique challenge as they may have minimal symptoms and yet transmit disease. Endoscopists face risk for infection with viruses like SARS-CoV-2, as the aerosol generating nature of endoscopy diffuses respiratory disease that can be spread via an airborne and droplet route. We describe our center's methodology for pediatric patient risk stratification to facilitate responsible use of endoscopic resources during this crisis. We also describe our recommendations for use of personal protective equipment by endoscopists, with the goal of ensuring the safety of ourselves, our anesthesiology and endoscopy staff, and our patients.

Hirschsprung disease in the U.S. associated Pacific Islands: more common than expected.

INTRODUCTION: Tripler Army Medical Center (TAMC) in Honolulu, Hawaii, is uniquely situated to serve patients from the United States Associated Pacific Islands (USAPIs) through the congressionally funded Pacific Island Health Care Project (PIHCP). Because of time differences and distance, a web-based store-and-forward consultation and referral network was established using the internet to more efficiently and economically facilitate patient care. Using both electronic and hard copy records, we sought to establish the incidence of Hirschsprung Disease (HD) in children from the USAPI and contrast it to that of the developed world. METHODS: PIHCP website records as well as all the inpatient and outpatient medical records of patients referred to TAMC fortreatment of HD from 1994 to 2002 were reviewed. A diagnosis of HD was confirmed in all cases with full thickness biopsy. Incidence figures for HD are based on this review and on the birth rates for these islands from the International Data Base of the U.S. Bureau of the Census. RESULTS: There were 14 cases of short-segment HD referred over a nine year study period. Nine patients came from the Federated States of Micronesia (FSM) with an average annual incidence of 1:3190, which is 1.5 to 2 times the reported incidence in Western nations. Remarkably, seven of these nine were from Pohnpei State, capital of the FSM (annual incidence of 1:1370 or3-5 times that in the West). Three patients came from the Republic of the Marshall Islands (RMI), and two came from American Samoa (AS). There were no reported consanguineous marriages, associated syndromes, or complications of surgery. CONCLUSION: HD was found to be up to 2-3 times more common among people from the FSM than has been reported in the developed world. Given the limitations of providing care and obtaining data from all the USAPls with a population that is spread over a massive expanse of ocean larger than the continental United States, this incidence is likely an underestimation of HD among Pacific Islanders. A secure web-based referral network developed in 1998 has been invaluable in collecting epidemiologic data from these islands as well as in providing health care workers in the USAPI with an efficient and inexpensive means to seek consultation from specialists and sub-specialists at a major tertiary care medical facility.

Microencapsulated subunit vaccine approach to enterotoxigenic Escherichia coli and other mucosal pathogens.

Infections of the intestinal, urogenital, and respiratory tracts are serious health problems worldwide from both a morbidity and mortality perspective. Mucosal pathogens attach to surfaces of mucosa as a prerequisite for colonization and subsequent pathogenesis. By expressing various surface adhesins (colonization factors, CF) they are able to bind to specific mucosal receptors. Enterotoxigenic Escherichia coli (ETEC) can express numerous CF that allow them to attach to a variety of hosts. Mucosal immunity directed against pathogenic microorganisms is critical in host protection with secretory IgA being particularly important in preventing microoganisms from colonizing host cells. M cells likewise have an important immunological function in the small intestines by binding and transporting antigens to lymphocytes and macrophages thus enhancing the immune response. The use of subunit vaccines, such as antigen encapsulated microspheres, can act to effectively deliver specific antigens so as to optimize their immunological response. With the threat of bioterrorism becoming a reality in recent years, the miroencapsulation of antigens from potential bioterrorist agents may be an effective method of delivery so as to induce a level of protection in at risk individuals. The encapsulation of ETEC colonization factors in microspheres and their subsequent administration in small animals and humans has been conducted for many years. Evidence suggests that this type of delivery system for ETEC antigens may enhance their immunogenicity and provide protection against this microorganism.

Murine antibody response to intranasally administered enterotoxigenic Escherichia coli colonization factor CS6.

Enterotoxigenic Escherichia coli (ETEC) is the most common cause of bacterial diarrhea worldwide and is an important cause of infant morbidity and mortality in developing nations. ETEC colonization factors (CF) are virulence determinants that appear to be protective antigens in humans and are the major target of vaccine efforts. One of the most prevalent CF, CS6, is expressed by about 30% of ETEC worldwide. This study was designed to compare the immunogenicity between encapsulated CS6 (CS6-PLG) and unencapsulated CS6. Recombinant CS6 was purified and encapsulated in biodegradable poly(DL-lactide-co-glycolide) (PLG) microspheres using current Good Manufacturing Practices (cGMP). CS6-PLG and CS6 were administered intranasally (IN) to BALB/c mice in three vaccinations 4 weeks apart. Enzyme linked immunosorbent assay (ELISA) was used to measure the anti-CS6 response in serum and mucosal secretions following each of the three inoculations. Mice vaccinated with two or three doses of CS6-PLG demonstrated a significantly greater rise in serum anti-CS6 IgG and mucosal IgA titer values than those immunized with two or three doses of CS6 alone. Three doses of CS6-PLG led to anti-CS6 serum IgG and mucosal IgA titer values 14-fold and 4.4-fold greater, respectively, than three doses of CS6 (P<0.02). IN administered CS6 to mice is safe and highly immunogenic either alone or when encapsulated in microspheres. PLG microsphere encapsulation of CS6 significantly augments the antibody response to that antigen when administered to a mucosal surface.