Surgical Necrotizing Enterocolitis and Spontaneous Intestinal Perforation Lead to Severe Growth Failure in Infants.

OBJECTIVE: We aimed to determine the incidence of growth failure in infants with necrotizing enterocolitis (NEC) or spontaneous intestinal perforation (SIP) and whether initial laparotomy versus peritoneal drainage (PD) impacted the likelihood of growth failure. SUMMARY BACKGROUND DATA: Infants with surgical NEC and SIP have high mortality, and most have neurodevelopmental impairment and poor growth. Existing literature on growth outcomes for these infants is limited. METHODS: This is a preplanned secondary study of the Necrotizing Enterocolitis Surgery Trial dataset. The primary outcome was growth failure (Z-score for weight <-2.0) at 18 to 22 months. We used logistic regression, including diagnosis and treatment, as covariates. Secondary outcomes were analyzed using the Fisher exact or Pearson χ2 test for categorical variables and the Wilcoxon rank sum test or one-way ANOVA for continuous variables. RESULTS: Among 217 survivors, 207 infants (95%) had primary outcome data. Growth failure at 18 to 22 months occurred in 24/50 (48%) of NEC infants versus 65/157 (42%) SIP (P=0.4). The mean weight-for-age Z-score at 18 to 22 months in NEC infants was -2.05±0.99 versus -1.84±1.09 SIP (P=0.2), and the predicted mean weight-for-age Z-score SIP (Beta -0.27; 95% CI: -0.53, -0.01; P=0.041). Median declines in weight-for-age Z-score between birth and 18 to 22 months were significant in all infants but most severe (>2) in NEC infants (P=0.2). CONCLUSIONS: This first ever prospective study of growth outcomes in infants with surgical NEC or SIP demonstrates that growth failure is very common, especially in infants with NEC, and persists at 18-22 months.

Are We Enhancing Recovery After Neonatal Surgery? Assessment of Enhanced Recovery After Surgery Principles for Ostomy Takedown.

INTRODUCTION: Enhanced Recovery After Surgery (ERAS) guidelines in adults have demonstrated reduced complications, length of stay, and cost. However, neonatal ERAS studies are limited and translation of adult ERAS guidelines to neonates is challenging. Furthermore, the knowledge, perception, and practice of neonatal ERAS guidelines is largely unknown. Our aim is to address this practice gap by determining current practice of the 2020 neonatal intestinal surgery ERAS guidelines at our institution and evaluating postoperative outcomes. METHODS: A retrospective study was conducted of patients <1 y who underwent elective ostomy takedown at a single-center tertiary children's hospital between 2013 and 2023. A 13-point ERAS score was developed. Demographics, clinical course, pain management, nutrition, ERAS scores, and outcomes were analyzed using descriptive statistics, logistic and negative binomial regression. RESULTS: One hundred eighty-six patients met the inclusion criteria. At surgery, the median age was 124 d (interquartile range [IQR] 81-220) and median weight was 4360 g (IQR 2920-7200). The median ERAS score was 6 (IQR 5-7). The highest scores were for appropriate (97.9%) and timely (91.9%) prophylactic antibiotics, and the lowest for preventing intraoperative hypothermia (14.5%), limiting opioids (9.1%), and early enteral feeding postoperatively (24.7%). Surgical site infection occurred in 14.5% and median length of stay was 28 (IQR 5-127) d. CONCLUSIONS: Our institution's current practice of the 2020 neonatal intestinal surgery ERAS guidelines was poor. We identified opportunities for improvement including postoperative antibiotic administration, prevention of intraoperative hypothermia, nutrition, and pain management. Future studies will focus on implementation of neonatal ERAS guidelines at our institution and evaluation of adherence and outcomes.

Comparison of two formulations of intravenous lipid emulsions in pediatric intestinal failure.

PURPOSE: The effect of different types of lipid emulsion may guide therapy of patients with intestinal failure (IF) to limit morbidity such as intestinal failure-associated liver disease (IFALD). METHODS: A retrospective chart review of pediatric patients with IF who received soybean oil lipid emulsion (SL) or mixed oil lipid emulsion (ML) was performed. Data over 1 year were collected. RESULTS: Forty-five patients received SL and 34 received ML. There were no differences in the incidence (82 versus 74%, P = 0.35) or resolution (86 versus 92%, P = 0.5) of IFALD between the cohorts. The median dose of ML was higher compared to SL (2 versus 1 g/kg/day, P < 0.001). If resolved, IFALD resolved rapidly in the ML cohort compared to the SL cohort (67 versus 37 days, P = 0.01). Weight gain was higher in the ML compared to the SL cohort at resolution of IFALD or 1 year from diagnosis of IF (P = 0.009). CONCLUSION: The administration of ML did not alter the incidence or resolution of IFALD compared to SL in pediatric IF. There was rapid resolution of IFALD and enhanced weight gain in the ML cohort compared to SL in pediatric IF.

Investigation of murine host sex as a biological variable in epithelial barrier function and muscle contractility in human intestinal organoids.

Intestinal failure (IF) occurs when intestinal surface area or function is not sufficient to support digestion and nutrient absorption. Human intestinal organoid (HIO)-derived tissue-engineered intestine is a potential cure for IF. Research to date has demonstrated successful HIO transplantation (tHIO) into mice with significant in vivo maturation. An area lacking in the literature is exploration of murine host sex as a biological variable (SABV) in tHIO function. In this study, we investigate murine host SABV in tHIO epithelial barrier function and muscle contractility. HIOs were generated in vitro and transplanted into nonobese diabetic, severe combined immunodeficiency gamma chain deficient male and female mice. tHIOs were harvested after 8-12 weeks in vivo. Reverse transcriptase polymerase chain reaction and immunohistochemistry were conducted to compare tight junctions and contractility-related markers in tHIOs. An Ussing chamber and contractility apparatus were used to evaluate tHIO epithelial barrier and muscle contractile function, respectively. The expression and morphology of tight junction and contractility-related markers from tHIOs in male and female murine hosts is not significantly different. Epithelial barrier function as measured by transepithelial resistance, short circuit current, and fluorescein isothiocyanate-dextran permeability is no different in tHIOs from male and female hosts, although these results may be limited by HIO epithelial immaturity and a short flux time. Muscle contractility as measured by total contractile activity, amplitude, frequency, and tension is not significantly different in tHIOs from male and female hosts. The data suggest that murine host sex may not be a significant biological variable influencing tHIO function, specifically epithelial barrier maintenance and muscle contractility, though limitations exist in our model.

Sodium Bicarbonate Locks May Be a Safe and Effective Alternative in Pediatric Intestinal Failure: A Pilot Study.

This was a retrospective study that compared outcomes in pediatric intestinal failure (IF) patients that were switched from ethanol lock therapy (ELT) to sodium bicarbonate lock therapy (SBLT). The primary outcome was rate of catheter-related blood stream infections (CRBSI). The secondary outcomes were number of hospitalizations, emergency room (ER) visits, central venous catheter (CVC)-related complications. In 4 patients, median rates of CRBSI were 2.77 (interquartile range [IQR] 0.6-5.6) on ELT versus 0 on SBLT per 1000 catheter days ( P = 0.17). The median rates of hospitalizations and ER visits for CVC-related complications were 6.1 (IQR 3.2-10.2) on ELT versus 0 on SBLT (IQR 0-0; P = 0.11) and 2.8 (IQR 2-3.6) on ELT versus 1.8 (IQR 0-3.7) on SBLT per 1000 catheter days ( P = 0.50), respectively. Rates of CVC-related complications were similar. No adverse events were reported. SBLT may be safe and effective for pediatric IF.

Evaluation of Murine Host Sex as a Biological Variable in Transplanted Human Intestinal Organoid Development.

BACKGROUND: Human intestinal organoids (HIOs), when transplanted into immunocompromised mice (tHIOs), demonstrate significant growth and maturation. While both male and female mice are reported to be viable hosts for these experiments, a direct comparison of sex-related differences in tHIO structure and development has not been performed. AIMS: We sought to identify host sex-related differences in tHIO engraftment, morphology, and epithelial and mesenchymal development. METHODS: HIOs were generated in vitro and transplanted beneath the kidney capsule of NSG male and female mice. tHIOs were harvested at 8-9 weeks. Anthropometric measurements were captured. tHIOs were divided in half and histology or RT-qPCR performed. Morphology was evaluated and epithelial architecture graded on a scale of 1 (absence of crypts/villi) to 4 (elongated crypt-villus axis). RT-qPCR and immunofluorescence microscopy were performed for epithelial and mesenchymal differentiation markers. RESULTS: Host survival and tHIO engraftment were equivalent in male and female hosts. tHIO weight and length were also equivalent between groups. The number of lumens per tHIOs from male and female hosts was similar, but the mean lumen circumference was larger for tHIOs from male hosts. tHIOs from male hosts were more likely to demonstrate higher grades of epithelial development. However, both groups showed similar differentiation into secretory and absorptive epithelial lineages. Markers for intestinal identity, mesenchymal development, and brush border enzymes were also expressed similarly between groups. CONCLUSIONS: While male host sex was associated with larger tHIO lumen size and mucosal maturation, tHIOs from both groups had similar engraftment, growth, and epithelial and mesenchymal cytodifferentiation.

Bioengineering of the digestive tract: approaching the clinic.

The field of regenerative medicine is developing technologies that, in the near future, will offer alternative approaches to either cure diseases affecting the gastrointestinal tract or slow their progression by leveraging the intrinsic ability of our tissues and organs to repair after damage. This article will succinctly illustrate the three technologies that are closer to clinical translation-namely, human intestinal organoids, sphincter bioengineering and decellularization, whereby the cellular compartment of a given segment of the digestive tract is removed to obtain a scaffold consisting of the extracellular matrix. The latter will be used as a template for the regeneration of a functional organ, whereby the newly generated cellular compartment will be obtained from the patient's own cells. Although clinical application of this technology is approaching, product development challenges are being tackled to warrant safety and efficacy.

In Vivo Transplantation of Human Intestinal Organoids Enhances Select Tight Junction Gene Expression.

BACKGROUND: Short bowel syndrome is a potentially fatal condition with inadequate management options. Tissue-engineered small intestine (TESI) is a promising solution, but confirmation of TESI function will be crucial before human application. We sought to define intestinal epithelial barrier function in human intestinal organoid (HIO)-derived TESI. MATERIALS AND METHODS: HIOs were generated in vitro from human embryonic stem cells. After 1 mo, HIOs were collected for analysis or transplanted into the kidney capsule of immunocompromised mice. Transplanted HIOs (tHIOs) were harvested for analysis at 4 or 8 wk. Reverse transcription quantitative polymerase chain reaction and immunofluorescent staining were performed for tight junction components: claudin 3 (CLDN3), claudin 15 (CLDN15), occludin (OCLN), and zonula occludens-1, or tight junction protein-1 (TJP1/ZO-1). RESULTS: Four-week-old tHIOs demonstrated significantly (P < 0.05) higher levels of CLDN15 (6x), OCLN (4x), and TJP1/ZO-1 (3x) normalized to GAPDH than in vitro HIOs. Eight-week-old tHIOs demonstrated significantly (P < 0.05) higher expression levels of CLDN3 (26x), CLDN15 (29x), OCLN (4x), and TJP1/ZO-1 (5x) than in vitro HIOs. There was no significant difference in expression of these tight junction components between 4- and 8-week-old tHIOs. Immunofluorescent staining revealed the presence of claudin 3, claudin 15, occludin, and zonula occludens-1 in both in vitro HIOs and tHIOs; however, the morphology appeared more mature in tHIOs. CONCLUSIONS: In vitro HIOs have lower levels of tight junction mRNA, and tight junction proteins appear morphologically immature. Transplantation facilitates maturation of the HIOs and enhances select tight junction gene expression.

Tunneled central venous catheters in pediatric intestinal failure: a single-center experience.

BACKGROUND: Parenteral nutrition for intestinal failure (IF) often requires a tunneled central venous catheter (CVC). The purpose of this study was to characterize complications after CVC placement and contributors to line loss in pediatric IF patients. METHODS: An institutional review board-approved retrospective review of pediatric (<18 y) IF patients who had a silicone tunneled CVC newly inserted or exchanged from 2012 to 2016 in an IF center was conducted. Patient demographics, procedure service (surgery versus interventional radiology), procedure type (new versus exchange), vessel, and complications related to CVCs were evaluated. Complications included dislodgement, infection, break, occlusion/malfunction, and others. An ethanol-lock protocol for silicone CVCs in IF patients was instituted in January 2012. RESULTS: Twenty-nine IF patients with tunneled CVCs were identified with 182 lines and 18,534 line d. Median age at line insertion was 17.1 mo (interquartile range [IQR] 7.6-31.5) with a median of five catheters (IQR 2-8) per patient. There were 19.2 complications per 1000 line d. Occlusions/malfunctions were the most common complication (6.0/1000 line d) followed by breaks (5.6/1000 line d). Median life of catheters was 51.5 d (IQR 21-129). On regression, adjusting for age, insertion service, and procedure type, shorter line life was associated with younger age (P = 0.04) and placement by interventional radiology (P < 0.01). Dislodgement was associated with newly placed lines relative risk 6.5 (95% CI 2.2-28.8). CONCLUSIONS: CVCs in pediatric IF patients have frequent complications and short line lifetimes. Dislodgement of CVC was an unexpectedly common complication with loss of access in newly placed lines. There may be modifiable processes to mitigate CVC complications.

The Association for Academic Surgery 2011-present: standing on the shoulders of giants.

The Association for Academic Surgery (AAS), which is a society dedicated to inspiring and developing young academic surgeons, recently celebrated its 50th anniversary. Each decade since its inception has seen incredible growth. This most recent decade, from 2011 to present, has been characterized by: (1) reevaluation and clarification of the society's vision, mission, core values and organizational structure; (2) diversification of the membership and leadership; (3) support for international outreach and global surgery research; (4) expansion of its impact through social media; and (5) adaptability to a changing political climate.

Human and mouse tissue-engineered small intestine both demonstrate digestive and absorptive function.

Short bowel syndrome (SBS) is a devastating condition in which insufficient small intestinal surface area results in malnutrition and dependence on intravenous parenteral nutrition. There is an increasing incidence of SBS, particularly in premature babies and newborns with congenital intestinal anomalies. Tissue-engineered small intestine (TESI) offers a therapeutic alternative to the current standard treatment, intestinal transplantation, and has the potential to solve its biggest challenges, namely donor shortage and life-long immunosuppression. We have previously demonstrated that TESI can be generated from mouse and human small intestine and histologically replicates key components of native intestine. We hypothesized that TESI also recapitulates native small intestine function. Organoid units were generated from mouse or human donor intestine and implanted into genetically identical or immunodeficient host mice. After 4 wk, TESI was harvested and either fixed and paraffin embedded or immediately subjected to assays to illustrate function. We demonstrated that both mouse and human tissue-engineered small intestine grew into an appropriately polarized sphere of intact epithelium facing a lumen, contiguous with supporting mesenchyme, muscle, and stem/progenitor cells. The epithelium demonstrated major ultrastructural components, including tight junctions and microvilli, transporters, and functional brush-border and digestive enzymes. This study demonstrates that tissue-engineered small intestine possesses a well-differentiated epithelium with intact ion transporters/channels, functional brush-border enzymes, and similar ultrastructural components to native tissue, including progenitor cells, whether derived from mouse or human cells.

Vitrification preserves murine and human donor cells for generation of tissue-engineered intestine.

BACKGROUND: Short bowel syndrome causes significant morbidity and mortality. Tissue-engineered intestine may serve as a viable replacement. Tissue-engineered small intestine (TESI) has previously been generated in the mouse model from donor cells that were harvested and immediately reimplanted; however, this technique may prove impossible in children who are critically ill, hemodynamically unstable, or septic. We hypothesized that organoid units (OU), multicellular clusters containing epithelium and mesenchyme, could be cryopreserved for delayed production of TESI. METHODS: OU were isolated from <3 wk-old mouse or human ileum. OU were then cryopreserved by either standard snap freezing or vitrification. In the snap freezing protocol, OU were suspended in cryoprotectant and transferred directly to -80°C for storage. The vitrification protocol began with a stepwise increase in cryoprotectant concentration followed by liquid supercooling of the OU solution to -13°C and nucleation with a metal rod to induce vitrification. Samples were then cooled to -80°C at a controlled rate of -1°C/min and subsequently plunged into liquid nitrogen for long-term storage. OU from both groups were maintained in cryostorage for at least 72 h and thawed in a 37°C water bath. Cryoprotectant was removed with serial sucrose dilutions and OU were assessed by Trypan blue assay for post-cryopreservation viability. Via techniques previously described by our laboratory, the thawed murine or human OU were either cultured in vitro or implanted on a scaffold into the omentum of a syngeneic or irradiated Nonobese Diabetic/Severe Combined Immunodeficiency, gamma chain deficient adult mouse. The resultant TESI was analyzed by histology and immunofluorescence. RESULTS: After cryopreservation, the viability of murine OU was significantly higher in the vitrification group (93 ± 2%, mean ± standard error of the mean) compared with standard freezing (56 ± 6%) (P < 0.001, unpaired t-test, n = 25). Human OU demonstrated similar viability after vitrification (89 ± 2%). In vitro culture of thawed OU produced expanding epithelial spheres supported by a layer of mesenchyme. TESI was successfully generated from the preserved OU. Hematoxylin and eosin staining demonstrated a mucosa composed of a simple columnar epithelium whereas immunofluorescence staining confirmed the presence of both progenitor and differentiated epithelial cells. Furthermore, beta-2-microglobulin confirmed that the human TESI epithelium originated from human cells. CONCLUSIONS: We demonstrated improved multicellular viability after vitrification over conventional cryopreservation techniques and the first successful vitrification of murine and human OU with subsequent TESI generation. Clinical application of this method may allow for delayed autologous implantation of TESI for children in extremis.

Bioengineering of Intestinal Grafts.

Intestinal failure manifests as an impaired capacity of the intestine to sufficiently absorb vital nutrients and electrolytes essential for growth and well-being in pediatric and adult populations. Although parenteral nutrition remains the mainstay therapeutic approach, the pursuit of a definitive and curative strategy, such as regenerative medicine, is imperative. Substantial advancements in the field of engineered intestinal tissues present a promising avenue for addressing intestinal failure; nevertheless, extensive research is still necessary for effective translation from experimental benchwork to clinical bedside applications.

Infant and Adult Human Intestinal Enteroids are Morphologically and Functionally Distinct.

Background & Aims: Human intestinal enteroids (HIEs) are gaining recognition as physiologically relevant models of the intestinal epithelium. While HIEs from adults are used extensively in biomedical research, few studies have used HIEs from infants. Considering the dramatic developmental changes that occur during infancy, it is important to establish models that represent infant intestinal characteristics and physiological responses. Methods: We established jejunal HIEs from infant surgical samples and performed comparisons to jejunal HIEs from adults using RNA sequencing (RNA-Seq) and morphologic analyses. We validated differences in key pathways through functional studies and determined if these cultures recapitulate known features of the infant intestinal epithelium. Results: RNA-Seq analysis showed significant differences in the transcriptome of infant and adult HIEs, including differences in genes and pathways associated with cell differentiation and proliferation, tissue development, lipid metabolism, innate immunity, and biological adhesion. Validating these results, we observed a higher abundance of cells expressing specific enterocyte, goblet cell and enteroendocrine cell markers in differentiated infant HIE monolayers, and greater numbers of proliferative cells in undifferentiated 3D cultures. Compared to adult HIEs, infant HIEs portray characteristics of an immature gastrointestinal epithelium including significantly shorter cell height, lower epithelial barrier integrity, and lower innate immune responses to infection with an oral poliovirus vaccine. Conclusions: HIEs established from infant intestinal tissues reflect characteristics of the infant gut and are distinct from adult cultures. Our data support the use of infant HIEs as an ex-vivo model to advance studies of infant-specific diseases and drug discovery for this population.

Insights into Human Norovirus Cultivation in Human Intestinal Enteroids.

Human noroviruses (HuNoVs) are a significant cause of epidemic and sporadic acute gastroenteritis worldwide. The lack of a reproducible culture system hindered the study of HuNoV replication and pathogenesis for almost a half-century. This barrier was overcome with our successful cultivation of multiple HuNoV strains in human intestinal enteroids (HIEs), which has significantly advanced HuNoV research. We optimized culture media conditions and generated genetically-modified HIE cultures to enhance HuNoV replication in HIEs. Building upon these achievements, we now present new insights to this culture system, which involve testing different media, unique HIE lines, and additional virus strains. HuNoV infectivity was evaluated and compared in new HIE models, including HIEs generated from different intestinal segments of individual adult organ donors, HIEs from human intestinal organoids produced from directed differentiation of human embryonic stem cells into intestinal organoids that were transplanted and matured in mice before making enteroids (H9tHIEs), genetically-engineered (J4 FUT2 knock-in [ KI ], J2 STAT1 knock-out [ KO ]) HIEs, as well as HIEs derived from a patient with common variable immunodeficiency (CVID) and from infants. Our findings reveal that small intestinal HIEs, but not colonoids, from adults, H9tHIEs, HIEs from a CVID patient, and HIEs from infants support HuNoV replication with segment and strain-specific differences in viral infection. J4 FUT2-KI HIEs exhibit the highest susceptibility to HuNoV infection, allowing the cultivation of a broader range of GI and GII HuNoV strains than previously reported. Overall, these results contribute to a deeper understanding of HuNoVs and highlight the transformative potential of HIE cultures in HuNoV research. Importance: HuNoVs cause global diarrheal illness and chronic infections in immunocompromised patients. This manuscript reports approaches for cultivating HuNoVs in secretor positive human intestinal enteroids (HIEs). HuNoV infectivity was compared in new HIE models, including ones from i) different intestinal segments of single donors, ii) human embryonic stem cell-derived organoids transplanted into mice, iii) genetically-modified lines, and iv) a patient with chronic variable immunodeficiency disease. HIEs from small intestine, but not colon, support HuNoV replication with donor, segment and strain-specific variations. Unexpectedly, HIEs from one donor are resistant to GII.3 infection. The genetically-modified J4 FUT2-KI HIEs enable cultivation of a broad range of GI and GII genotypes. New insights into strain-specific differences in HuNoV replication in HIEs support this platform for advancing understanding of HuNoV biology and developing potential therapeutics.

Infant and adult human intestinal enteroids are morphologically and functionally distinct.

Human intestinal enteroids (HIEs) are gaining recognition as physiologically relevant models of the intestinal epithelium. While HIEs from adults are used extensively in biomedical research, few studies have used HIEs from infants. Considering the dramatic developmental changes that occur during infancy, it is important to establish models that represent infant intestinal characteristics and physiological responses. We established jejunal HIEs from infant surgical samples and performed comparisons to jejunal HIEs from adults using RNA sequencing (RNA-Seq) and morphologic analyses. We then validated differences in key pathways through functional studies and determined whether these cultures recapitulate known features of the infant intestinal epithelium. RNA-Seq analysis showed significant differences in the transcriptome of infant and adult HIEs, including differences in genes and pathways associated with cell differentiation and proliferation, tissue development, lipid metabolism, innate immunity, and biological adhesion. Validating these results, we observed a higher abundance of cells expressing specific enterocyte, goblet cell, and enteroendocrine cell markers in differentiated infant HIE monolayers, and greater numbers of proliferative cells in undifferentiated 3D cultures. Compared to adult HIEs, infant HIEs portray characteristics of an immature gastrointestinal epithelium including significantly shorter cell height, lower epithelial barrier integrity, and lower innate immune responses to infection with an oral poliovirus vaccine. HIEs established from infant intestinal tissues reflect characteristics of the infant gut and are distinct from adult cultures. Our data support the use of infant HIEs as an ex vivo model to advance studies of infant-specific diseases and drug discovery for this population. IMPORTANCE: Tissue or biopsy stem cell-derived human intestinal enteroids are increasingly recognized as physiologically relevant models of the human gastrointestinal epithelium. While enteroids from adults and fetal tissues have been extensively used for studying many infectious and non-infectious diseases, there are few reports on enteroids from infants. We show that infant enteroids exhibit both transcriptomic and morphological differences compared to adult cultures. They also differ in functional responses to barrier disruption and innate immune responses to infection, suggesting that infant and adult enteroids are distinct model systems. Considering the dramatic changes in body composition and physiology that begin during infancy, tools that appropriately reflect intestinal development and diseases are critical. Infant enteroids exhibit key features of the infant gastrointestinal epithelium. This study is significant in establishing infant enteroids as age-appropriate models for infant intestinal physiology, infant-specific diseases, and responses to pathogens.

Insights into human norovirus cultivation in human intestinal enteroids.

Human noroviruses (HuNoVs) are a significant cause of epidemic and sporadic acute gastroenteritis worldwide. The lack of a reproducible culture system hindered the study of HuNoV replication and pathogenesis for almost a half-century. This barrier was overcome with our successful cultivation of multiple HuNoV strains in human intestinal enteroids (HIEs), which has significantly advanced HuNoV research. We optimized culture media conditions and generated genetically modified HIE cultures to enhance HuNoV replication in HIEs. Building upon these achievements, we now present new insights into this culture system, which involve testing different media, unique HIE lines, and additional virus strains. HuNoV infectivity was evaluated and compared in new HIE models, including HIEs generated from different intestinal segments of individual adult organ donors, HIEs from human intestinal organoids produced from directed differentiation of human embryonic stem cells that were then transplanted and matured in mice before making enteroids (H9tHIEs), genetically engineered (J4FUT2 knock-in [KI], J2STAT1 knockout [KO]) HIEs, as well as HIEs derived from a patient with common variable immunodeficiency (CVID) and from infants. Our findings reveal that small intestinal HIEs, but not colonoids, from adults, H9tHIEs, HIEs from a CVID patient, and HIEs from infants support HuNoV replication with segment and strain-specific differences in viral infection. J4FUT2-KI HIEs exhibit the highest susceptibility to HuNoV infection, allowing the cultivation of a broader range of genogroup I and II HuNoV strains than previously reported. Overall, these results contribute to a deeper understanding of HuNoVs and highlight the transformative potential of HIE cultures in HuNoV research.IMPORTANCEHuman noroviruses (HuNoVs) cause global diarrheal illness and chronic infections in immunocompromised patients. This paper reports approaches for cultivating HuNoVs in secretor positive human intestinal enteroids (HIEs). HuNoV infectivity was compared in new HIE models, including ones from (i) different intestinal segments of single donors, (ii) human embryonic stem cell-derived organoids transplanted into mice, (iii) genetically modified lines, and (iv) a patient with common variable immunodeficiency disease. HIEs from small intestine, but not colon, support HuNoV replication with donor, segment, and strain-specific variations. Unexpectedly, HIEs from one donor are resistant to GII.3 infection. The genetically modified J4FUT2 knock-in (KI) HIEs enable cultivation of a broad range of GI and GII genotypes. New insights into strain-specific differences in HuNoV replication in HIEs support this platform for advancing understanding of HuNoV biology and developing potential therapeutics.

Delayed family reunification of pediatric disaster survivors increases mortality and inpatient hospital costs: a simulation study.

BACKGROUND: Disasters occur randomly and can severely tax the health care delivery system of affected and surrounding regions. A significant proportion of disaster survivors are children, who have unique medical, psychosocial, and logistical needs after a mass casualty event. Children are often transported to specialty centers after disasters for a higher level of pediatric care, but this can also lead to separation of these survivors from their families. In a recent theoretical article, we showed that the availability of a pediatric trauma center after a mass casualty event would decrease the time needed to definitively treat the pediatric survivor cohort and decrease pediatric mortality. However, we also found that if the pediatric center was too slow in admitting and discharging patients, these benefits were at risk of being lost as children became "trapped" in the slow center. We hypothesized that this effect could result in further increased mortality and greater costs. METHODS: Here, we expand on these ideas to test this hypothesis via mathematical simulation. We examine how a delay in discharge of part of the pediatric cohort is predicted to affect mortality and the cost of inpatient care in the setting of our model. RESULTS: We find that mortality would increase slightly (from 14.2%-16.1%), and the cost of inpatient care increases dramatically (by a factor of 21) if children are discharged at rates consistent with reported delays to reunification after a disaster from the literature. CONCLUSIONS: Our results argue for the ongoing improvement of identification technology and logistics for rapid reunification of pediatric survivors with their families after mass casualty events.

Mesenchymal-specific inhibition of vascular endothelial growth factor (VEGF) attenuates growth in neonatal mice.

BACKGROUND: Vascular endothelial growth factor (VEGF) is a key mediator of angiogenesis and vasculogenesis. However, the role of VEGF in the regulation of neonatal mouse development is not completely defined. We sought to determine the effect of VEGF inhibition on the development of the neonatal mouse using a transgenic approach. MATERIALS AND METHODS: We generated triple transgenic mice that express the soluble VEGF receptor, (sFlt-1), specifically in the mesenchyme (dermo-1(Cre)- tetracycline reverse transcriptional activator (rtTA)(flox/flox)-tet(0)-sflt-1). Mothers of the pups (transgenic and littermate controls) were fed doxycycline chow at birth for transgene activation via breast milk, and the pups were sacrificed at various time points. To test reversibility of the phenotype, mice from both groups (n = 6) were switched to normal chow at P50 and monthly weights were measured for 9 mo. RESULTS: Dermo-1(Cre)-rtTA(flox/flox)-tet(0)-sflt-1 mice were smaller compared with littermate controls at P21. There was a significant reduction in tissue VEGF levels following sFlt-1 expression. The rate of growth was reduced but did not impact overall survival after 1 y. A significant reduction in organ size as a percentage body weight was seen in the kidney and stomach, whereas the weight of the colon and spleen were relatively increased; however, no gross histologic difference was observed. After 6 mo on normal diet, the dermo-1(Cre)-rtTA(flox/flox)-tet(0)-sflt-1 mouse's weight doubled, indicating reversibility of phenotype. CONCLUSION: Mesenchymal-specific inhibition of VEGF in neonatal mice results in a severe but reversible arrest in somatic growth that does not affect overall survival at 1 yr. This mouse is a useful tool to test the function of VEGF in somatic growth.

Regenerative medicine technologies applied to transplant medicine. An update.

Regenerative medicine (RM) is changing how we think and practice transplant medicine. In regenerative medicine, the aim is to develop and employ methods to regenerate, restore or replace damaged/diseased tissues or organs. Regenerative medicine investigates using tools such as novel technologies or techniques, extracellular vesicles, cell-based therapies, and tissue-engineered constructs to design effective patient-specific treatments. This review illustrates current advancements in regenerative medicine that may pertain to transplant medicine. We highlight progress made and various tools designed and employed specifically for each tissue or organ, such as the kidney, heart, liver, lung, vasculature, gastrointestinal tract, and pancreas. By combing both fields of transplant and regenerative medicine, we can harbor a successful collaboration that would be beneficial and efficacious for the repair and design of de novo engineered whole organs for transplantations.