A foundational approach to culture and analyze malnourished organoids.

The gastrointestinal (GI) epithelium plays a major role in nutrient absorption, barrier formation, and innate immunity. The development of organoid-based methodology has significantly impacted the study of the GI epithelium, particularly in the fields of mucosal biology, immunity, and host-microbe interactions. Various effects on the GI epithelium, such as genetics and nutrition, impact patients and alter disease states. Thus, incorporating these effects into organoid-based models will facilitate a better understanding of disease progression and offer opportunities to evaluate therapeutic candidates. One condition that has a significant effect on the GI epithelium is malnutrition, and studying the mechanistic impacts of malnutrition would enhance our understanding of several pathologies. Therefore, the goal of this study was to begin to develop methodology to generate viable malnourished organoids with accessible techniques and resources that can be used for a wide array of mechanistic studies. By selectively limiting distinct macronutrient components of organoid media, we were able to successfully culture and evaluate malnourished organoids. Genetic and protein-based analyses were used to validate the approach and confirm the presence of known biomarkers of malnutrition. Additionally, as proof-of-concept, we utilized malnourished organoid-derived monolayers to evaluate the effect of malnourishment on barrier formation and the ability of the bacterial pathogen Shigella flexneri to infect the GI epithelium. This work serves as the basis for new and exciting techniques to alter the nutritional state of organoids and investigate the related impacts on the GI epithelium.

A Versatile Human Intestinal Organoid-Derived Epithelial Monolayer Model for the Study of Enteric Pathogens.

Gastrointestinal infections cause significant morbidity and mortality worldwide. The complexity of human biology and limited insights into host-specific infection mechanisms are key barriers to current therapeutic development. Here, we demonstrate that two-dimensional epithelial monolayers derived from human intestinal organoids, combined with in vivo-like bacterial culturing conditions, provide significant advancements for the study of enteropathogens. Monolayers from the terminal ileum, cecum, and ascending colon recapitulated the composition of the gastrointestinal epithelium, in which several techniques were used to detect the presence of enterocytes, mucus-producing goblet cells, and other cell types following differentiation. Importantly, the addition of receptor activator of nuclear factor kappa-B ligand (RANKL) increased the presence of M cells, critical antigen-sampling cells often exploited by enteric pathogens. For infections, bacteria were grown under in vivo-like conditions known to induce virulence. Overall, interesting patterns of tissue tropism and clinical manifestations were observed. Shigella flexneri adhered efficiently to the cecum and colon; however, invasion in the colon was best following RANKL treatment. Both Salmonella enterica serovars Typhi and Typhimurium displayed different infection patterns, with S. Typhimurium causing more destruction of the terminal ileum and S. Typhi infecting the cecum more efficiently than the ileum, particularly with regard to adherence. Finally, various pathovars of Escherichia coli validated the model by confirming only adherence was observed with these strains. This work demonstrates that the combination of human-derived tissue with targeted bacterial growth conditions enables powerful analyses of human-specific infections that could lead to important insights into pathogenesis and accelerate future vaccine development. IMPORTANCE While traditional laboratory techniques and animal models have provided valuable knowledge in discerning virulence mechanisms of enteric pathogens, the complexity of the human gastrointestinal tract has hindered our understanding of physiologically relevant, human-specific interactions; and thus, has significantly delayed successful vaccine development. The human intestinal organoid-derived epithelial monolayer (HIODEM) model closely recapitulates the diverse cell populations of the intestine, allowing for the study of human-specific infections. Differentiation conditions permit the expansion of various cell populations, including M cells that are vital to immune recognition and the establishment of infection by some bacteria. We provide details of reproducible culture methods and infection conditions for the analyses of Shigella, Salmonella, and pathogenic Escherichia coli in which tissue tropism and pathogen-specific infection patterns were detected. This system will be vital for future studies that explore infection conditions, health status, or epigenetic differences and will serve as a novel screening platform for therapeutic development.

Missed Opportunities in the Preparticipation Physical Examination for High School Athletes.

The preparticipation sports examination (PPE) is required for US high school athletes. We queried members of the Illinois Chapter of the American Academy of Pediatrics (ICAAP) about what they actually include in a PPE, allotted PPE time, and counseling about full contact sports participation. Of 2300 ICAAP members contacted electronically, 228 (10%) responded, yielding 205 usable surveys. When performing a PPE, 34 (25%) always complete all of 6 selected AAP-recommended PPE components and 102 (74%) always get a concussion history. In contrast, 29 (21%) always complete all of the 6 selected AAP PPE components and 70 (49%) always get a concussion history as part of well-child checkup. Main interferences with clinician proficiency are lack of time and training cited by 49% and 37%, respectively. Pediatricians were evenly divided about whether or not to counsel against full contact sports, but were more likely to do so after the athlete sustained a first concussion.

Public Attitudes and Knowledge About Youth Sports Participation and Concussion Risk in an Urban Area.

BACKGROUND: Every year, millions of children in the United States participate in youth full-contact sports, which carry concussion risks-the long-term sequelae of which are not well understood. We examined the attitudes and knowledge of adults in Chicago about youth sports participation, concussion risk, and whether physicians should counsel against youth participation in full-contact sports. METHODS: An anonymous paper survey featuring 13 attitudinal, 13 demographic, and 9 knowledge questions was distributed to a convenience sample of adults ≥18 years in hospital waiting areas and four Chicago parks. Participants were asked to hypothetically consider themselves the parent of a 10-year-old child regarding attitudes towards full-contact sports participation. RESULTS: Between June 13 and July 27, 2016, 1091 partial or complete valid surveys were collected. Almost half (46%) of respondents would not allow a hypothetical 10-year-old son to play tackle football. The majority (74%) of respondents agreed that it was appropriate for physicians to counsel against youth participation in full-contact sports. Respondents obtained information about concussions from, on average, 2-3 sources, although only 34% received information from physicians. Respondents demonstrated a high concussion knowledge level (average: 6.75 of 9 questions). However, only 39% of respondents correctly answered that the following statement was false: "After a mild concussion, there are usually visible changes on medical imaging". CONCLUSIONS: Overall, respondents are well-informed about concussions. They are divided about the participation of youth in full-contact sports and are amenable to physician counseling against youth participation in full-contact sports.

Attitudes and Counseling Practices of Pediatricians Regarding Youth Sports Participation and Concussion Risks.

OBJECTIVE: To examine attitudes and practices of pediatricians toward sports-related head trauma and youth participation in tackle football and ice hockey. STUDY DESIGN: A respondent-anonymous electronic survey was distributed 3 times to members of the American Academy of Pediatrics Section of Bioethics, Council on Injury, Violence, and Poison Prevention, and Council on Sports Medicine and Fitness. RESULTS: Of 791 eligible pediatricians, 227 (29%) responded. Most respondents (189/223; 85%) treat sports-related concussions, among whom 83% (137/165) reported access to an established return-to-play protocol within their practice. Virtually all (160/166; 96%) reported increased parental awareness/concern regarding concussions and 85% (139/163) reported increased visits for head trauma. Overall, 77% (140/183) would not allow their son to play tackle football and 35% (64/181) and 34% (63/184) would not allow their son or daughter, respectively, to participate in ice hockey. Most respondents endorsed limiting or eliminating tackling (143/176; 81%) and checking (144/179; 80%) from practice. Respondents were evenly divided in their support for counseling against youth participation in full-contact sports, with 48% in favor (87/180). CONCLUSIONS: Most respondents would not allow their own child to play tackle football and endorsed limiting or eliminating tackling in practice. The American Academy of Pediatrics should consider recommending restrictions on tackling in football to support the current concussion concerns of its members.

Viral Hepatitis in Children: A Through E.

Hepatitis is defined as inflammation of the liver. This inflammation can be acute and self-limited, chronic (leading to cirrhosis and an increased risk for hepatocellular carcinoma), or fulminant (requiring lifesaving liver transplantation). Although there are many causes of hepatitis, this article focuses on the main childhood viral hepatidities: types A, B, C, D, and E. This review discusses the main characteristics of each virus, including salient epidemiology, clinical characteristics, diagnosis, treatment, and prevention strategies. [Pediatr Ann. 2016;45(12):e420-e426.].

Long-term venous complications after full-size and segmental pediatric liver transplantation.

OBJECTIVE: To assess the long-term incidence of venous complications, including portal vein and hepatic vein stenoses, in both whole cadaveric and reduced-size cadaveric and living related liver transplants in a pediatric population, and to assess the therapeutic modalities in the treatment of these lesions. SUMMARY BACKGROUND DATA: A shortage in appropriate-sized liver grafts for pediatric patients led to the use of segmental liver grafts, which became the predominant graft used in 325 of 600 (54%) transplants at the authors' institution. To assess the long-term impact of this strategy, the authors examined the incidence of late (>90 days) venous complications and the efficacy of all therapeutic interventions. METHODS: Six hundred pediatric liver transplants were performed in 325 patients, with reduced-size or split (RSS; n = 207), living related (LRD; n = 118), or full-size cadaveric grafts (FS; n = 275) from 1988 to 2000. All transplants identified with late portal vein or vena caval stenoses or thromboses from a cohort of 524 grafts with survival greater than 90 days were reviewed for demographics, symptoms, therapeutic intervention, recurrence, morbidity, and mortality. RESULTS: Fifty lesions were identified in 49 patients (38 portal vein and 12 hepatic vein-cava stenoses). Sex distribution was similar between portal vein and hepatic vein to cava, as was the mean patient age. Portal vein stenoses occurred in 32 LRD, 3 RSS, and 3 FS, while hepatic vein-cava stenoses occurred in 2 LRD, 8 RSS, and 2 FS. In the 38 portal vein stenoses, 9 had prior perioperative portal vein and/or 5 hepatic artery thrombectomies. Portal vein stenoses were identified after bleeding (17/38), ascites (6/38), increased liver function tests (6/38), splenomegaly (5/38), or screening ultrasound (4/38). Portal vein stenosis was associated most often with cryopreserved vein for portal conduits. Excluding conduits, the incidence of late portal vein complications was reduced to 1%. Lesions became symptomatic at a mean of 50.8 +/- 184.2 months posttransplant. All patients underwent venous angioplasty with a 66% (25/38) success rate, while 7 of 25 required further angioplasty and stenting. In the 13 unsuccessful angioplasties, 8 required surgical shunts for complete portal vein thrombosis. Recurrence occurred in 9 patients: all were amenable to stenting. Nine patients (24%) eventually died of sepsis (4) and surgical deaths at shunt or retransplant (5). Hepatic vein-cava stenoses occurred after a mean of 37.2 +/- 35.2 months, presenting with ascites (n = 10), increased liver function tests (n = 2), and splenomegaly (n = 2). All patients were diagnosed by venogram and managed by balloon dilatation alone (n = 6) or stented (n = 4), with an 80% (10/12) success, with two late recurrences amenable to repeat angioplasty or stenting. Long-term survival was 80% at 1 year. CONCLUSIONS: The use of segmental grafts without venous conduits is not associated with a significant rate of long-term venous complication. When late venous complications do occur, venous angioplasty and stenting are both a safe and effective management modality. If necessary, venous angioplasty may be repeated with the placement of a stent. When this is required, care must be taken to place the stent in a position where the metallic object will not interfere with future surgical manipulations should retransplantation be necessary.

Thrombocytopenia in children with severe iron deficiency.

PURPOSE: Thrombocytopenia has been reported in some children with severe iron deficiency anemia, but the validity of the association and the mechanism of the thrombocytopenia are not well established. Six children with severe iron deficiency and thrombocytopenia are described, and the literature is reviewed. PATIENTS AND METHODS: Clinical, hematologic, and morphologic data were collected and analyzed for six patients referred for evaluation of severe microcytic anemia and thrombocytopenia. RESULTS: The children ranged in age from 14 months to 17 years (median age 27 months) and were otherwise healthy. The iron deficiency was nutritional in four patients younger than 3 years of age and resulted from menstrual blood loss in two teenage girls. The mean initial hemoglobin was 2.5 g/dL (range 1.6-4.7) and the mean initial platelet count was 64 x 109/L (range 11-102). Bone marrow examinations were performed in three patients and showed increased numbers of megakaryocytes. After treatment with therapeutic doses of oral iron, all the patients showed rapid increases in their platelet counts. CONCLUSIONS: These observations validate and extend previous reports of an association between severe iron deficiency and thrombocytopenia. The increased numbers of megakaryocytes and the extremely rapid increase in platelet counts after initiation of iron therapy suggest an essential role for iron in a late stage of thrombopoiesis.