Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Background Vitamin D deficiency and low bone mineral density (BMD) are complications of inflammatory bowel disease. Vitamin D deficiency is more prevalent among individuals of color compared to Caucasians. There is little data comparing differences in serum 25-hydroxyvitamin D (25OHD) concentrations and BMD between African American and Caucasian children with Crohn’s Disease (CD). Methods We compared serum 25OHD concentrations of African American children with CD (n=52) to Caucasian children with CD (n=64) and healthy African American controls (n=40). We also analyzed BMD using dual energy x-ray absorptiometry (DXA) results from our pediatric CD population. Results African American children with CD had lower serum 25OHD concentrations [16.1 (14.5-17.9, 95%CI) ng/mL] than Caucasians with CD [22.3 (20.2-24.6, 95%CI) ng/mL; p<0.001]. African Americans with CD and controls exhibited similar serum 25OHD concentration [16.1 (14.5-17.9, 95%CI) vs 16.3 (14.4-18.4, 95%CI) ng/mL; NS]. African Americans with CD exhibited no difference in serum 25OHD concentration when controlling for seasonality, disease severity and surgical history, though serum 25OHD concentration was significantly decreased in overweight children (BMI≥85%, p =0.003). Multiple regression analysis demonstrated that obese African American females with CD had the lowest serum 25OHD concentrations [9.6 (6.8-13.5, 95%CI) ng/mL]. BMD was comparable between African American and Caucasian children with CD (Z score −0.4 ± 0.9 vs −0.7 ± 1.2; NS). Conclusions African American children with CD are more likely to have vitamin D deficiency compared to Caucasian with CD, but have similar BMD. CD disease severity and history of surgery do not affect serum 25OHD concentrations among African American children with CD. African American children have low serum 25OHD concentrations, independent of CD, compared to Caucasian children. Future should research should focus on how race affects vitamin D status and BMD in children with CD.

Background Crohn’s disease (CD) is highly heritable. NOD2 has emerged as the main susceptibility gene among individuals of European ancestry; however, NOD2 does not appear to contribute to CD susceptibility among many non-European populations. Today’s African American (AA) population represents an admixture of West African (80%) and European (20%) ancestry. Since genotype-based tools are becoming increasingly available for CD, it is important that we validate the risk variants in different populations, such as admixed AAs. Methods We analyzed the NOD2 variants among admixed AAs (n=321, 240 with CD and 111 healthy controls) and non-admixed West Africans (n=40) by genotyping for 4 known disease-causing NOD variants. We extracted the publically available 1000 Genomes data on NOD2 variants from 500 subjects of West African origin. Association with disease was evaluated by logistic regression. Results An association with CD was found for the classical SNP 1007fs (2.6% CD, 0% HC, p=0.012); there was no association when the genotypic and allelic frequencies of the risk alleles were compared for SNPs R702W and G908R. No known NOD2 risk alleles were seen in either the West African cohort or in subjects of African ancestry from the 1000 Genomes project. Conclusions The NOD2 gene is a risk for CD in AAs, although the allele frequencies and the attributable risk are much lower compared to Caucasians. The risk alleles are not seen in the West African population suggesting the risk for CD contributed by NOD2 among AAs is due exclusively to recent European admixture

Previous studies have demonstrated the safety of performing endoscopic retrograde cholangiopancreatography (ERCP) in the pediatric population; however, few have addressed the outcomes of children undergoing ERCP during acute pancreatitis (AP). We hypothesize that ERCP performed in the setting of AP can be executed with similar technical success and adverse event profiles to those in pediatric patients without pancreatitis. Using the Pediatric ERCP Database Initiative, a multi-national and multi-institutional prospectively collected dataset, we analyzed 1124 ERCPs. One hundred and ninety-four (17%) of these procedures were performed in the setting of AP. There were no difference in the procedure success rate, procedure time, cannulation time, fluoroscopy time, or American Society of Anesthesiology class despite patients with AP having higher American Society of Gastrointestinal Endoscopy grading difficulty scores. This study suggests that ERCP can be safely and efficiently performed in pediatric patients with AP when appropriately indicated.

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens 1,2 . Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota 3-14 . Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described 6,10,15-24 , many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.