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Tolerance to innocuous antigens from the diet and the commensal microbiota is a fundamental process essential to health. Why tolerance is efficiently induced to substances arising from the hostile environment of the gut lumen is incompletely understood but may be related to how these antigens are encountered by the immune system. We observed that goblet cell associated antigen passages (GAPs), but not other pathways of luminal antigen capture, correlated with the acquisition of luminal substances by lamina propria (LP) antigen presenting cells (APCs) and with the sites of tolerance induction to luminal antigens. Strikingly this role extended beyond antigen delivery. The GAP function of goblet cells facilitated maintenance of pre-existing LP T regulatory cells (Tregs), imprinting LP-dendritic cells with tolerogenic properties, and facilitating LP macrophages to produce the immunomodulatory cytokine IL-10. Moreover, tolerance to dietary antigen was impaired in the absence of GAPs. Thus, by delivering luminal antigens, maintaining pre-existing LP Tregs, and imprinting tolerogenic properties on LP-APCs GAPs support tolerance to substances encountered in the hostile environment of the gut lumen.

The intestinal immune system samples luminal contents to induce adaptive immune responses that include tolerance in the steady state and protective immunity during infection. How luminal substances are delivered to the immune system has not been fully investigated. Goblet cells have an important role in this process by delivering luminal substances to the immune system through the formation of goblet cell-associated antigen passages (GAPs). Soluble antigens in the intestinal lumen are transported across the epithelium transcellularly through GAPs and delivered to dendritic cells for presentation to T cells and induction of immune responses. GAPs can be identified and quantified by using the ability of GAP-forming goblet cells to take up fluorescently labeled dextran. Here, we describe a method to visualize GAPs and other cells that have the capacity to take up luminal substances by intraluminal injection of fluorescent dextran in mice under anesthesia, tissue sectioning for slide preparation and imaging with fluorescence microscopy. In contrast to in vivo two-photon imaging previously used to identify GAPs, this technique is not limited by anatomical constraints and can be used to visualize GAP formation throughout the length of the intestine. In addition, this method can be combined with common immunohistochemistry protocols to visualize other cell types. This approach can be used to compare GAP formation following different treatments or changes to the luminal environment and to uncover how sampling of luminal substances is altered in pathophysiological conditions. This protocol requires 8 working hours over 2-3 d to be completed.

Purpose The study aimed to investigate associations between maternal vitamin D status during pregnancy and molar incisor hypomineralisation (MIH) and hypomineralised second primary molars (HSPM) among children. Methods The study had a longitudinal design using prospectively collected data from 176 mother and child pairs. Mothers were initially recruited in a randomised controlled trial to assess a pregnancy exercise programme. Along with the 7-year follow-up, we invited the children to a dental examination. The exposure variable was maternal serum 25-hydroxyvitamin D in gestational weeks 18–22 and 32–36, categorised as insufficient ( Results Among the children (7–9 years old), 32% and 22% had at least one tooth with MIH or HSPM, respectively. A significant association was found between insufficient maternal vitamin D measured in gestational weeks 18–22 and the number of affected teeth among those with MIH at 7–9 years (adjusted RR = 1.82, 95% CI 1.13–2.93). Conclusion Considering any limitations of the present study, it has been shown that insufficient maternal serum vitamin D at mid-pregnancy was associated with a higher number of affected teeth among the offspring with MIH at 7–9 years of age. Further prospective studies are needed to investigate whether this finding is replicable and to clarify the role of maternal vitamin D status during pregnancy and MIH, as well as HSPM, in children.

The study aimed to investigate associations between maternal vitamin D status during pregnancy and molar incisor hypomineralisation (MIH) and hypomineralised second primary molars (HSPM) among children.The study had a longitudinal design using prospectively collected data from 176 mother and child pairs. Mothers were initially recruited in a randomised controlled trial to assess a pregnancy exercise programme. Along with the 7-year follow-up, we invited the children to a dental examination. The exposure variable was maternal serum 25-hydroxyvitamin D in gestational weeks 18-22 and 32-36, categorised as insufficient ( 50 nmol/l) and sufficient (≥ 50 nmol/l). Negative binomial hurdle models were used to analyse potential associations between the exposure variables and MIH or HSPM. The models were adjusted for potential confounders.Among the children (7-9 years old), 32% and 22% had at least one tooth with MIH or HSPM, respectively. A significant association was found between insufficient maternal vitamin D measured in gestational weeks 18-22 and the number of affected teeth among those with MIH at 7-9 years (adjusted RR = 1.82, 95% CI 1.13-2.93).Considering any limitations of the present study, it has been shown that insufficient maternal serum vitamin D at mid-pregnancy was associated with a higher number of affected teeth among the offspring with MIH at 7-9 years of age. Further prospective studies are needed to investigate whether this finding is replicable and to clarify the role of maternal vitamin D status during pregnancy and MIH, as well as HSPM, in children.

Background Vitamin D insufficiency is common in pregnant women worldwide. Regular prenatal exercise is considered beneficial for maternal and fetal health. There is a knowledge gap regarding the impact of prenatal exercise on maternal vitamin D levels. The objective of this study was to investigate whether a prenatal exercise program influenced serum levels of total, free and bioavailable 25-hydroxyvitamin D (25(OH)D) and related parameters. This is a post hoc analysis of a randomized controlled trial with gestational diabetes as the primary outcome. Methods Healthy, pregnant women from two Norwegian cities (Trondheim and Stavanger) were randomly assigned to a 12-week moderate-intensity exercise program (Borg perceived rating scale 13–14) or standard prenatal care. The intervention group (n = 429) underwent exercise at least three times weekly; one supervised group training and two home based sessions. The controls (n = 426) received standard prenatal care, and exercising was not denied. Training diaries and group training was used to promote compliance and evaluate adherence. Serum levels of 25(OH)D, parathyroid hormone, calcium, phosphate, magnesium and vitamin D-binding protein were measured before (18–22 weeks′ gestation) and after the intervention (32–36 weeks′ gestation). Free and bioavailable 25(OH)D concentrations were calculated. Regression analysis of covariance (ANCOVA) was applied to assess the effect of the training regime on each substance with pre-intervention levels as covariates. In a second model, we also adjusted for study site and sampling month. Intention-to-treat principle was used. Results A total of 724 women completed the study. No between-group difference in serum 25(OH)D and related parameters was identified by ANCOVA using baseline serum levels as covariates. The second model revealed a between-group difference in levels of 25(OH)D (1.9, 95% CI 0.0 to 3.8 nmol/L; p = 0.048), free 25(OH)D (0.55, 95% CI 0.10 to 0.99 pmol/L; p = 0.017) and bioavailable 25(OH)D (0.15 95% CI 0.01 to 0.29 nmol/L; p = 0.036). No serious adverse events related to regular exercise were seen. Conclusion This study, a post hoc analysis, indicates that exercise may affect vitamin D status positively, and emphasizes that women with uncomplicated pregnancies should be encouraged to perform regular exercise. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Our current knowledge of the different immune cells in neuroblastoma is based on in vitro and in vivo studies mainly focusing on a single cell type. Different studies have conveyed conflicting results and despite the use of anti-GD2 immunotherapy in the clinic, a comprehensive immune cell overview at the single-cell level is still missing and understanding the complete composition of immune cells neuroblastoma will be crucial for the development of novel immunotherapeutics against the disease. In this study, we performed single-cell RNA-sequencing on human neuroblastoma coupled with multiplex immunohistochemistry and survival analysis to provide a complete cellular and molecular immune cell landscape of human neuroblastoma. Further, we contrasted the neuroblastoma data with single-cell RNA-sequencing data from normal fetal adrenal gland to characterize cell-state changes from normal tissue to neuroblastoma. Our analysis revealed 27 immune cell subtypes including distinct subpopulations of myeloid, NK, B and T cells that were associated with a survival benefit. Multiple subtypes of B and NK cells, not seen in neuroblastoma before, were identified. We propose the presence of tertiary lymphoid structures and showed that Active NK cells correlated with improved survival. In contrast to adult cancers, we detected no difference in cytotoxicity and exhaustion score for cytotoxic T cells, nor Treg activity. However, we demonstrated an increase in inflammatory monocyte signature score from normal to tumor derived myeloid cells. Finally, we performed receptor-ligand interaction analysis between tumor, stroma and immune cells, where we highlight several interactions that we suggest for future studies of how to exploit immune cells as a therapeutic option in neuroblastoma. Our findings significantly broaden the understanding of the immune composition in neuroblastoma and provides a resource for the development of novel immunotherapeutics. Citation Format: Bronte Manouk Verhoeven, Shenglin Mei, Thale K. Olsen, Karin K. Gustafsson, Anders Valind, Axel Lindström, David G. Nord, Shahrzad S. Fard, Catharina Hagerling, Peter V. Kharchenko, Per Kogner, John I. Johnsen, Ninib Baryawno. The immune cell atlas of human neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6215.

Given the scarcity of data on diabetes prevalence and associated risk factors among women in rural Nepal, we aimed to examine this, using glycated hemoglobin (HbA1c) as a diagnostic tool. A cross-sectional survey addressing reproductive health and non-communicable diseases was conducted in 2012–2013 among non-pregnant, married women in Bolde, a rural district of Nepal. HbA1c ≥ 6.5% (48 mmol/mol) was used as diagnostic criterion for diabetes, a cut-off of 7.0% (53 mmol/mol) was used to increase the specificity. HbA1c was measured in 757 women (17–86 years). The prevalence of diabetes and prediabetes was 13.5% and 38.5%, respectively. When using 7.0% as a cut-off, the prevalence of diabetes was 5.8%. Aging, intake of instant noodles and milk and vegetarian food (ns) were associated with increased risk for diabetes. Waist circumference was higher among women with diabetes, although not significant. The women were uneducated (87.6%), and only 12% had heard about diabetes. In conclusion, we observed a higher prevalence of diabetes and prediabetes than anticipated among rural, Nepalese women. The increased risk was mainly attributed to dietary factors. In contrast to most previous studies in Nepal, we used HbA1c as diagnostic criterion.

Hypovitaminosis D is prevalent worldwide, and especially in South-Asia. According to the Institute of Medicine (IOM), 25(OH)D levels below 30 nmol/L are defined as vitamin D deficiency (VDD) and levels between 30–50 nmol/L as insufficiency (VDI). Besides its role in calcium homeostasis, it has been postulated that vitamin D is involved in metabolic syndrome. Given the scarcity of data on vitamin D status in Nepal, we aimed to examine the prevalence of VDD and VDI, as well as the determinants and association with metabolic parameters (lipids, HbA1c), in a cohort of women in rural Nepal. Altogether, 733 women 48.5 ± 11.7 years of age were included. VDD and VDI were observed in 6.3 and 42.4% of the participants, respectively, and the prevalence increased by age. Women reporting intake of milk and eggs > 2 times weekly had higher 25(OH)D levels than those reporting intake < 2 times weekly. Women with vitamin D levels < 50 nmol/L displayed higher levels of cholesterol, LDL-cholesterol, triglycerides, and HbA1c. Additionally, a regression analysis showed a significant association between hypovitaminosis D, dyslipidemia, and HbA1c elevation. In conclusion, VDI was prevalent and increased with age. Milk and egg intake > 2 times weekly seemed to decrease the risk of VDI. Moreover, hypovitaminosis D was associated with an adverse metabolic profile.