A novel <i>in vitro</i> model of the small intestinal epithelium in co-culture with ‘gut-like’ dendritic cells

Abstract Cross-talk between dendritic cells (DCs) and the intestinal epithelium is important in the decision to mount a protective immune response to a pathogen or to regulate potentially damaging responses to food antigens and the microbiota. Failures in this decision-making process contribute to the development of intestinal inflammation, making the molecular signals that pass between DCs and intestinal epithelial cells potential therapeutic targets. Until now, in vitro models with sufficient complexity to understand these interactions have been lacking. Here, we outline the development of a co-culture model of in vitro differentiated ‘gut-like’ DCs with small intestinal organoids (enteroids). Sequential exposure of murine bone marrow progenitors to Flt3L, granulocyte macrophage colony-stimulating factor (GM-CSF) and all-trans-retinoic acid (RA) resulted in the generation of a distinct population of conventional DCs expressing CD11b+SIRPα+CD103+/− (cDC2) exhibiting retinaldehyde dehydrogenase (RALDH) activity. These ‘gut-like’ DCs extended transepithelial dendrites across the intact epithelium of enteroids. ‘Gut-like’ DC in co-culture with enteroids can be utilized to define how epithelial cells and cDCs communicate in the intestine under a variety of different physiological conditions, including exposure to different nutrients, natural products, components of the microbiota, or pathogens. Surprisingly, we found that co-culture with enteroids resulted in a loss of RALDH activity in ‘gut-like’ DCs. Continued provision of GM-CSF and RA during co-culture was required to oppose putative negative signals from the enteroid epithelium. Our data contribute to a growing understanding of how intestinal cDCs assess environmental conditions to ensure appropriate activation of the immune response.</jats:p

NF-κB2 signalling in enteroids modulates enterocyte responses to secreted factors from bone marrow-derived dendritic cells

Alternative pathway NF-κB signalling regulates susceptibility towards developing inflammatory bowel disease (IBD), colitis-associated cancer and sepsis-associated intestinal epithelial cell apoptosis and shedding. However, the cell populations responsible for the perturbed alternative pathway NF-κB signalling in intestinal mucosal pathology remain unclear. In order to investigate the contribution of the epithelial compartment, we have tested whether NF-κB2 regulated transcription in intestinal epithelial cells controls the intestinal epithelial response to cytokines that are known to disrupt intestinal barrier permeability. Enteroids were generated from the proximal, middle and distal regions of small intestine (SI) from C57BL/6J wild-type mice and displayed region-specific morphology that was maintained during sub-culture. Enteroids treated with 100 ng/mL TNF were compared with corresponding regions of SI from C57BL/6J mice treated systemically with 0.33 mg/kg TNF for 1.5 h. TNF-induced apoptosis in all regions of the intestine in vitro and in vivo but resulted in Paneth cell degranulation only in proximal tissue-derived SI and enteroids. TNF also resulted in increased enteroid sphericity (quantified as circularity from two-dimensional bright field images). This response was dose and time-dependent and correlated with active caspase-3 immunopositivity. Proximal tissue-derived enteroids generated from Nfκb2−/− mice showed a significantly blunted circularity response following the addition of TNF, IFNγ, lipopolysaccharide (LPS) activated C57BL/6J-derived bone marrow-derived dendritic cells (BMDC) and secreted factors from LPS-activated BMDCs. However, Nfκb1−/− mouse-derived enteroids showed no significant changes in response to these stimuli. In conclusion, the selection of SI region is important when designing enteroid studies as region-specific identity and response to stimuli such as TNF are maintained in culture. Intestinal epithelial cells are at least partially responsible for regulating their own fate by modulating NF-κB2 signalling in response to stimuli known to be involved in multiple intestinal and systemic diseases. Future studies are warranted to investigate the therapeutic potential of intestinal epithelial NF-κB2 inhibition

Co-culture of placental explants with isolated CD4 and CD8 T cells: a functional model to define the consequences of placental inflammation

Appropriate placental function is essential for successful pregnancy and placental dysfunction is associated with fetal growth restriction (FGR) and stillbirth. Villitis of unknown etiology (VUE) and chronic intervillositis of unknown etiology (CIUE) are immune-mediated conditions characterised by placental infiltrates of macrophages, CD4 and CD8 T cells. VUE and CIUE occur more frequently in the placentas of pregnancies complicated by FGR. The mechanisms by which this inflammation induces placental dysfunction are yet to be defined. We aimed to develop an in vitro model of placental inflammation to investigate functional consequences of immune cells in the placental environment. Fragments of placental tissue were co-cultured with CD4 and CD8 T cells isolated from whole blood. CellTrackerTM fluorescence was used to identify T cells in cultured explants. Tissue histology, endocrine and nutrient transport function was assessed using established methods. This novel preparation will enable future investigations into immune cell interactions with placenta

Intra-individual variations in the bifurcation of the radial nerve and the length of the posterior interosseous nerve

Anatomical literature on the radial nerve predominantly features inter-individual variations, with comparatively few studies investigating intra-individual variations. The radial nerve has a complex and variable course, particularly in relation to the location at which the nerve bifurcates to form the superficial branch of the radial nerve and the posterior interosseous nerve. Variations of the radial nerve may change the way the nerve and its branches, their blood supply and nerve transmission respond to forces. This study investigated the presence of intra-individual differences in the bifurcation point of the radial nerve and the length of the posterior interosseous nerve from the bifurcation to the radial tunnel. Eighteen embalmed human cadavers were dissected to reveal the radial nerve. Measurements were taken from the level of the lateral humeral epicondyle to the bifurcation of the radial nerve, and from the bifurcation to the radial tunnel. All cadavers presented with intra-individual variations between the left and right limbs. Significant differences were found between the left and right limbs for the measurement from the lateral humeral epicondyle to the bifurcation (median difference _ 18.0 mm; p _ 0.016) but not for the measurement from the bifurcation to the radial tunnel (median difference _ 7.0 mm; p _ 0.396). In conclusion, the location of the radial nerve bifurcation is subject to both intra- and inter-individual variations. Its specific relationship to the lateral humeral epicondyle also varies, occurring both distal and proximal to the level of the epicondyle. Clinical implications of these findings warrant further investigation.sch_die17pub5268pub

Advanced maternal age and adverse pregnancy outcomes: A systematic review and meta-analysis

<div><p>Background</p><p>Advanced maternal age (AMA; ≥35 years) is an increasing trend and is reported to be associated with various pregnancy complications.</p><p>Objective</p><p>To determine the risk of stillbirth and other adverse pregnancy outcomes in women of AMA.</p><p>Search strategy</p><p>Embase, Medline (Ovid), Cochrane Database of Systematic Reviews, ClinicalTrials.gov, LILACS and conference proceedings were searched from ≥2000.</p><p>Selection criteria</p><p>Cohort and case-control studies reporting data on one or more co-primary outcomes (stillbirth or fetal growth restriction (FGR)) and/or secondary outcomes in mothers ≥35 years and <35 years.</p><p>Data collection and analysis</p><p>The effect of age on pregnancy outcome was investigated by random effects meta-analysis and meta-regression. Stillbirth rates were correlated to rates of maternal diabetes, obesity, hypertension and use of assisted reproductive therapies (ART).</p><p>Main results</p><p>Out of 1940 identified titles; 63 cohort studies and 12 case-control studies were included in the meta-analysis. AMA increased the risk of stillbirth (OR 1.75, 95%CI 1.62 to 1.89) with a population attributable risk of 4.7%. Similar trends were seen for risks of FGR, neonatal death, NICU unit admission restriction and GDM. The relationship between AMA and stillbirth was not related to maternal morbidity or ART.</p><p>Conclusions</p><p>Stillbirth risk increases with increasing maternal age. This is not wholly explained by maternal co-morbidities and use of ART. We propose that placental dysfunction may mediate adverse pregnancy outcome in AMA. Further prospective studies are needed to directly test this hypothesis.</p></div

Forest plot of odds ratios of stillbirth stratified by maternal age group (≥35, 35–39, ≥40, 40–49, ≥45 and ≥50 years of age) weighted from random effects analysis shows increased risk of stillbirth in AMA population (OR 1.75; 95%CI 1.62–1.89).

<p>Heterogeneity was classified as severe (Cochran’s χ²_, I² = 95.0%).</p