A novel in vitro model of the small intestinal epithelium in co-culture with 'gut-like' dendritic cells.

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.

Mummified embalmed head skin: SR-FTIR microspectroscopic exploration.

This case report details the examination of the skin of an Egyptian mummified head with a possible skin disorder. The head, thought to be dated in the first half of the 18th Dynasty, New Kingdom (1570-1400 BCE) belongs to the Museum of Forensic Anthropology, University of Madrid. Initial histological examination demonstrated evidence of chronic inflammation, which was confirmed by immunohistochemistry and Transmission Electron Microscopy (TEM). However, confirmation of pathology could be confounded by both the age of the specimen and the process of preservation by mummification. In this case report, Synchrotron Radiation Fourier Transform Microspectroscopy (SR-µFTIR) was used to add novel insights into embalmed mummified tissue. More precisely, FTIR is used for the first time on the specific specimens, while no other similar studies have been performed on these samples priorly. Additionally, modern skin tissue was examined too, in order to compare the amount of degradation to the mummified one. Whilst the FTIR results confirmed the results from the initial histological study, they also showed a biochemical modification of the mummified skin that could be indicative of tissue degradation. The latter was supported by comparing it to FTIR results of the modern tissue used.

An Open-Format Enteroid Culture System for Interrogation of Interactions Between Toxoplasma gondii and the Intestinal Epithelium.

When transmitted through the oral route, Toxoplasma gondii first interacts with its host at the small intestinal epithelium. This interaction is crucial to controlling initial invasion and replication, as well as shaping the quality of the systemic immune response. It is therefore an attractive target for the design of novel vaccines and adjuvants. However, due to a lack of tractable infection models, we understand surprisingly little about the molecular pathways that govern this interaction. The in vitro culture of small intestinal epithelium as 3D enteroids shows great promise for modeling the epithelial response to infection. However, the enclosed luminal space makes the application of infectious agents to the apical epithelial surface challenging. Here, we have developed three novel enteroid-based techniques for modeling T. gondii infection. In particular, we have adapted enteroid culture protocols to generate collagen-supported epithelial sheets with an exposed apical surface. These cultures retain epithelial polarization, and the presence of fully differentiated epithelial cell populations. They are susceptible to infection with, and support replication of, T. gondii. Using quantitative label-free mass spectrometry, we show that T. gondii infection of the enteroid epithelium is associated with up-regulation of proteins associated with cholesterol metabolism, extracellular exosomes, intermicrovillar adhesion, and cell junctions. Inhibition of host cholesterol and isoprenoid biosynthesis with Atorvastatin resulted in a reduction in parasite load only at higher doses, indicating that de novo synthesis may support, but is not required for, parasite replication. These novel models therefore offer tractable tools for investigating how interactions between T. gondii and the host intestinal epithelium influence the course of infection.

Developing a 3D intestinal epithelium model for livestock species.

The in vitro 3D culture of intestinal epithelium is a valuable resource in the study of its function. Organoid culture exploits stem cells' ability to regenerate and produce differentiated epithelium. Intestinal organoid models from rodent or human tissue are widely available whereas large animal models are not. Livestock enteric and zoonotic diseases elicit significant morbidity and mortality in animal and human populations. Therefore, livestock species-specific models may offer novel insights into host-pathogen interactions and disease responses. Bovine and porcine jejunum were obtained from an abattoir and their intestinal crypts isolated, suspended in Matrigel, cultured, cryopreserved and resuscitated. 'Rounding' of crypts occurred followed by budding and then enlargement of the organoids. Epithelial cells were characterised using immunofluorescent staining and confocal microscopy. Organoids were successfully infected with Toxoplasma gondii or Salmonella typhimurium. This 3D organoid model offers a long-term, renewable resource for investigating species-specific intestinal infections with a variety of pathogens.

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

BACKGROUND: Advanced maternal age (AMA; ≥35 years) is an increasing trend and is reported to be associated with various pregnancy complications. OBJECTIVE: To determine the risk of stillbirth and other adverse pregnancy outcomes in women of AMA. SEARCH STRATEGY: Embase, Medline (Ovid), Cochrane Database of Systematic Reviews, ClinicalTrials.gov, LILACS and conference proceedings were searched from ≥2000. SELECTION CRITERIA: 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. DATA COLLECTION AND ANALYSIS: 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). MAIN RESULTS: 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. CONCLUSIONS: 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.

A novel in vitro model of villitis of unknown etiology demonstrates altered placental hormone and cytokine profile.

PROBLEM: Placental dysfunction is present over 50% of cases of stillbirth and fetal growth restriction (FGR). Villitis of unknown etiology (VUE), an inflammatory condition of the placenta characterized by maternal T cell infiltrates in the villous stroma and dysregulation of inflammatory cytokines, is more frequent in FGR and stillbirth. METHOD OF STUDY: A novel in vitro model of placental inflammation was developed to test the hypothesis that inflammatory cells seen in VUE and/or cytokines impair placental function. RESULTS: Coculture of placental explants with maternal leukocytes resulted in increased leukocytes in villous tissue and elevated concentrations of IL-1ß, IL-1Ra, IL-6, IL-10, and IFN-γ (P≤.05). Human chorionic gonadotrophin secretion was reduced following coculture with leukocytes (P≤.01) and cytokines (P≤.05). CONCLUSION: These observations support the hypothesis that altered placental inflammation has deleterious effects on placental function. This model could be used to further understanding about the pathophysiology of VUE and to test potential therapies.

Sampling and Definitions of Placental Lesions: Amsterdam Placental Workshop Group Consensus Statement.

CONTEXT: -The value of placental examination in investigations of adverse pregnancy outcomes may be compromised by sampling and definition differences between laboratories. OBJECTIVE: -To establish an agreed-upon protocol for sampling the placenta, and for diagnostic criteria for placental lesions. Recommendations would cover reporting placentas in tertiary centers as well as in community hospitals and district general hospitals, and are also relevant to the scientific research community. DATA SOURCES: -Areas of controversy or uncertainty were explored prior to a 1-day meeting where placental and perinatal pathologists, and maternal-fetal medicine specialists discussed available evidence and subsequently reached consensus where possible. CONCLUSIONS: -The group agreed on sets of uniform sampling criteria, placental gross descriptors, pathologic terminologies, and diagnostic criteria. The terminology and microscopic descriptions for maternal vascular malperfusion, fetal vascular malperfusion, delayed villous maturation, patterns of ascending intrauterine infection, and villitis of unknown etiology were agreed upon. Topics requiring further discussion were highlighted. Ongoing developments in our understanding of the pathology of the placenta, scientific bases of the maternofetoplacental triad, and evolution of the clinical significance of defined lesions may necessitate further refinements of these consensus guidelines. The proposed structure will assist in international comparability of clinicopathologic and scientific studies and assist in refining the significance of lesions associated with adverse pregnancy and later health outcomes.

Characterizing Villitis of Unknown Etiology and Inflammation in Stillbirth.

Villitis of unknown etiology (VUE) is an enigmatic inflammatory condition of the placenta associated with fetal growth restriction and stillbirth. Greater understanding of this condition is essential to understand its contribution to adverse outcomes. Our aim was to identify and quantify the cells in VUE in cases of stillbirth and to characterize immune responses specific to this condition. Immunohistochemistry was performed on placentas from stillborn infants whose cause of death was recorded as VUE to identify CD45(+) leukocytes, CD163(+) macrophages, CD4(+) and CD8(+) T cells, neutrophils, and proinflammatory and anti-inflammatory cytokines. Images were quantified with HistoQuest software. CD45(+) leukocytes comprised 25% of cells in VUE lesions: macrophages (12%) and CD4 T cells (11%) being predominant cell types; CD8 T cells were observed in all lesions. Leukocytes and macrophages were increased throughout the placenta in stillbirths; pan-placental CD4(+) and CD8(+) T cells outside VUE lesions were increased in stillbirth with VUE. There was increased IL-2 and IL-12 and reduced IL-4 immunostaining in VUE lesions. Our results suggest VUE in stillbirth has a similar immune cell profile to live birth. Pan-placental macrophages, CD4 and CD8 T cells indicate a wider inflammatory response unrestricted to VUE lesions. The cytokine profile observed suggests a skew towards inappropriate Th1 immune responses. Full characterisation VUE lesion phenotype confirms its immunological origins and provides foundations to develop novel investigations.

Circulating cytokines and alarmins associated with placental inflammation in high-risk pregnancies.

PROBLEM: Inflammation during pregnancy has devastating consequences for the placenta and fetus. These events are incompletely understood, thereby hampering screening and treatment. METHOD OF STUDY: The inflammatory profile of villous tissue was studied in pregnancies at high-risk of placental dysfunction and compared to uncomplicated pregnancies. The systemic inflammatory profile was assessed in matched maternal serum samples in cases of reduced fetal movements (RFM). RESULTS: Placentas from RFM pregnancies had a unique inflammatory profile characterized by increased interleukin (IL)-1 receptor antagonist and decreased IL-10 expression, concomitant with increased numbers of placental macrophages. This aberrant cytokine profile was evident in maternal serum in RFM, as were increased levels of alarmins (uric acid, HMGB1, cell-free fetal DNA). CONCLUSION: This distinct inflammatory profile at the maternal-fetal interface, mirrored in maternal serum, could represent biomarkers of placental inflammation and could offer novel therapeutic options to protect the placenta and fetus from an adverse maternal environment.

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.