Fetal Intestine Research Articles

In utero human intestine harbors unique metabolome, including bacterial metabolites.

Symbiotic microbial colonization through the establishment of the intestinal microbiome is critical to many intestinal functions, including nutrient metabolism, intestinal barrier integrity, and immune regulation. Recent studies suggest that education of intestinal immunity may be ongoing in utero. However, the drivers of this process are unknown. The microbiome and its byproducts are one potential source. Whether a fetal intestinal microbiome exists is controversial, and whether microbially derived metabolites are present in utero is unknown. Here, we aimed to determine whether bacterial DNA and microbially derived metabolites can be detected in second trimester human intestinal samples. Although we were unable to amplify bacterial DNA from fetal intestines, we report a fetal metabolomic intestinal profile with an abundance of bacterially derived and host-derived metabolites commonly produced in response to microbiota. Though we did not directly assess their source and function, we hypothesize that these microbial-associated metabolites either come from the maternal microbiome and are vertically transmitted to the fetus to prime the fetal immune system and prepare the gastrointestinal tract for postnatal microbial encounters or are produced locally by bacteria that were below our detection threshold.

Retinopathy of prematurity and neonatal gut microbiome: An interview with Professor Dimitra Skondra, Associate Professor of Ophthalmology and Vitreoretinal Surgeon at The University of Chicago (USA).

Retinopathy of prematurity (ROP) is a sight-threatening disorder of the retina affecting neonates of very low birth weight and gestational age, and is characterized by the development of abnormal blood vessel growth. According to Dr Dimitra Skondra, Associate Professor of Ophthalmology and Vitreoretinal Surgeon at the University of Chicago School of Medicine in Chicago, USA, the neonatal gut microbiome may be implicated in the neoangiogenesis process in the neonatal retina and this role may be one of the missing links in the pathogenesis of ROP. The human gut microbiome consists of bacteria, viruses, protozoa and fungi, which colonize the sterile fetal intestine, and differ depending on gestational age, mode of delivery, type of neonatal feeding, the usage of antibiotics and the requirement of neonatal intensive care. To date, it has been related to multiple nutritive, metabolic and immunological functions and has been implicated in the pathogenesis of several human diseases, such as the inflammatory bowel diseases, autoimmune and neurogenerative disorders, metabolic syndrome, cardiovascular diseases and various types of malignant neoplasias. Recent research has proposed that the neonatal gut microbiome profile in high-risk neonates who develop ROP is significantly enriched with Enterobacteriacaea species several weeks prior to the diagnosis of ROP. Further research using animal models is required to prove the causative or secondary role of the microbiome composition in the development and clinical course of ROP. If this role is proven, the gut microbiome could then be a target of intervention for personalized medicine in the prevention and therapeutic management of ROP in neonates.

The effect of intestinal microbiota on pregnancy and baby's health

Introduction: The intestinal microflora (IM) is a heterogeneous complex of all microorganisms inhabiting the human body, both commensal, symbiotic and pathogenic. It consists mainly of bacteria, but also of viruses, archaea, fungi and protozoa. IM has an important metabolic and immunological role and participates in the course of pathological processes. The composition of a newborn and baby’s IM changes dynamically in the first year of life. Just in the first days of birth, the newborn is exposed to many different strains of bacteria, including Staphylococcus sp. and Enterococcus sp.Aim: To purpose of systematic review was to collect and analyze material of effect of the mother's gut microbiota on the pregnancy and of the health of baby.Material and methods: Review of the literature published in 2008-2020.Conclusion: The uterus environment was originally considered sterile and the newborn's intestines were to be colonized only during and after delivery. However, recent studies indicate that placenta and amniotic fluid are involved in microbiota colonization of fetal intestines during prenatal life. This means that the fetus has an initial stock of microorganisms before birth. In addition, a significant effect of maternal diet and probiotic supplementation during pregnancy on the composition of microorganisms found in the amniotic fluid has been demonstrated. There is also a relationship between microbiotic programming in the aspect of civilization diseases. Summary: The education of women of reproductive age on the subject of proper nutrition and supplementation during pregnancy and the puerperium is extremely important. Undertaking pro-health activities will affect the child's future health and development.

Effects of nutrient restriction and melatonin supplementation from mid-to-late gestation on maternal and fetal small intestinal carbohydrase activities in sheep

The objective of this experiment was to evaluate the effects of nutrient restriction and melatonin supplementation during mid-to-late gestation on maternal and fetal small intestinal carbohydrase activities in sheep. Ewes were randomly assigned to one of 4 dietary treatments arranged in a 2 × 2 factorial design. Ewes were fed to provide 100% (adequate; ADQ) or 60% (restricted; RES) of nutrient recommendations, and diets were supplemented with either no melatonin (control; CON) or 5 mg melatonin/d (melatonin; MEL). This resulted in 4 treatment groups: CON-ADQ (n = 7), CON-RES (n = 8), MEL-ADQ (n = 8), MEL-RES (n = 8). Treatments began on day 50 of gestation, and ewes were euthanized on day 130 for tissue collection. The maternal and fetal small intestine were collected and assayed for small intestinal carbohydrase activities. Data were analyzed using the GLM procedure of SAS with fetal sex, melatonin, nutrition, and the melatonin by nutrition interaction included in the model statement. There were no melatonin by nutrition interactions for maternal or fetal small intestinal protein concentration or carbohydrase activities (P ≥ 0.11). Dietary melatonin supplementation decreased (P = 0.03) maternal small intestinal protein concentration by 22.7% and increased (P = 0.03) maternal small intestinal glucoamylase, isomaltase, and maltase activity per gram protein by 45.5%, 41.3%, and 40.6%, respectively. Nutrient restriction from mid-to-late gestation did not influence (P ≥ 0.46) maternal small intestinal protein concentration, or maltase, isomaltase, and lactase activity. Maternal glucoamylase activity per gram intestine increased (P = 0.05) with nutrient restriction by 49.1%. Melatonin supplementation and maternal nutrient restriction did not influence (P ≥ 0.15) fetal small intestinal protein concentration, or glucoamylase, isomaltase, and lactase activity. Maternal nutrient restriction from mid-to-late gestation decreased (P = 0.05) fetal maltase activity per gram intestine by 20.5% but did not influence fetal maltase activity per gram protein. These data indicate that some maternal and fetal carbohydrases are influenced by nutrient restriction and melatonin supplementation in sheep. More information is needed to understand how nutritional and hormonal factors regulate digestive enzyme activity in ruminants to design improved maternal nutrition programs to optimize fetal growth and development while maintaining maternal productivity.

The unique applicability of the human placenta to the Adverse Outcome Pathway (AOP) concept: the placenta provides fundamental insights into human organ functions at multiple levels of biological organization

Despite the short lifespan of the human placenta, the proper formation and function of the organ is of crucial importance for fetal development. Placental dysfunction increases the risk of complications for mother and child during pregnancy and childbirth and beyond as it predisposes to fetal programming. The placenta is an upstream organ of the fetus. It performs the functions of fetal lungs, liver, intestines, kidneys and glands as long as these organs are not fully functional. Furthermore, it is the only human organ that is non-invasively available either after elective abortion or after birth. This is a crucial point given that the conceptual framework of Adverse Outcome Pathway (AOP) requires data on organ function. In vitro and ex vivo placental studies, combined with epidemiological and clinical data on pregnant women, newborns, and infants can uniquely cover all levels of information needed to develop new AOPs and complement existing AOPs related to reproductive toxicity and beyond. To stimulate further research in this area and to support researchers in future studies dealing with the development of AOPs related to the placenta, this review first gives a brief description of placental structure, placental development and relevant pregnancy diseases. The state of knowledge about the available placental models, their particularities and limitations are briefly discussed. Finally, the use of placental research for the development of AOPs is presented with an illustrative example.

The Melatonin and Enriched Environment Ameliorated Low Protein-Induced Intrauterine Growth Retardation by IGF-1 And mtor Signaling Pathway and Autophagy Inhibition in Rats.

Sprague-Dawley rats were randomly allocated to 3 groups: control (C), model (M) and EE+MEL group. Animals were housed in an enriched environment (EE+MEL group) or remained in a standard environment (C group, M group). IUGR rat model was built by feeding a low protein diet during pregnancy. MEL was administered by gavaging. At day 1 post-birth, the baseline characteristics and serum biochemical parameters, morphology of liver and small intestine, enzyme activities, and mRNA expression levels of fetal rats were determined. The autophagy marker LC3 and Beclin1 were determined by western blot analysis. EE+MEL markedly improved the baseline characteristics, hepatic and intestinal morphology of IUGR fetuses. In addition, the lactase activities in the fetal intestine were markedly increased by the EE+MEL. The levels of serum somatostatin (SST), Growth hormone (GH), GH releasing hormone (GHRH), Insulin-like Growth Factor 1 (IGF-1), triiodothyronine (T3), and tetraiodothyronine (T4) were found to be recovered by EE+MEL. In addition, the EE+MEL significantly ameliorated the mRNA expression of SST, GHRH, and GHRH receptor (GHRHR), GH, GHR, IGF-1, and IGF-1 receptor (IGF1R), IGF binding protein-1 (IGFBP1), mammalian target of rapamycin (mTOR), S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4EBP1) in fetuses. In IUGR fetal livers, LC3 and Beclin1 were found to be increased at birth, while LC3 and Beclin1 were observed to be significantly decreased in the EE+MEL group. EE+MEL could improve fetal rats' baseline characteristics, serum biochemical parameters, birth weight, intestinal and hepatic morphology and enzyme activities. These effects could be explained by the activation of the IGF-1/IGFBP1 and IGF-1/mTOR/S6K1/4EBP1 signaling pathway and autophagy inhibition.

A 34-Marker Panel for Imaging Mass Cytometric Analysis of Human Snap-Frozen Tissue.

Imaging mass cytometry (IMC) is able to quantify the expression of dozens of markers at sub-cellular resolution on a single tissue section by combining a novel laser ablation system with mass cytometry. As such, it allows us to gain spatial information and antigen quantification in situ, and can be applied to both snap-frozen and formalin-fixed, paraffin-embedded (FFPE) tissue sections. Herein, we have developed and optimized the immunodetection conditions for a 34-antibody panel for use on human snap-frozen tissue sections. For this, we tested the performance of 80 antibodies. Moreover, we compared tissue drying times, fixation procedures and antibody incubation conditions. We observed that variations in the drying times of tissue sections had little impact on the quality of the images. Fixation with methanol for 5 min at −20°C or 1% paraformaldehyde (PFA) for 5 min at room temperature followed by methanol for 5 min at −20°C were superior to fixation with acetone or PFA only. Finally, we observed that antibody incubation overnight at 4°C yielded more consistent results as compared to staining at room temperature for 5 h. Finally, we used the optimized method for staining of human fetal and adult intestinal tissue samples. We present the tissue architecture and spatial distribution of the stromal cells and immune cells in these samples visualizing blood vessels, the epithelium and lamina propria based on the expression of α-smooth muscle actin (α-SMA), E-Cadherin and Vimentin, while simultaneously revealing the colocalization of T cells, innate lymphoid cells (ILCs), and various myeloid cell subsets in the lamina propria of the human fetal intestine. We expect that this work can aid the scientific community who wish to improve IMC data quality.

Regulatory role of l-proline in fetal pig growth and intestinal epithelial cell proliferation.

l-proline (Pro) is a precursor of ornithine, which is converted into polyamines via ornithine decarboxylase (ODC). Polyamines plays a key role in the proliferation of intestinal epithelial cells. The study investigated the effect of Pro on polyamine metabolism and cell proliferation on porcine enterocytes in vivo and in vitro. Twenty-four Huanjiang mini-pigs were randomly assigned into 1 of 3 groups and fed a basal diet that contained 0.77% alanine (Ala, iso-nitrogenous control), 1% Pro or 1% Pro + 0.0167% α-difluoromethylornithine (DFMO) from d 15 to 70 of gestation. The fetal body weight and number of fetuses per litter were determined, and the small and large intestines were obtained on d 70 ± 1.78 of gestation. The in vitro study was performed in intestinal porcine epithelial (IPEC-J2) cells cultured in Dulbecco's modified Eagle medium-high glucose (DMEM-H) containing 0 μmol/L Pro, 400 μmol/L Pro, or 400 μmol/L Pro + 10 mmol/L DFMO for 4 d. The results showed that maternal dietary supplementation with 1% Pro increased fetal weight; the protein and DNA concentrations of the fetal small intestine; and mRNA levels for potassium voltage-gated channel, shaker-related subfamily, member 1 (Kv1.1) in the fetal small and large intestines (P < 0.05). Supplementing Pro to either gilts or IPEC-J2 cells increased ODC protein abundances and polyamine concentrations in the fetal intestines and IPEC-J2 cells (P < 0.05). In comparison with the Pro group, the combined administration of Pro and DFMO reduced the expression of ODC protein and spermine concentration in the fetal intestine, as well as the concentrations of putrescine, spermidine and spermine in IPEC-J2 cells (P < 0.05). Meanwhile, the percentage of cells in the S-phase and the mRNA levels of proto-oncogenes c-fos and c-myc were increased in response to Pro supplementation, whereas depletion of cellular polyamines with DFMO increased tumor protein p53 (p53) mRNA levels (P < 0.05). Taken together, dietary supplementation with Pro improved fetal pig growth and intestinal epithelial cell proliferation via enhancing polyamine synthesis.

Special Issue No. – 10, June, 2020 Journal &gt; Special Issue &gt; Special Issue No. – 10, June, 2020 &gt; Page 5 “Quantative Methods in Modern Science” organized by Academic Paper Ltd, Russia MORPHOLOGICAL AND ANATOMICAL FEATURES OF THE GENUS GAGEA SALISB., GROWING IN THE EAST KAZAKHSTAN REGION Authors: Zhamal T. Igissinova,Almash A. Kitapbayeva,Anargul S. Sharipkhanova,Alexander L. Vorobyev,Svetlana F. Kolosova,Zhanat

Special Issue No. – 10, June, 2020 Journal &gt; Special Issue &gt; Special Issue No. – 10, June, 2020 &gt; Page 5 “Quantative Methods in Modern Science” organized by Academic Paper Ltd, Russia MORPHOLOGICAL AND ANATOMICAL FEATURES OF THE GENUS GAGEA SALISB., GROWING IN THE EAST KAZAKHSTAN REGION Authors: Zhamal T. Igissinova,Almash A. Kitapbayeva,Anargul S. Sharipkhanova,Alexander L. Vorobyev,Svetlana F. Kolosova,Zhanat

Comparison of protease and aminopeptidase activities in meconium: A pilot study.

The successive accumulation of proteases and aminopeptidases in meconium are important physiological components of the intrauterine environment in which a fetus develops. The aim of the present study was to assess the changes in the activities of these enzymes in meconium of healthy infants, and to investigate whether there were any statistically significant associations between activity of the enzymes of interest and the mode of delivery. The activities of proteases and aminopeptidases were determined in meconium portions (n=110) using the substrates BODIPY FL casein and L-leucine-7-amido-4-methylcoumarin hydrochloride, respectively. Serial meconium samples (2-5 per neonate) were collected from healthy infants born vaginally (n=14), and by a cesarean section (n=16). Protease activity (104 RFU/h) was lower in the first meconium sample compared with the final sample from the same infant (3.99±2.03 vs. 5.76±2.24, respectively, mean ± standard deviation; P=0.004). Conversely, there was no significant difference in aminopeptidase activity (103 nM/l/h) between consecutive meconium samples (P=0.702). The ratios of the first-meconium sample enzyme activity to the last-meconium sample enzyme activity were lower for proteases compared with aminopeptidases (0.76±0.48 vs. 1.35±1.04, respectively mean ± standard deviation; P=0.014), and sustained in the infants born by a cesarean section (P=0.008). Spearman's correlation coefficient analysis between the first and last meconium samples showed the correlation increased in the infants born vaginally compared with the rest of the infants (proteases, R=0.618 vs. R=0.314; aminopeptidases, R=0.688 vs. R=0.566). Aminopeptidase activity did not exhibit any notable dynamic changes during meconium accumulation in the fetal intestine. In infants born vaginally compared with those born by a cesarean section, the activity of both proteases and aminopeptidases in the first meconium sample showed an improved correlation with the activity of the final meconium sample. This may suggest that in the intrauterine environment, during accumulation of meconium in the digestive tract of the fetus, the activity and/or levels of these enzymes and the substrates they catalyze were more stable in newborns born vaginally compared with infants born by caesarean section.

Lack of Evidence for Microbiota in the Placental and Fetal Tissues of Rhesus Macaques.

The prevailing paradigm in obstetrics has been the sterile womb hypothesis. However, some are asserting that the placenta, intra-amniotic environment, and fetus harbor microbial communities. The objective of this study was to determine whether the fetal and placental tissues of rhesus macaques harbor bacterial communities. Fetal, placental, and uterine wall samples were obtained from cesarean deliveries without labor (∼130/166 days gestation). The presence of bacteria in the fetal intestine and placenta was investigated through culture. The bacterial burden and profiles of the placenta, umbilical cord, and fetal brain, heart, liver, and colon were determined through quantitative real-time PCR and DNA sequencing. These data were compared with those of the uterine wall as well as to negative and positive technical controls. Bacterial cultures of fetal and placental tissues yielded only a single colony of Cutibacterium acnes This bacterium was detected at a low relative abundance (0.02%) in the 16S rRNA gene profile of the villous tree sample from which it was cultured, yet it was also identified in 12/29 background technical controls. The bacterial burden and profiles of fetal and placental tissues did not exceed or differ from those of background technical controls. By contrast, the bacterial burden and profiles of positive controls exceeded and differed from those of background controls. Among the macaque samples, distinct microbial signals were limited to the uterine wall. Therefore, using multiple modes of microbiologic inquiry, there was not consistent evidence of bacterial communities in the fetal and placental tissues of rhesus macaques.IMPORTANCE Microbial invasion of the amniotic cavity (i.e., intra-amniotic infection) has been causally linked to pregnancy complications, especially preterm birth. Therefore, if the placenta and the fetus are typically populated by low-biomass microbial communities, current understanding of the role of microbes in reproduction and pregnancy outcomes will need to be fundamentally reconsidered. Could these communities be of benefit by competitively excluding potential pathogens or priming the fetal immune system for the microbial bombardment it will experience upon delivery? If so, what properties (e.g., microbial load and community membership) of these microbial communities preclude versus promote intra-amniotic infection? Given the ramifications of the in utero colonization hypothesis, critical evaluation is required. In this study, using multiple modes of microbiologic inquiry (i.e., culture, quantitative real-time PCR [qPCR], and DNA sequencing) and controlling for potential background DNA contamination, we did not find consistent evidence for microbial communities in the placental and fetal tissues of rhesus macaques.

Ureaplasma parvum induces preterm birth by triggering immune responses in the fetus and at the maternal-fetal interface

Abstract Preterm birth is the leading cause of neonatal morbidity and mortality worldwide. One of every four preterm births is due to intra-amniotic infection, most commonly associated with Ureaplasma species. However, a causal link between Ureaplasma species and adverse pregnancy outcomes, as well as the triggered host immune responses, has not been investigated. Molecular microbiological surveys first revealed that Ureaplasma parvum is the most common bacterium in women with intra-amniotic infection. Ultrasound-guided intra-amniotic injection of U. parvum in pregnant mice resulted in preterm birth and neonatal death. Specific qPCR revealed that U. parvum invaded specific fetal (lung, intestine, spleen, fetal membranes, and placenta) and maternal (decidua, uterus, and cervix) compartments. Consistently, U. parvum induced a strong inflammatory response in the amniotic cavity, fetal membranes, placenta, and fetal lung and spleen, as well as at the maternal-fetal interface. However, U. parvum neither invaded the non-reproductive maternal organs nor induced a maternal systemic inflammatory response. An ex vivo model of intra-amniotic infection showed that U. parvum elicits pro-inflammatory responses in human amnion epithelial cells. Lastly, the treatment of U. parvum-injected mice with clarithromycin, a clinically relevant macrolide, prevented preterm birth and neonatal death. Collectively, this investigation provides a causal link between U. parvum and adverse pregnancy outcomes, and yields insights into the host immune responses triggered by this genital mycoplasma. Importantly, these data support the use of clarithromycin in the treatment of Ureaplasma-associated intra-amniotic infection leading to preterm birth.

Viable bacterial colonization is limited in the human intestine in utero

Abstract Mucosal immunity develops in the human fetal intestine by 11–14 weeks gestation, yet whether viable microbes exist in utero and interact with intestinal immunity is unknown. Structures consistent with coccoid bacterial morphology, embedded in fetal meconium were evident before mid-gestation by high-resolution scanning electron microscopy (n=4). Molecular methods indicated extremely low bacterial burden and simple profiles in fetal meconium (n=40 of 50) compared to controls (n=87). A subset of Micrococcaceae-dominated (n=9) meconium associated with proportions of lamina propria PLZF+ CD161+ CD4+ T cells and divergent intestinal epithelial transcriptomes. Fetal Micrococcus luteus was isolated only in the presence of a monocyte feeder cell line. This strain grew on placental hormones, remained viable within fetal antigen presenting cells, exhibited species-specific immunomodulatory capacity and genomic features indicating fetal adaptation. Thus, viable bacteria are highly limited and inconsistently detectable in human fetal meconium at mid-gestation.

Mechanisms of Fetal T Cell Tolerance and Immune Regulation.

The developing human fetus generates both tolerogenic and protective immune responses in response to the unique requirements of gestation. Thus, a successful human pregnancy depends on a fine balance between two opposing immunological forces: the semi-allogeneic fetus learns to tolerate both self- and maternal- antigens and, in parallel, develops protective immunity in preparation for birth. This critical window of immune development bridges prenatal immune tolerance with the need for postnatal environmental protection, resulting in a vulnerable neonatal period with heightened risk of infection. The fetal immune system is highly specialized to mediate this transition and thus serves a different function from that of the adult. Adaptive immune memory is already evident in the fetal intestine. Fetal T cells with pro-inflammatory potential are born in a tolerogenic environment and are tightly controlled by both cell-intrinsic and -extrinsic mechanisms, suggesting that compartmentalization and specialization, rather than immaturity, define the fetal immune system. Dysregulation of fetal tolerance generates an inflammatory response with deleterious effects to the pregnancy. This review aims to discuss the recent advances in our understanding of the cellular and molecular composition of fetal adaptive immunity and the mechanisms that govern T cell development and function. We also discuss the tolerance promoting environment that impacts fetal immunity and the consequences of its breakdown. A greater understanding of fetal mechanisms of immune activation and regulation has the potential to uncover novel paradigms of immune balance which may be leveraged to develop therapies for transplantation, autoimmune disease, and birth-associated inflammatory pathologies.

HT-29 and Caco2 Cell Lines Are Suitable Models for Studying the Role of Arachidonic Acid-Metabolizing Enzymes in Intestinal Cell Differentiation

Introduction: Cytochrome (CYP) epoxygenases (CYP2C and CYP2J) and soluble epoxide hydrolase (sEH) participate in the metabolism of arachidonic acid and may also have a potential role in enterocyte differentiation. The first critical step in the study of intestinal cell differentiation is the determination of a suitable in vitro model, which must be as similar as possible to the conditions of a living organism. It is known that HT-29 and Caco2 cell lines derived from human colorectal carcinomas can differentiate into enterocyte-like cells in appropriate culture conditions. Material and Methods: We tested 4 different approaches of enterocyte-like differentiation and determined the most appropriate culture conditions for each model. Subsequently, the changes in the expression of CYP epoxygenases and sEH in undifferentiated and differentiated cells were measured by In-Cell ELISA. These results were compared with immunohistochemical profiles of expression of CYP epoxygenases and sEH in samples of human embryonic and fetal intestines as well as adult duodenum and colon. Results: Our results show that sodium butyrate (NaBt)-differentiated HT-29 cells and spontaneously differentiated Caco2 cells resemble CYP epoxygenases and sEH profiles, corresponding with different types of intestines. Conclusion: Our study revealed that the most suitable models for the study of the role of CYP epoxygenases and sEH expression in differentiation of intestinal epithelium are NaBt-differentiated HT-29 cells and spontaneously differentiated Caco2 cells.

Prenatal low-dose endotoxin exposure prolongs intestinal epithelial activation after birth and contributes to necrotizing enterocolitis

PurposeTo investigate the effects of low dose endotoxin on transcriptional activity in intestinal epithelium, and its role in necrotizing enterocolitis (NEC). MethodsLipopolysaccharides (LPS) were injected into the amniotic cavity of pregnant mice under ultrasound guidance. The effects of LPS on fetal and neonatal intestines were determined. Mouse pups were exposed to low dose LPS (0.01 μg per fetus) prenatally and subjected to experimental NEC after birth. The incidence and severity of NEC, as well as intestinal permeability, NF-κB activation, and IL-6 expression were studied. The signaling pathways in the intestinal epithelial cells (IECs) that were activated by LPS were also investigated. ResultsLow dose LPS did not increase apoptosis, myeloperoxidase activity, histological injury or NF-κB activity in fetal intestines. However, prenatal low dose LPS exposure disturbed the transient and self-limited activation of NF-κB in neonatal intestines after birth. Importantly, it increased the incidence and severity of experimental NEC in neonatal mice. In primary IECs, low dose LPS induced IRAK-1 expression via activation of GSK3β. Elevated IRAK-1 levels prolonged the activation of IECs upon stimulation by high dose LPS. ConclusionPrenatal low dose endotoxin exposure disturbs self-limited postnatal epithelial cell activation and predisposes the neonatal intestine to NEC. Level of evidenceNot applicable (experimental animal study).

Viable bacterial colonization is highly limited in the human intestine in utero.

Mucosal immunity develops in the human fetal intestine by 11–14 weeks gestation, yet whether viable microbes exist in utero and interact with the intestinal immune system is unknown. Bacterial-like morphology was identified in pockets of human fetal meconium at mid-gestation by scanning electron microscopy (n=4) and a sparse bacterial signal was detected by 16S rRNA sequencing (n=40 of 50) compared to environmental controls (n=87). Eighteen taxa were enriched in fetal meconium with Micrococcaceae (n=9) and Lactobacillus (n=6) the most abundant. Fetal intestines dominated by Micrococcaceae exhibited distinct patterns of T cell composition and epithelial transcription. Fetal Micrococcus luteus, isolated only in the presence of monocytes, grew on placental hormones, remained viable within antigen presenting cells, limited inflammation ex vivo, and possessed genomic features linked with survival in the fetus. Thus, viable bacteria are highly limited in the fetal intestine at mid-gestation, though strains with immunomodulatory capacity are detected in subsets of specimens.

Short chain fatty acids produced by colonizing intestinal commensal bacterial interaction with expressed breast milk are anti-inflammatory in human immature enterocytes.

Necrotizing enterocolitis (NEC) is a devastating intestinal emergency that affects ten percent of very low birth weight premature babies and costs society in both expense and heartache. It is probably caused by an inappropriate interaction of colonizing bacteria with an immature intestine. A possible preventative measure is to feed prematures their mother’s expressed breast milk in conjunction with a probiotic. This synbiotic prevention reduces the severity and incidence of this condition. This study was designed to determine the mechanism of the synbiotic effect in human and mouse fetal intestine. Breast milk interacting with a NEC preventative probiotic such as Bifidobacterium infantis can produce increased levels of short chain fatty acids (acetate, propionate and butyrate) (SCFAs). SCFAs are known to be anti-inflammatory in mature enterocytes and immunocytes. Very little is known about their role in immature intestine. When exposed to a human fetal cell line, fetal intestinal organoids and fetal mouse intestine, these SCFAs were anti-inflammatory. Their mechanism of anti-inflammation differed from those reported for mature cells by involving the G-protein coupled receptor (GPR 109A) and inhibiting histone deacetylase 4 and 5. These bacterial metabolites may help explain the synbiotic anti-inflammatory effect of breast milk and probiotics given to premature infants at risk for NEC.

Gastrointestinal organoid technology advances studies of enteric virus biology.

The historical lack of models recapitulating the complexity of the human intestinal epithelium has hindered studies into many aspects of human enteric virus biology. Immortalized and transformed cell lines are typically limited by the presence of only one cell type, whereas susceptibility in animal models often requires infection routes that differ from humans or necessitates modification of immune system components [1, 2]. In addition, comparing animals from different species remains a confounding factor when trying to infer how findings may apply to human health. Thus, the development of human gastrointestinal organoids to study virus–host interactions marks a significant advance. They provide a physiologically relevant ex vivo platform in which human enteric microorganisms can be studied interacting with the human intestinal epithelium. Furthermore, their intermediate complexity, falling between cell lines and animal models, adds an additional tool to better understand these viruses. Here, we highlight how gastrointestinal organoids have provided new insights into the biology of human enteric viruses and the potential of this technology for advancing the field. The different flavors of gastrointestinal organoids Gastrointestinal organoids are three-dimensional (3D) structures derived from primary tissues (i.e., patient biopsy) containing intestinal stem/progenitor cells or from human pluripotent stem cells (hPSCs) [3]. They contain multiple intestinal epithelial cell types that perform critical functions that are also observed in the human intestine (e.g., absorption, barrier function, differentiation). Induced pluripotent stem cell–derived human intestinal organoids (HIOs) most closely resemble the human fetal intestine [4] and may encompass an epithelium alone [5] or also contain a mesenchyme [6]. However, most infectious disease laboratories work with human intestinal enteroids (HIEs), patient-derived, 3D epithelium-only structures that can be transitioned to a 2D monolayer on plates or transwells. HIEs maintain the physiological and genetic characteristics of their sources for long periods [7–10]. They can be differentiated from the crypt-like state into villus-like state by withdrawing growth factors required to maintain stem cells (i.e., WNT3A) from the culture media [11–13]. Cellular differentiation of specific cell lineages can be further achieved by pharmacologic or genetic means. For example, secretory cells are enriched following dibenzazepine (DBZ) treatment to block NOTCH signaling [12] and enteroendocrine cells by overexpression of NEUROGENIN-3 [14]. In addition, receptor activator of NF-κB ligand (RANKL) treatment of HIE with and without tumor necrosis factor alpha (TNF-α) addition has been used to drive microfold cell development [15, 16]. However, HIE and HIO do not completely mimic the intestinal epithelium in vivo. For example, they do not entirely reproduce the cellularity observed in vivo as few Paneth cells and no Tuft cells are present [17]. Furthermore, the lack of villi and genetic or pharmacologic skewing of differentiation may also affect the proportionality of individual cell lineages.

Effects of Nutrient Restriction During Midgestation to Late Gestation on Maternal and Fetal Postruminal Carbohydrase Activities in Sheep.

To examine the effects of nutrient restriction during midgestation to late gestation on maternal and fetal digestive enzyme activities, 41 singleton ewes (48.3 ± 0.6 kg of BW) were randomly assigned to dietary treatments: 100% (control; CON; n = 20) or 60% of nutrient requirements (restricted; RES; n = 21) from day 50 until day 90 (midgestation). At day 90, 14 ewes (CON, n = 7; RES, n = 7) were euthanized. The remaining ewes were subjected to treatments of nutrient restriction or remained on a control diet from day 90 until day 130 (late gestation): CON-CON (n = 6), CON-RES (n = 7), RES-CON (n = 7), and RES-RES (n = 7) and were euthanized on day 130. The fetal and maternal pancreas and small intestines were weighed, subsampled, and assayed for digestive enzyme activity. One unit (U) of enzyme activity is equal to 1 µmol of product produced per minute for amylase, glucoamylase, lactase, and trypsin and 0.5 µmol of product produced per minute for maltase and isomaltase. Nutrient restriction during midgestation and late gestation decreased (P < 0.05) maternal pancreatic and small intestinal mass but did not affect fetal pancreatic or small intestinal mass. Maternal nutrient restriction during late gestation decreased (P = 0.03) fetal pancreatic trypsin content (U/pancreas) and tended to decrease (P < 0.08) fetal pancreatic trypsin concentration (U/g), specific activity (U/g protein), and content relative to BW (U/kg of BW). Nutrient restriction of gestating ewes decreased the total content of α-amylase (P = 0.04) and tended to decrease total content of trypsin (P = 0.06) and protein (P = 0.06) in the maternal pancreas on day 90. Nutrient restriction during midgestation on day 90 and during late gestation on day 130 decreased (P = 0.04) maternal pancreatic α-amylase-specific activity. Sucrase activity was undetected in the fetal and maternal small intestine. Nutrient restriction during late gestation increased (P = 0.01) maternal small intestinal maltase and lactase concentration and tended to increase (P = 0.06) isomaltase concentration. Realimentation during late gestation after nutrient restriction during midgestation increased lactase concentration (P = 0.04) and specific activity (P = 0.05) in the fetal small intestine. Fetal small intestinal maltase, isomaltase, and glucoamylase did not respond to maternal nutrient restriction. These data indicate that some maternal and fetal digestive enzyme activities may change in response to maternal nutrient restriction.