Impact of Maternal Malnutrition on Gut Barrier Defense: Implications for Pregnancy Health and Fetal Development

Small intestinal Paneth cells, enteric glial cells (EGC), and goblet cells maintain gut mucosal integrity, homeostasis, and influence host physiology locally and through the gut-brain axis. Little is known about their roles during pregnancy, or how maternal malnutrition impacts these cells and their development. Pregnant mice were fed a control diet (CON), undernourished by 30% vs. control (UN), or fed a high fat diet (HF). At day 18.5 (term = 19), gut integrity and function were assessed by immunohistochemistry and qPCR. UN mothers displayed reduced mRNA expression of Paneth cell antimicrobial peptides (AMP; Lyz2, Reg3g) and an accumulation of villi goblet cells, while HF had reduced Reg3g and mucin (Muc2) mRNA and increased lysozyme protein. UN fetuses had increased mRNA expression of gut transcription factor Sox9, associated with reduced expression of maturation markers (Cdx2, Muc2), and increased expression of tight junctions (TJ; Cldn-7). HF fetuses had increased mRNA expression of EGC markers (S100b, Bfabp, Plp1), AMP (Lyz1, Defa1, Reg3g), and TJ (Cldn-3, Cldn-7), and reduced expression of an AMP-activator (Tlr4). Maternal malnutrition altered expression of genes that maintain maternal gut homeostasis, and altered fetal gut permeability, function, and development. This may have long-term implications for host-microbe interactions, immunity, and offspring gut-brain axis function

Host Immune Response to ZIKV in an Immunocompetent Embryonic Mouse Model of Intravaginal Infection

Zika virus (ZIKV) only induces mild symptoms in adultshowever, it can cause congenital Zika syndrome (CZS), including microcephaly. Most of the knowledge on ZIKV pathogenesis was gained using immunocompromised mouse models, which do not fully recapitulate human pathology. Moreover, the study of the host immune response to ZIKV becomes challenging in these animals. Thus, the main goal of this study was to develop an immunocompetent mouse model to study the ZIKV spread and teratogeny. FVB/NJ immune competent dams were infected intravaginally with ZIKV during the early stage of pregnancy. We found that the placentae of most fetuses were positive for ZIKV, while the virus was detected in the brain of only about 42% of the embryos. To investigate the host immune response, we measured the expression of several inflammatory factors. Embryos from ZIKV-infected dams had an increased level of inflammatory factors, as compared to Mock. Next, we compared the gene expression levels in embryos from ZIKV-infected dams that were either negative or positive for ZIKV in the brain. The mRNA levels of viral response genes and cytokines were increased in both ZIKV-positive and negative brains. Interestingly, the levels of chemokines associated with microcephaly in humans, including CCL2 and CXCL10, specifically increased in embryos harboring ZIKV in the embryo brains

Malaria in pregnancy regulates P‐glycoprotein (P‐gp/ Abcb1a ) and ABCA1 efflux transporters in the Mouse Visceral Yolk Sac

Malaria in pregnancy (MiP) induces intrauterine growth restriction (IUGR) and preterm labour (PTL). However, its effects on yolk sac morphology and function are largely unexplored. We hypothesized that MiP modifies yolk sac morphology and efflux transport potential by modulating ABC efflux transporters. C57BL/6 mice injected with Plasmodium berghei ANKA (5 × 105 infected erythrocytes) at gestational day (GD) 13.5 were subjected to yolk sac membrane harvesting at GD 18.5 for histology, qPCR and immunohistochemistry. MiP did not alter the volumetric proportion of the yolk sac's histological components. However, it increased levels of Abcb1a mRNA (encoding P-glycoprotein) and macrophage migration inhibitory factor (Mif chemokine), while decreasing Abcg1 (P < 0.05); without altering Abca1, Abcb1b, Abcg2, Snat1, Snat2, interleukin (Il)-1β and C-C Motif chemokine ligand 2 (Ccl2). Transcripts of Il-6, chemokine (C-X-C motif) ligand 1 (Cxcl1), Glut1 and Snat4 were not detectible. ABCA1, ABCG1, breast cancer resistance protein (BCRP) and P-gp were primarily immunolocalized to the cell membranes and cytoplasm of endodermic epithelium but also in the mesothelium and in the endothelium of mesodermic blood vessels. Intensity of P-gp labelling was stronger in both endodermic epithelium and mesothelium, whereas ABCA1 labelling increased in the endothelium of the mesodermic blood vessels. The presence of ABC transporters in the yolk sac wall suggests that this fetal membrane acts as an important protective gestational barrier. Changes in ABCA1 and P-gp in MiP may alter the biodistribution of toxic substances, xenobiotics, nutrients and immunological factors within the fetal compartment and participate in the pathogenesis of malaria-induced IUGR and PTL

Differential expression of follistatin and FLRG in human breast proliferative disorders

<p>Abstract</p> <p>Background</p> <p>Activins are growth factors acting on cell growth and differentiation. Activins are expressed in high grade breast tumors and they display an antiproliferative effect inducing G0/G1 cell cycle arrest in breast cancer cell lines. Follistatin and follistatin- related gene (FLRG) bind and neutralize activins. In order to establish if these activin binding proteins are involved in breast tumor progression, the present study evaluated follistatin and FLRG pattern of mRNA and protein expression in normal human breast tissue and in different breast proliferative diseases.</p> <p>Methods</p> <p>Paraffin embedded specimens of normal breast (NB - n = 8); florid hyperplasia without atypia (FH - n = 17); fibroadenoma (FIB - n = 17); ductal carcinoma <it>in situ </it>(DCIS - n = 10) and infiltrating ductal carcinoma (IDC - n = 15) were processed for follistatin and FLRG immunohistochemistry and <it>in situ </it>hybridization. The area and intensity of chromogen epithelial and stromal staining were analyzed semi-quantitatively.</p> <p>Results</p> <p>Follistatin and FLRG were expressed both in normal tissue and in all the breast diseases investigated. Follistatin staining was detected in the epithelial cytoplasm and nucleus in normal, benign and malignant breast tissue, with a stronger staining intensity in the peri-alveolar stromal cells of FIB at both mRNA and protein levels. Conversely, FLRG area and intensity of mRNA and protein staining were higher both in the cytoplasm and in the nucleus of IDC epithelial cells when compared to NB, while no significant changes in the stromal intensity were observed in all the proliferative diseases analyzed.</p> <p>Conclusion</p> <p>The present findings suggest a role for follistatin in breast benign disease, particularly in FIB, where its expression was increased in stromal cells. The up regulation of FLRG in IDC suggests a role for this protein in the progression of breast malignancy. As activin displays an anti-proliferative effect in human mammary cells, the present findings indicate that an increased FST and FLRG expression in breast proliferative diseases might counteract the anti-proliferative effects of activin in human breast cancer.</p

Expressão e localização de A/Bativina, seus receptores ActRIB e ActRIIA, de -inibinae de folistatina na glândula mamária bovina gestacional

Exportado OPUSMade available in DSpace on 2019-08-12T16:46:13Z (GMT). No. of bitstreams: 2 disserta__o_resultados_enrico.pdf: 1376771 bytes, checksum: 03288ef565e6d6884f6ddf1755e04886 (MD5) disserta__o_enrico.pdf: 550727 bytes, checksum: e4e0caca63f611a431e59ab3c9c19cbd (MD5) Previous issue date: 27Ativinas são fatores fatores de crescimento pertencentes a superfamília de fatores de crescimento â (TGF-â) e agem através dos receptores ActRIB e ActRIIA e seus efeitos são antagonizados pelas inibinas e folistatinas. As ativinas estão relacionadas ao crescimento ductal mamário e à diferenciação lóbulo-alveolar de camundongos. Por esse motivo, o presente trabalho avaliou a expressão gênica e protéica de âA/âB, ActRIB e ActRIIA, inibina e folistatina na glândula mamária de novilhas gestantes e não gestantes. As glândulas mamárias foram obtidas a partir de novilhas da raça nelore não gestantes (NG, n=9) e gestantes na 1o fase (GP, n=9) e 2o fase (GS n=9) do desenvolvimento mamário gestacional. O tecido mamário foi analisado por imunohistoquímica e PCR em tempo real. Os anticorpos primários utilizados foram os policlonais de coelho anti- âA (1:400), anti-ActRIB (1:200), anti-ActRIIA (1:200) e anti-FS (1:200) e os policlonais de cabra anti-B (1:200) e anti- (1:200), seguidos pela técnica de coloração por streptavidina-peroxidase. A imunocoloração ductal e lóbulo-alveolar de âA, ActRIB e ActRIIA, foi mais intensa no grupo GP que nos grupos NG e GS (p<0.05, ANOVA, Kruskal-Wallis e teste de Dunn). A subunidade âB ativina, a FS e a INB foram localizadas nas células epiteliais alveolares e ductais com intensidade de imunocoloração similar em todas as fases. A região estromal foi imunopositiva para todas as proteínas nas 3 fases estudadas. Os mRNAS das subunidades âA/âB, FS e ActRIIA foram similarmente expressos nas 3 fases estudadas. Contudo, o mRNA da cadeia -INH foi mais expresso durante a fase gestacional e o receptor ActRIB foi mais expresso somente durante a lactogenesis. Concluindo, este estudo demonstra pela primeira vez que as subunidades âA e âB da ativina, os receptores ActRIB e ActRIIA, á-inibina e FS são expressos nas estruturas estromais ductais e lobulares durante o desenvolvimento e diferenciação da glândula mamária bovina.Gestational Expression of â Activin, ActRIB and ActRIIA Receptors, -Inhibin and Follistatin in Bovine Mammary Gland.Activins belong to the transforming growth factor superfamily and bind selectively to ActRIB and ActRIIA receptors. Their effects are antagonized by inhibins (INH) and follistatins (FS). Since activins have been related to mammary ductal elongation and lobular-alveolar differentiation in mice, we evaluated gene and protein expression of Activin-A/B subunits, ActRIB and ActRIIA receptors, FS and -INH in the mammary gland of nulliparous and pregnant heifers. Mammary glands were obtained from beef heifers slaughtered in nulliparous (NP; n=9), pregnant in the 1st (60-120 days - mammogenesis; n=9) and in the 2nd stages of gestation (150-210 days lactogenesis; n=9). Mammary tissue was analyzed by immunohistochemistry and Real Time PCR. We found a higher intensity of ductal and lobular immunostaining for A, ActRIB and ActRIIA in the mammogenesis group when compared to NP and lactogenesis groups, however the stromal cells displayed less immunostaining for A and ActRIB during lactogenesis (p<0.05, Kruskal-Wallis ANOVA and Dunns test). Activin B subunit, FS and -INH proteins were localized in ductal, lobular and stromal cells with similar intensity during all the stages. The mRNA transcripts of Activin-A/B subunits, FS and ActRIIA were expressed equally in the mammary tissue of non-pregnant and pregnant heifers. However -INH mRNA expression was up regulated during gestation and ActRIB mRNA was up regulated only during lactogenesis. In conclusion, this study firstly shows that A/B activin, ActRIB, ActRIIA, -INH and FS are expressed in stromal, ductal and lobular structures in the bovine mammary gland during non-gestational and gestational mammary gland development and differentiation

Functional Expression of Multidrug-Resistance (MDR) Transporters in Developing Human Fetal Brain Endothelial Cells

There is little information about the functional expression of the multidrug resistance (MDR) transporters P-glycoprotein (P-gp, encoded by ABCB1) and breast cancer resistance protein (BCRP/ABCG2) in the developing blood–brain barrier (BBB). We isolated and cultured primary human fetal brain endothelial cells (hfBECs) from early and mid-gestation brains and assessed P-gp/ABCB1 and BCRP/ABCG2 expression and function, as well as tube formation capability. Immunolocalization of the von Willebrand factor (marker of endothelial cells), zonula occludens-1 and claudin-5 (tight junctions) was detected in early and mid-gestation-derived hfBECs, which also formed capillary-like tube structures, confirming their BEC phenotype. P-gp and BCRP immunostaining was detected in capillary-like tubes and in the cytoplasm and nucleus of hfBECs. P-gp protein levels in the plasma membrane and nuclear protein fractions, as well as P-gp protein/ABCB1 mRNA and BCRP protein levels decreased (p < 0.05) in hfBECs, from early to mid-gestation. No differences in P-gp or BCRP activity in hfBECs were observed between the two age groups. The hfBECs from early and mid-gestation express functionally competent P-gp and BCRP drug transporters and may thus contribute to the BBB protective phenotype in the conceptus from early stages of pregnancy

TGF-β1 regulation of multidrug resistance P-glycoprotein in the developing male blood-brain barrier

P-glycoprotein (P-gp), an efflux transporter encoded by the abcb1 gene, protects the developing fetal brain. Levels of P-gp in endothelial cells of the blood-brain barrier (BBB) increase dramatically during the period of peak brain growth. This is coincident with increased release of TGF-β1 by astrocytes and neurons. Although TGF-β1 has been shown to modulate P-gp activity in a number of cell types, little is known about how TGF-β1 regulates brain protection. In the present study, we hypothesized that TGF-β1 increases abcb1 expression and P-gp activity in fetal and postnatal BBB in an age-dependent manner. We found TGF-β1 to potently regulate abcb1 mRNA and P-gp function. TGF-β1 increased P-gp function in brain endothelial cells (BECs) derived from fetal and postnatal male guinea pigs. These effects were more pronounced earlier in gestation when compared with BECs derived postnatally. To investigate the signaling pathways involved, BECs derived at gestational day 50 and postnatal day 14 were exposed to ALK1 and ALK5 inhibitors and agonists. Through inhibition of ALK5, we demonstrated that ALK5 is required for the TGF-β1 effects on P-gp function. Activation of ALK1, by the agonist BMP-9, produced similar results to TGF-β1 on P-gp function. However, TGF-β1 signaling through the ALK1 pathway is age-dependent as dorsomorphin, an ALK1 inhibitor, attenuated TGF-β1-mediated effects in BECs derived at postnatal day 14 but not in those derived at gestational day 50. In conclusion, TGF-β1 regulates P-gp at the fetal and neonatal BBB and both ALK5 and ALK1 pathways are implicated in the regulation of P-gp function. Aberrations in TGF-β1 levels at the developing BBB may lead to substantial changes in fetal brain exposure to P-gp substrates, triggering consequences for brain development.This study was funded by the Canadian Institutes for Health Research (FRN-57746 to S.G.M. and W.G.)

Impact of bacterial and viral challenge on multidrug resistance in first- and third-trimester human placenta

The ABC transporters P-glycoprotein (P-gp, official gene symbol ABCB1) and breast cancer resistance protein (BCRP, official gene symbol ABCG2) protect the conceptus from exposure to toxins and xenobiotics present in the maternal circulation. Viral or bacterial challenges alter expression of placental multidrug transporters in rodents. We hypothesized that exposure to lipopolysaccharide (LPS, bacterial antigen) and polyinosinic-polycytidylic acid (poly(I:C), viral antigen) would decrease P-gp and BCRP in the human placenta. Placental explants from first and third trimesters were challenged with 0.1 to 10 μg/mL LPS or 1 to 50 μg/mL poly(I:C) for 4 or 24 hours; mRNA levels, protein expression, and localization were assessed by quantitative real-time PCR, Western blot analysis, and immunohistochemistry, respectively. Toll-like receptor (TLR)-3 and TLR-4 mRNA expression increased from the first to third trimester (P < 0.01), and the receptors localized to cytotrophoblasts in the first trimester and to syncytiotrophoblasts in the third trimester. LPS exposure in first-trimester explants decreased (P < 0.001) ABCB1 and ABCG2 mRNA and protein levels. In contrast, poly(I:C) decreased (P < 0.05) ABCB1, TLR-3, and TLR-4 mRNA levels in the third trimester but not first trimester. LPS and poly(I:C) treatments increased (P < 0.01) IL-8 and chemokine ligand 2. Results suggest that bacterial infections likely alter exposure of the conceptus to toxins and drugs during early pregnancy, whereas viral infections may disrupt fetal protection in later stages of pregnancy