Veillonella and Bacteroides are associated with gestational diabetes mellitus exposure and gut microbiota immaturity.

BackgroundDysbiosis during childhood impacts the configuration and maturation of the microbiota. The immaturity of the infant microbiota is linked with the development of inflammatory, allergic, and dysmetabolic diseases.AimsTo identify taxonomic changes associated with age and GDM and classify the maturity of the intestinal microbiota of children of mothers with GDM and children without GDM (n-GDM).MethodsNext-generation sequencing was used to analyze the V3-V4 region of 16S rRNA gene. QIIME2 and Picrust2 were used to determine the difference in the relative abundance of bacterial genera between the study groups and to predict the functional profile of the intestinal microbiota.ResultsAccording to age, the older GDM groups showed a lower alpha diversity and different abundance of Enterobacteriaceae, Veillonella, Clostridiales, and Bacteroides. Regarding the functional profile, PWY-7377 and K05895 associated with Vitamin B12 metabolism were reduced in GDM groups. Compared to n-GDM group, GDM offspring had microbiota immaturity as age-discriminatory taxa in random forest failed to classify GDM offspring according to developmental age (OOB error 81%). Conclusion. Offspring from mothers with GDM have a distinctive taxonomic profile related to taxa associated with gut microbiota immaturity

Simultaneous evaluation of metabolomic and inflammatory biomarkers in children with different body mass index (BMI) and waist-to-height ratio (WHtR).

Metabolic disturbances and systemic pro-inflammatory changes have been reported in children with obesity. However, it is unclear the time-sequence of metabolic or inflammatory modifications during children obesity evolution. Our study aimed to quantify simultaneously metabolomic and inflammatory biomarkers in serum from children with different levels of adiposity. For this purpose, a cross-sectional study was used to perform targeted metabolomics and inflammatory cytokines measurements. Serum samples from children between six to ten years old were analyzed using either body mass index (BMI) or waist-to-height ratio (WHtR) classifications. One hundred and sixty-eight school-aged children were included. BMI classification in children with overweight or obesity showed altered concentrations of glucose and amino acids (glycine and tyrosine). Children classified by WHtR exhibited imbalances in amino acids (glycine, valine, and tyrosine) and lipids (triacyl glycerides and low-density lipoprotein) compared to control group. No differences in systemic inflammation biomarkers or in the prevalence of other results were found in these children. Abnormal arterial blood pressure was found in 32% of children with increased adiposity. In conclusion, obesity in school-aged children is characterized by significant metabolic modifications that are not accompanied by major disturbances in circulating concentrations of inflammatory biomarkers

Transmigration of Neural Stem Cells across the Blood Brain Barrier Induced by Glioma Cells

<div><p>Transit of human neural stem cells, ReNcell CX, through the blood brain barrier (BBB) was evaluated in an <i>in vitro</i> model of BBB and in nude mice. The BBB model was based on rat brain microvascular endothelial cells (RBMECs) cultured on Millicell inserts bathed from the basolateral side with conditioned media (CM) from astrocytes or glioma C6 cells. Glioma C6 CM induced a significant transendothelial migration of ReNcells CX in comparison to astrocyte CM. The presence in glioma C6 CM of high amounts of HGF, VEGF, zonulin and PGE₂, together with the low abundance of EGF, promoted ReNcells CX transmigration. In contrast cytokines IFN-α, TNF-α, IL-12p70, IL-1β, IL-6, IL-8 and IL-10, as well as metalloproteinases -2 and -9 were present in equal amounts in glioma C6 and astrocyte CMs. ReNcells expressed the tight junction proteins occludin and claudins 1, 3 and 4, and the cell adhesion molecule CRTAM, while RBMECs expressed occludin, claudins 1 and 5 and CRTAM. Competing CRTAM mediated adhesion with soluble CRTAM, inhibited ReNcells CX transmigration, and at the sites of transmigration, the expression of occludin and claudin-5 diminished in RBMECs. In nude mice we found that ReNcells CX injected into systemic circulation passed the BBB and reached intracranial gliomas, which overexpressed HGF, VEGF and zonulin/prehaptoglobin 2.</p></div

Matrix Metalloproteinase-3 (MMP-3) Is an Endogenous Activator of the MMP-9 Secreted by Placental Leukocytes: Implication in Human Labor.

BACKGROUND:The activity of matrix degrading enzymes plays a leading role in the rupture of the fetal membranes under normal and pathological human labor, and matrix metalloproteinase-9 (MMP-9) it is considered a biomarker of this event. To gain further insight into local MMP-9 origin and activation, in this study we analyzed the contribution of human placental leukocytes to MMP-9 secretion and explored the local mechanisms of the pro-enzyme activation. METHODS:Placental blood leukocytes were obtained from women at term gestation without labor and maintained in culture up to 72 h. MMP-9 activity in the culture supernatants was determined by zymography and using a specific substrate. The presence of a potential pro-MMP-9 activator in the culture supernatants was monitored using a recombinant biotin-labeled human pro-MMP-9. To characterize the endogenous pro-MMP-9 activator, MMP-1, -3, -7 and -9 were measured by multiplex assay in the supernatants, and an inhibition assay of MMP-9 activation was performed using an anti-human MMP-3 and a specific MMP-3 inhibitor. Finally, production of MMP-9 and MMP-3 in placental leukocytes obtained from term pregnancies with and without labor was assessed by immunofluorescence. RESULTS:Placental leukocytes spontaneously secreted pro-MMP-9 after 24 h of culture, increasing significantly at 48 h (P≤0.05), when the active form of MMP-9 was detected. Culture supernatants activated the recombinant pro-MMP-9 showing that placental leukocytes secrete the activator. A significant increase in MMP-3 secretion by placental leukocytes was observed since 48 h in culture (P≤0.05) and up to 72 h (P≤0.001), when concentration reached its maximum value. Specific activity of MMP-9 decreased significantly (P≤0.005) when an anti-MMP-3 antibody or a specific MMP-3 inhibitor were added to the culture media. Placental leukocytes from term labor produced more MMP-9 and MMP-3 compared to term non-labor cells. CONCLUSIONS:In this work we confirm that placental leukocytes from human term pregnancies are able to secrete large amounts of MMP-9, and that the production of the enzyme it is enhanced by labor. We also demonstrate for the first time that endogenous MMP-3 plays a major role in MMP-9 activation process. These findings support the contribution of placental leukocytes to create the collagenolytic microenvironment that induces the rupture of the fetal membranes during human labor

Zonulin present in glioma C6 CM, opens the BBB and favors ReNcells CX transmigration.

<p>A) Zonulin is detected by Western blot in glioma C6 CM and not in astrocyte CM. B) ReNcells CX transmigration assay across RBMECs done by placing in the basal compartment of the Millicell, astrocyte or glioma C6 CM with or without zonulin. N = 6, F(3,20) = 228.2; *** p<0.001; as assessed by one-way ANOVA followed by Bonferroni's post hoc test. C) Elimination of zonulin from glioma C6 CM reverses the decrease in TEER exerted by glioma C6 CM. N = 3, F(3,30) = 5.981; **P<0.01, ***P<0.001 with respect to glioma C6 CM; as assessed by two-way ANOVA followed by Bonferroni's post hoc test.</p

HGF, VEGF and the lack of EGF in glioma C6 CM induce NSCs transmigration across RBMECs.

<p>A) The amount of HGF and VEGF determined by ELISA is higher in glioma C6 CM than in astrocyte CM, while the opposite is found for EGF. IP of HGF, VEGF or EGF reduces the amount of the growth factor present in CM. HGF: N = 4, F(3,12) = 26.54; VEGF: N = 3, F(3,8) = 20.98; EGF: N = 3, F(3,8) = 44.52; **P<0.01, ***P<0.001, ns = not significant; as assessed by one-way ANOVA followed by Bonferroni's post hoc test. B) ReNcells CX transmigration assay across RBMECs done by placing in the basal compartment of the Millicell, DMEM with HGF or EGF; or DMEM with neutralizing antibodies against HGF, VEGF or EGF; or astrocyte or glioma C6 CM with or without neutralizing antibodies against HGF, VEGF and EGF. Control corresponds to the transmigration induced by glioma C6 CM without the neutralizing antibodies against HGF (upper panel), VEGF (middle panel) and EGF (lower panel), and is considered as 100% transmigration. HGF: N = 3, F(6,14) = 124.7; VEGF: N = 3, F(4,10) = 275.5; EGF: N = 3, F(7,16) = 30.77; **P<0.01, ***P<0.001, ns = not significant; as assessed by one-way ANOVA followed by Bonferroni's post hoc test. C) RBMECs cultured for 8 h in DMEM with HGF and EGF have a TEER similar to that obtained with astrocyte CM. IP of VEGF reverses the decrease in TEER induced by glioma C6 CM whereas IP of EGF from astrocyte CM decreases TEER in a manner similar to that obtained with glioma C6 CM. HGF: N = 3, F(2,16) = 5.668; **P<0.01 with respect to astrocytes CM; VEGF: N = 3, F(2,20) = 14.64; *P<0.05 with respect to astrocytes CM; #P<0.05 with respect to glioma C6 CM; EGF: N = 3, F(3,30) = 23.07; *P<0.05, **P<0.01, ***P<0.001 with respect to astrocytes CM; ^#P<0.05, ^##P<0.01, ^###P<0.001 with respect to glioma C6 CM; ⁺P<0.05, ⁺⁺P<0.01, ⁺⁺⁺P<0.001 with respect to astrocytes CM + α-EGF; as assessed by one-way ANOVA followed by Bonferroni's post hoc test.</p

The presence of PGE₂ and the lack of EGF in glioma C6 CM promote a leakier BBB.

<p>A) Quantitation of PGE₂ by ELISA in astrocyte CM and in glioma C6 CM derived from cells treated or not with COX-2 inhibitor NS398. N = 3, F(2,6) = 161.9; **P<0.01, ***P<0.001; as assessed by one-way ANOVA followed by Bonferroni's post hoc test. B) TEER of RBMECs cultures incubated in the basal compartment with astrocytes or glioma C6 CM derived from control or NS398 treated cells. N = 3, F(2,20) = 53.56; *P<0.05, **P<0.01 with respect to astrocytes CM; ^##P<0.01, ^###P<0.001 with respect to glioma C6 CM; as assessed by two-way ANOVA followed by Bonferroni's post hoc test. C) Quantitative analysis of cytokines IFN-α, TNF-α, IL-12p70, IL-1β, IL-6, IL-8 and IL-10 present in astrocyte and glioma C6 CM. N = 3, df = 2, as assessed by Student's t-test.</p

Expression of TJ proteins in transmigrating cells favors their passage across RBMECs.

<p>A) Analysis by Western blots of the expression of occludin, claudins 1 to 5 and CRTAM in NIH-3T3 and L-fibroblasts, ReNcells CX and RBMECs. B) The transmigration of NIH-3T3 and L-fibroblasts across RBMECs is independent of the CM present in the basal compartment, but is significantly higher in NIH-3T3 fibroblasts that express claudin-2 than in L-fibroblast that do not express TJ proteins. N = 3, F(1,6) = 232.22, ***P<0.001; F(2,8) = 0.0895, ns = not significant; as assessed by two-way ANOVA followed by Bonferroni's post hoc test. C) Exogenous expression of occludin in L-fibroblasts enhances their transmigration across RBMECs monolayers. N = 9, t = 6.411, df = 8; ***P<0.001 as assessed by Student's t-test. D) Competing CRTAM mediated adhesion with soluble human CRTAM (CRTAM-Fc) added to the upper compartment of a Millicell insert, reduces transmigration of ReNcells CX. N = 4, F(2,6) = 73.86; ***P<0.001, ns = not significant; as assessed by one-way ANOVA followed by Bonferroni's post hoc test.</p

Glioma C6 CM induces transmigration of NSCs across RBMECs.

<p>A) Transmigration of ReNcells CX across RBMECs induced by astrocyte or glioma C6 CM. Light microscopy image of toluidine blue stained cells on the basal surface of a filter and scheme illustrating each assay. B) Graphed data. N = 3, F(1,12) = 343.8; *** p<0.001, ns = not significant; as assessed by two-way ANOVA followed by Bonferroni's post hoc test. C) In an 8 h transmigration assay, a similar degree of ReNcells CX transmigration is attained with glioma C6 cells or glioma C6 CM placed in the basal compartment. N = 10, F(3,36) = 60.74; ***P<0.001, ns = not significant; as assessed by one-way ANOVA followed by Bonferroni's post hoc test. D) Representative light microscopy image of toluidine blue stained cells on the basal surface of the filter and scheme illustrating each assay.</p