Journal: Molecular Human Reproduction

Article Title: ADAM8 localizes to extravillous trophoblasts within the maternal–fetal interface and potentiates trophoblast cell line migration through a β1 integrin-mediated mechanism

doi: 10.1093/molehr/gay034

Figure Lengend Snippet: ADAM8 preferentially localizes to anchoring columns of placental villi. (A) Representative immunofluorescence, immunohistochemistry and RNAScope microscopy images of serially sectioned first trimester placental villi (6–11 weeks gestation; n = 5) and decidua (10–12 weeks gestation; n = 4) showing ADAM8 mRNA transcript localization (pink punctate signal) within trophoblast subtypes. Proximal and distal column, as well as interstitial EVT subtypes of trophoblasts are identified by immunostaining of cytokeratin (KRT7) and HLA-G. ‘PCT’ indicates proximal column trophoblast; ‘DCT’ indicates distal column trophoblast; ‘VT’ indicates villous trophoblast; ‘SynT’ indicates syncytiotrophoblast; ‘MC’ indicates placenta mesenchymal core; ‘iEVT’ indicates interstitial extravillous trophoblast; ‘GC’ indicates trophoblast giant cell. The perforated white box indicates enlarged inset image. Black arrowheads denote ADAM8 signal. Bars = 100 μm. (B) Fluorescence activated cell-sorting (FACS) plots demonstrate the trophoblast isolation strategy used to purify mesencymal core cells (MC), distal column trophoblasts (DCT) and villous/proximal column trophoblasts (VT). Live cells, depleted of CD31+ (endothelial) and CD45+ (immune) cells using immuno-magnetic beads, were positively gated by 7AAD exclusion. Cells were further segregated by excluding CD45+ immune cells and by cell surface labeling of HLA-G and CD49f. Cell subtype proportions are indicated within each gated population (percent of cells within FACS plot). (C) ADAM8 mRNA levels in FACS-purified MCs, VTs and DCTs. Trophoblast subtype purity was assessed by qPCR analysis targeting the pan-trophoblast marker KRT7, the EVT-marker HLA-G and the mesenchymal lineage marker VIM. GAPDH was used for normalization. Results are presented as mean ± SD in bar graphs from four distinct placental villi specimens (n = 4); results were analyzed by one-way ANOVA and Dunn’s multiple comparisons test. (D) Immunoblot showing protein levels of pro- and active-ADAM8, HLA-G, and EGFR in MC, VT and DCT isolated from placental villi using immuno-magnetic beads. Molecular weights (kDa) are shown to the left and β-actin indicates loading control.

Article Snippet: Contaminating immune cells were removed from the cell admixture by EasySep CD45 immuno-magnetic bead purification (StemCell Technologies) where remaining cells were plated onto plastic culture plates for 30 min to remove stromal cells.

Techniques: Immunofluorescence, Immunohistochemistry, RNAscope, Microscopy, Immunostaining, Fluorescence, FACS, Isolation, Magnetic Beads, Labeling, Purification, Marker, Western Blot, Control

Journal: Journal of Crohn's & Colitis

Article Title: TREM-1 + Macrophages Define a Pathogenic Cell Subset in the Intestine of Crohn’s Disease Patients

doi: 10.1093/ecco-jcc/jjab022

Figure Lengend Snippet: The intestinal microenvironment of Crohn’s disease patients promotes an inflammatory transcriptome in blood monocytes. [A–C] Differentially expressed transcriptomic pathways in blood CD14 + monocytes from healthy donors treated for 5 h with [A] TREM-1 agonist antibody [5 µg/mL], [B] LP-CM from three controls or [C] LP-CM from three Crohn’s disease patients. Quantification of gene expression was done with the NanoString nCounter Human Myeloid Innate Immunity V2 Panel. Pathway signature scores condense each sample’s gene expression profile into a small set of pathway scores. An experiment can then be explored through the lens of pathway signature scores instead of in the much higher-dimension lens of gene expression values. Pathway signature scores are fit using the first principal component of each gene set’s data. They are orientated such that an increasing score corresponds to mostly increasing expression [specifically, each pathway score has positive weights for at least half its genes]. Covariates plots compare pathway signature scores to covariates. Red triangles and green triangles help to show the relative increase and decrease in pathways, respectively, compared to the control condition. [D] The 38 molecules that increased by a minimum two-fold in LP-CM from the three Crohn’s disease patients compared to LP-CM from the three controls. The fold change is based on the mean of the three values per group. [E] LP-CM inflammation score for each sample. This score is the mean of the linear NPX values [see Section 2.6] of the 38 proteins shown in D as a way to get a relative quantification of the inflammation status of the individuals analysed. [F] Correlations between the LP-CM inflammation scores and the top ten increased genes in human blood CD14 + monocytes treated with the Crohn’s disease LP-CM compared to cells treated with the control LP-CM [see ]. [G] Correlations between the LP-CM inflammation scores and the frequency of cells in the same individual determined by flow cytometry [gated as in ]. [F, G] Correlations were assessed by Spearman’s test. Abbreviations: Ctr, controls; LP-CM, lamina propria conditioned media; Mfs, macrophages.

Article Snippet: After washing in PBS and removal of erythrocytes as described above, CD14 + monocytes were isolated using CD14 + magnetic beads (EasySep Human CD14 Positive Selection Kit II [Stemcell Technologies, Canada]).

Techniques: Gene Expression, Expressing, Control, Quantitative Proteomics, Flow Cytometry

Journal: Journal of Crohn's & Colitis

Article Title: TREM-1 + Macrophages Define a Pathogenic Cell Subset in the Intestine of Crohn’s Disease Patients

doi: 10.1093/ecco-jcc/jjab022

Figure Lengend Snippet: The intestinal microenvironment of Crohn’s disease patients promotes an inflammatory transcriptome in blood monocytes, which is partly reduced by TREM-1 blockade.

Article Snippet: After washing in PBS and removal of erythrocytes as described above, CD14 + monocytes were isolated using CD14 + magnetic beads (EasySep Human CD14 Positive Selection Kit II [Stemcell Technologies, Canada]).

Techniques:

Journal: Journal of Crohn's & Colitis

Article Title: TREM-1 + Macrophages Define a Pathogenic Cell Subset in the Intestine of Crohn’s Disease Patients

doi: 10.1093/ecco-jcc/jjab022

Figure Lengend Snippet: IL-6 induced in blood monocytes by Crohn’s disease LP-CM is partially reduced by TREM-1/TNF double blockade. Differentially expressed [A] transcriptomic pathways or [B] CCL3 , CCL4 and IL-6 expression in blood CD14 + monocytes from a healthy donor treated for 5 h with LP-CM from three Crohn’s disease patients plus TREM-1 antagonist antibody [1 µg/mL]. Quantification of gene expression was done with the NanoString nCounter Human Myeloid Innate Immunity V2 Panel. Explanation of pathway signature scores is described in the legend to . [C–E] CCL3 , CCL4 and IL-6 expression, determined by RT-PCR, in human blood CD14 + monocytes treated for 5 h with LP-CM from six additional Crohn’s disease patients plus TREM-1 antagonist antibody alone [1 µg/mL], TNF adalimumab antibody alone [1 µg/mL], or both antibodies together. These blocking experiments were done with the appropriate isotype controls, IgG1 and IgG4, as described in sheet 1. [B–E] Each colour indicates a different individual. [B–E] Data are shown as median values. The difference between paired samples was assessed by a Wilcoxon test; * p vs adalimumab alone. Abbreviations: LP-CM, lamina propria conditioned media.

Article Snippet: After washing in PBS and removal of erythrocytes as described above, CD14 + monocytes were isolated using CD14 + magnetic beads (EasySep Human CD14 Positive Selection Kit II [Stemcell Technologies, Canada]).

Techniques: Expressing, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Blocking Assay