Journal: Journal of Clinical Medicine

Article Title: Endometrial Dysbiosis Is Related to Inflammatory Factors in Women with Repeated Implantation Failure: A Pilot Study

doi: 10.3390/jcm11092481

Figure Lengend Snippet: Nucleotide sequences and annealing temperature of the primers utilized in PCR experiments.

Article Snippet: A total of 14 DNA templates were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) or from Belgian Coordinated Collections of Micro-organisms (BCCM/LMG, Gent, Belgio), including templates of common microorganisms of the disease, Enterobacteriaceae species (LMG 2783), Enterococcus species (LMG 17117), Escherichia coli (LMG 2092), Gardnerella vaginalis (LMG 7832), Klebsiella pneumoniae (LMG 2095), Mycoplasma hominis (DSMZ 106704), Staphylococcus species (DSMZ 1798), Streptococcus species (LMG 8518), Pseudopropionibacterium rubrum (DSMZ 100122), Neisseria Subflava (LMG 5313), and Prevotella (DSMZ 15606), as well as templates of sexually transmitted disease pathogens, Chlamydia trachomatis (DSMZ 19440) and Neisseria gonorrhoeae (DSMZ 9188).

Techniques:

Journal: Journal of Clinical Medicine

Article Title: Endometrial Dysbiosis Is Related to Inflammatory Factors in Women with Repeated Implantation Failure: A Pilot Study

doi: 10.3390/jcm11092481

Figure Lengend Snippet: Descriptive statistics of microbial parameters for the two study groups. The data are expressed as mean ± SD. Statistical analysis was performed by unpaired t -test. **** p < 0.0001 vs. eubiosis patients.

Article Snippet: A total of 14 DNA templates were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) or from Belgian Coordinated Collections of Micro-organisms (BCCM/LMG, Gent, Belgio), including templates of common microorganisms of the disease, Enterobacteriaceae species (LMG 2783), Enterococcus species (LMG 17117), Escherichia coli (LMG 2092), Gardnerella vaginalis (LMG 7832), Klebsiella pneumoniae (LMG 2095), Mycoplasma hominis (DSMZ 106704), Staphylococcus species (DSMZ 1798), Streptococcus species (LMG 8518), Pseudopropionibacterium rubrum (DSMZ 100122), Neisseria Subflava (LMG 5313), and Prevotella (DSMZ 15606), as well as templates of sexually transmitted disease pathogens, Chlamydia trachomatis (DSMZ 19440) and Neisseria gonorrhoeae (DSMZ 9188).

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: Tumoral EHF predicts the efficacy of anti-PD1 therapy in pancreatic ductal adenocarcinoma

doi: 10.1084/jem.20180749

Figure Lengend Snippet: Tumor cells expressing low levels of EHF induce the conversion, expansion, and function in vitro of T reg cells and MDSCs. (A) Representative plots of T reg cell conversion from CD4 + CD25 − T cell induced by PANC-1-EHF cells relative to PANC-1-vector and T reg cell conversion induced by BxPC-3-EHF-KD cells relative to BxPC-3-scramble. TGFβ1 was used as positive control. RPMI 1640 was used as negative control. CD25 and FOXP3 expression was determined by flow cytometry after 3 d of coculture (left). Percentage of T reg cell conversion from CD4 + CD25 − T cells. CD4 was gated (right). (B) Representative histogram of T reg cell proliferation. Flow cytometry was performed after 5 d of coculture by gating on live cells to determine the percentage of T reg cells that diluted CFSE (left). Statistical analysis of the percentage of T reg cell division (right). (C) T reg cell suppression assay compared the percentage of CD8 + T cells division cocultured with PANC-1-EHF (BxPC-3-EHF-KD)–generated T reg cells and PANC-1-vector (BxPC-3-scramble)–generated T reg cells. Statistical analyses of the responder T cells division (right). (D) Human PBMCs were cultured in vitro under different conditions (negative control: RPMI 1640; positive control: IL6 and GM-CSF; experimental group: PANC-1-vector, PANC-1-EHF, BxPC-3-scramble, and BxPC-3-EHF-KD) and analyzed on day 6 to determine the MDSC marker expression by flow cytometry. Representative plots show the surface CD11b + and CD33 + costaining of MDSCs. HLA-DR − cells were gated (left). The frequency of CD11b + CD33 + cells among HLA-DR − cells was quantified in the bar graph (right). (E) Mouse BMDCs were cultured in vitro under different conditions (negative control: RPMI 1640; positive control: IL6 and GM-CSF; experimental group: PANC02-EHF and PANC02-vector) and analyzed on day 6 to determine the MDSC marker expression by flow cytometry. Representative plots show the surface CD45 + CD11b + Gr-1 + costaining of MDSCs (left). The frequencies of CD11b + Gr-1 + cells among CD45 + cells were quantified in the bar graph (right). (F) Representative histogram of MDSC expansion. IL-6 and GM-CSF were used as positive controls. Flow cytometry was performed after 3 d of coculture by gating on live cells to determine the percentage of MDSCs that diluted CFSE (left). Statistical analysis of the percentage of MDSCs division (right). (G) MDSCs induced by PANC02-vector or PANC02-EHF cells were isolated to evaluate their suppressive activities in vitro. Histograms were gated on CFSE + cells to determine the percentage of CD8 + T cells that diluted CFSE (left). The percentage of division of responder T cells is summarized and quantified in the bar graph (right). The coculture experiments (A–G) were repeated five times independently. Representative data are shown. Data are presented as mean ± SD. Paired Student’s t test was used for statistical analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Briefly, EHF stably transduced PANC-1 cells were immunoprecipitated with anti-EHF antibody (Thermo Fisher Scientific; PA5-30716).

Techniques: Expressing, In Vitro, Plasmid Preparation, Positive Control, Negative Control, Flow Cytometry, Suppression Assay, Generated, Cell Culture, Marker, Isolation

Journal: The Journal of Experimental Medicine

Article Title: Tumoral EHF predicts the efficacy of anti-PD1 therapy in pancreatic ductal adenocarcinoma

doi: 10.1084/jem.20180749

Figure Lengend Snippet: EHF negatively regulates TGFβ1 and GM-CSF expression in PDAC. (A) Changes in gene expression profiling upon EHF expression. qPCR was performed to detect the transcriptional change in the secreted factors that can induce T reg cell and MDSC accumulation. RNA was purified from PANC-1, PANC-1-vector, and PANC-1-EHF tumors. Relative expression is shown as fold change relative to GAPDH. (B) qPCR on EHF, TGFB1, and CSF2 was performed in the following cell lines: BxPC3, BxPC3-scramble, and BxPC3-EHF-KD; AsPC1, AsPC1-vector, and AsPC1-EHF; and CFPAC-1, CFPAC-1-scramble, and CFPAC-1-EHF-KD. (C) ELISA of TGFβ1 and GM-CSF in indicated cell lines. qPCR and ELISA experiments (A–C) were repeated three times independently. Paired Student’s t test was used as statistical analysis. *, P < 0.01; **, P < 0.01; ***, P < 0.001; n.s., not significant. (D) Western blot analysis of EHF, TGFβ1, and GM-CSF in indicated cell lines. Experiments were repeated three times independently. Representative data are shown. (E) Expression of EHF, TGFβ1, and GM-CSF in harvested subcutaneous mouse tumor tissues. P, PANC02-vector ( n = 7); E, PANC02-EHF ( n = 7). Experiments were repeated three times independently. Representative data are shown. (F) Representative IHC images of EHF, TGFβ1, and GM-CSF expression using human PDAC tissue ( n = 96). Bars, 100 µm. (G) Spearman rank correlation analysis was used to evaluate the correlation of tumor-specific EHF and TGFβ1/GM-CSF expression ( n = 96). The number at the right side of the plots represents the case number; plots without number at the right side represent only one case. All experimental data verified in three independent experiments. Data are presented as mean ± SD.

Article Snippet: Briefly, EHF stably transduced PANC-1 cells were immunoprecipitated with anti-EHF antibody (Thermo Fisher Scientific; PA5-30716).

Techniques: Expressing, Gene Expression, Purification, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: Tumoral EHF predicts the efficacy of anti-PD1 therapy in pancreatic ductal adenocarcinoma

doi: 10.1084/jem.20180749

Figure Lengend Snippet: EHF directly down-regulates the expression of TGFβ1 and GM-CSF in PDACs by binding to the EBS in their gene promoters. (A) EHF-scanned motif logo. (B and C) Predicted ETS binding sites (EBS) in the human TGFB1 (B) and CSF2 (C) promoters. Position relative to the transcription start site of the gene, sequence, and corresponding scores. (D and F) Binding of EHF to the promoters of TGFB1 (D) and CSF2 (F) in PANC-1-EHF determined by ChIP. Experiments were repeated three times independently. Representative data are shown. (E and G) The HEK293 (left) and PANC-1 cells (right) were transfected with either vector control or pCDH-EHF in conjunction with the luciferase reporter pGL3-empty vector, WT pGL3-TGFB1/CSF2-promoter, or pGL3-TGFB1/CSF2-promoter with EBS1 mutation (EBS1-mut), EBS2 mutation (EBS2-mut), or EBS1+2 mutation (EBS1+2-mut) vector. Results are expressed as fold induction relative to that of the corresponding cells transfected with the control vector after normalization of firefly luciferase activity according to Renilla luciferase activity. Experiments were repeated three times independently. Data are presented as mean ± SD. Paired Student’s t test was used for statistical analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s. not significant.

Article Snippet: Briefly, EHF stably transduced PANC-1 cells were immunoprecipitated with anti-EHF antibody (Thermo Fisher Scientific; PA5-30716).

Techniques: Expressing, Binding Assay, Sequencing, Transfection, Plasmid Preparation, Control, Luciferase, Mutagenesis, Activity Assay

Journal: The Journal of Experimental Medicine

Article Title: Tumoral EHF predicts the efficacy of anti-PD1 therapy in pancreatic ductal adenocarcinoma

doi: 10.1084/jem.20180749

Figure Lengend Snippet: Tumor EHF deficiency induces immune suppression through TGFβ1 and GM-CSF. (A) Statistical analysis of CD4 + CD25 − T cells to T reg cell conversion (left) and T reg cell proliferation (right) induced by PANC-1-EHF cells relative to PANC-1-vector with or without TGFβ1 neutralization. CD4 + CD25 − T cells cultured in RPMI 1640 were used as negative controls. (B) Statistical analysis of mouse BMDC to MDSC conversion and MDSC expansion induced by PANC02-vector cells relative to PANC02-EHF with or without GM-CSF neutralization. BMDCs cultured in RPMI 1640 were used as negative controls. Experiments were repeated three times independently. Paired Student’s t test was used for statistical analysis. (C) PANC02-vector or PANC02-EHF was injected subcutaneously in a Matrigel plug containing neutralizing anti–GM-CSF mAb and anti-TGFβ1 mAb (vs. isotype IgGs as control) into the flanks of C57BL/6 mice. After 8 d, TGFβ1 and GM-CSF antibodies or isotype IgGs were intratumorally injected at 20 µg/mouse two times a week. (D and E) Representative dot plots of the proportion of tumor infiltration by T reg cells (D, left) and MDSCs (E, left) in the PANC02-vector and PANC02-EHF groups with or without combined TGFβ1 and GM-CSF depletion. Statistical analysis of the proportion of tumor-infiltrating T reg cells (D, right) and MDSCs (E, right) in the PANC02-vector and PANC02-EHF groups with or without combined TGFβ1 and GM-CSF depletion. The mouse experiments were repeated three times independently, using seven mice per experimental group. Representative data are shown. Nonpaired Student’s t test was used for statistical analysis. (F) Schematic of the roles of EHF in tumor immune modulation. EHF decreased the tumor-infiltrating T reg cells and MDSCs by transcriptionally suppressing the expression of TGFβ1 and GM-CSF. Data are presented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant.

Article Snippet: Briefly, EHF stably transduced PANC-1 cells were immunoprecipitated with anti-EHF antibody (Thermo Fisher Scientific; PA5-30716).

Techniques: Plasmid Preparation, Neutralization, Cell Culture, Injection, Control, Expressing