Journal: PLoS Pathogens

Article Title: A Nasal Epithelial Receptor for Staphylococcus aureus WTA Governs Adhesion to Epithelial Cells and Modulates Nasal Colonization

doi: 10.1371/journal.ppat.1004089

Figure Lengend Snippet: Surface exposure of SREC-I on HNECs and CRNECs was monitored by FACS with the same anti-human SREC-I antibody and isotype control. Statistical evaluation on HNECs (A) and on CRNECs (B). Data represent means ± SD of 3 or 4 independent experiments. Statistical analysis was performed by Student's t-test. Significant differences are indicated by one ( P <0.05), two ( P <0.01), or three ( P <0.001) asterisks ( * ).

Article Snippet: Primary human nasal epithelial cells (HNECs) were purchased from Provitro (Berlin) and cultivated in airway epithelial growth medium (Provitro).

Techniques: Control

Journal: PLoS Pathogens

Article Title: A Nasal Epithelial Receptor for Staphylococcus aureus WTA Governs Adhesion to Epithelial Cells and Modulates Nasal Colonization

doi: 10.1371/journal.ppat.1004089

Figure Lengend Snippet: S. aureus adherence to confluent human nasal epithelial cells (HNECs) monitored under static and mild shear stress condition (0.5 dynes) in 24 well plates or flow chamber assays, respectively. Attachment of the S. aureus wild-type (wt), tagO , and a complemented tagO mutant ( tagO compl.) evaluated microscopically by counting at least 6 visible fields per well or chamber slide containing HNECs (A) or CRNECs (B). The data is expressed as means and SEM of 5 independent experiments (static) or of 6 independent experiments (flow). Statistical analysis was performed by one-way ANOVA with Bonferroni's multiple comparison test. Significant differences between groups are indicated by one ( P <0.05), two ( P <0.01), or three ( P <0.001) asterisks ( * ).

Article Snippet: Primary human nasal epithelial cells (HNECs) were purchased from Provitro (Berlin) and cultivated in airway epithelial growth medium (Provitro).

Techniques: Shear, Mutagenesis, Comparison

Journal: PLoS Pathogens

Article Title: A Nasal Epithelial Receptor for Staphylococcus aureus WTA Governs Adhesion to Epithelial Cells and Modulates Nasal Colonization

doi: 10.1371/journal.ppat.1004089

Figure Lengend Snippet: Inhibition of SREC-I dependent adhesion to HNECS by Fab 2 - fragments of an anti-human SREC-I antibody in 24 well microtiter plate assays under static conditions (A) and shear stress conditions in ibidi chamber slide assays (B). 10 µg/ml of the anti-SREC-I Fab 2 -fragments and isotype control were used in all assays. Attachment of the S. aureus wild-type (wt), and tagO mutant was evaluated microscopically by counting at least 6 visible fields per well or chamber slide containing HNECs. The data is expressed as the means and SEM of 5 independent experiments under static and flow conditions. Statistical analysis was performed by one-way ANOVA with Bonferroni's multiple comparison test (A and B). Fab 2 - fragments of an anti-human SREC-I antibody were also used to test the influence of SREC-I on bacterial adhesion under flow condition to CRNECs in ibidi chamber slide assays (C). 10 µg/ml of the SREC-I Fab 2 -fragments and isotype control were used in all assays. Attachment of the S. aureus wild-type (wt), and tagO mutant was evaluated microscopically by counting at least 6 visible fields per chamber slide containing CRNECs. The data represent mean and SEM of 5 independent experiments. Statistical analysis was performed by one-way ANOVA with Bonferroni's multiple comparison test. (C) We also tested the influence of SREC-I on the adhesion of other S. aureus strain backgrounds (D). Fab 2 - fragments of an anti-human SREC-I antibody were used under flow conditions in ibidi chamber slide assays with HNECS. 10 µg/ml of the SREC-I Fab 2 - fragments and isotype control were used in all assays. Attachment of the S. aureus Newman (wt), and SH1000 mutant was evaluated microscopically by counting at least 6 visible fields per chamber slide containing CRNECs. The data represent mean and SEM of 4 independent experiments. Statistical analysis was performed by one-way ANOVA with Bonferroni's multiple comparison test. (D) Significant differences between groups are indicated by one ( P <0.05), two ( P <0.01), or three ( P <0.001) asterisks ( * ).

Article Snippet: Primary human nasal epithelial cells (HNECs) were purchased from Provitro (Berlin) and cultivated in airway epithelial growth medium (Provitro).

Techniques: Inhibition, Shear, Control, Mutagenesis, Comparison

Journal: PLoS Pathogens

Article Title: A Nasal Epithelial Receptor for Staphylococcus aureus WTA Governs Adhesion to Epithelial Cells and Modulates Nasal Colonization

doi: 10.1371/journal.ppat.1004089

Figure Lengend Snippet: The anti-human SREC-I antibody was used to test the influence of SREC-I on adhesion of WTA-coated latex beads under static condition in a 24 well microtiter plate assays on HNECs (A) and CRNECs (B). A ratio of 20 beads per epithelial cells was used in all depicted assays. 10 µg/ml of the anti-SREC-I antibody and isotype control were used to test the impact of SREC-I. Adhesion of the beads coated with wt WTA (HNECs and CRNECs) and dltA WTA (HNECs only) or control beads containing no WTA (HNECs and CRNECs) was measured in a fluoreader and expressed as relative fluorescence. The data are expressed as the means and SEM of 6 independent experiments. Statistical analysis was performed by one-way ANOVA with Bonferroni's multiple comparison test (A and B). The dose dependency of WTA-coated latex beads was tested in assays with CRNECs (C). Adhesion of the beads coated with wt WTA and dltA WTA or control beads containing no WTA was measured in a fluoreader and expressed as relative fluorescence. The data are expressed as the means and SEM of 5 independent experiments (C). Significant differences between groups are indicated by one ( P <0.05), two ( P <0.01), or three ( P <0.001) asterisks ( * ).

Article Snippet: Primary human nasal epithelial cells (HNECs) were purchased from Provitro (Berlin) and cultivated in airway epithelial growth medium (Provitro).

Techniques: Control, Fluorescence, Comparison

Journal: Cell reports

Article Title: Identification of an Early Unipotent Neutrophil Progenitor with Pro-Tumoral Activity in Mouse and Human Bone Marrow

doi: 10.1016/j.celrep.2018.07.097

Figure Lengend Snippet: (A) Flow cytometry analysis of healthy human BM uncovers a heterogeneous Lin−CD66b+CD117+ hNeP fraction. Dump antibody cocktail contains antibodies against markers that are expressed by: HSC (CD90 (Thy1)), lymphocytes and their progenitors (CD3, CD19, CD56, CD161, CD7, CD127 (IL-7Rα)), Erythrocytes and their progenitors (CD41, CD235a (Glycophorin A)), Eosinophils/Basophils and their progenitors (Siglec 8, FcεRIα, , CD125 (IL-5Rα)), CMP/GMP and monocyte progenitors (CD123 (IL-3Rα)), DCs and macrophages (CD11c, CD169). (B) ScRNA-Seq analysis of Lin−CD66b+CD117+ hNeP cells reveals two major subpopulations, Subset A and Subset B. 20,000 hNeP cells were FACS sorted from healthy human BM for scRNA-Seq. Heatmap shows top 40 differentially expressed genes in each cluster. Log2 Fold Change of each gene expression is relative to the entire dataset. 2 biological triplicates, 2 technical replicates. (C) Lin−CD66b+CD117+ hNeP were divided into CD34+ subset and CD34−subset by flow cytometry. Confocal microscopy was used to detect Ki67 localization (red) within the nuclei (blue) in CD34+ subset and CD34−subset using antibodies to Ki67 and Hoechst. IgG stained cells served as negative control. Bar: 5µm.

Article Snippet: CD34 + hNeP, CD34 − hNeP, and mature neutrophils were FACS sorted from fresh human BM obtained from AllCells, Inc. (Alameda, CA).

Techniques: Flow Cytometry, Expressing, Confocal Microscopy, Staining, Negative Control

Journal: Cell reports

Article Title: Identification of an Early Unipotent Neutrophil Progenitor with Pro-Tumoral Activity in Mouse and Human Bone Marrow

doi: 10.1016/j.celrep.2018.07.097

Figure Lengend Snippet: (A) Scheme showing the experimental procedure. CD34+ hNeP and CD34−hNeP subsets identified in Figure 5C were sorted from healthy human BM, and adoptively transferred into NSG-M3 recipient mice. Each recipient mouse received 25,000 donor human hNeP progenitor cells. After the transfer, peripheral blood was collected from each recipient via saphenous vein for flow cytometry on Day (D) 5, 7, 14, 28 (D5, D7, D14, D28), respectively. (B) Representative plots show the appearance of monocytes (CD86+ CD66b−), neutrophils (CD86−Siglec 8−CD66b+), eosinophils (Siglec 8+), and lymphocytes (hLy+) in each recipient group at the time points indicated. hLy antibody cocktail contains CD3, CD19, and CD56. N = 10 mice for each group.

Article Snippet: CD34 + hNeP, CD34 − hNeP, and mature neutrophils were FACS sorted from fresh human BM obtained from AllCells, Inc. (Alameda, CA).

Techniques: Flow Cytometry

Journal: Cell reports

Article Title: Identification of an Early Unipotent Neutrophil Progenitor with Pro-Tumoral Activity in Mouse and Human Bone Marrow

doi: 10.1016/j.celrep.2018.07.097

Figure Lengend Snippet: (A) hNeP is increased in melanoma patient blood. hNeP frequency was detected by flow cytometry in peripheral blood collected from healthy donors (N = 5) and melanoma patients (N = 5). Error bars indicate mean ± SEM. (B) Left, scheme showing the experiment procedure. CD34+ hNeP subset, CD34−hNeP subset, and human cMoP were FACS sorted from healthy human BM. The 3 populations were adoptively transferred into NSG-M3 recipient mice. Each recipient mouse received 25,000 donor human progenitor cells. Blank control group received only PBS for adoptive transfer. The next day, 1 × 106 143B human osteosarcoma cells were SubQ injected into each recipient mouse. Right, the tumor volume in each recipient was measured at 10d post-injection. N = 5 mice in each group. Error bars indicate mean ± SEM. (C) CD34+ hNeP cells blunt T cell activation. FACS sorting strategies for CD34+ hNeP and CD34−hNeP are shown in Figure 5A and ​andCC using using flow cytometry; FACS sorting strategies for mature neutrophils is shown in Figure S9A using flow cytometry. Histogram overlay of CD69 for CD3+ T cells cultured with CD34+ hNeP, CD34−hNeP, and mature neutrophils, in the presence of anti-CD3 for 24 hours. CD3+ T cells alone cultured without anti-CD3 served as the negative control group, and CD3+ T cells alone cultured with anti-CD3, CD28 served as the positive control group for T cell activation.

Article Snippet: CD34 + hNeP, CD34 − hNeP, and mature neutrophils were FACS sorted from fresh human BM obtained from AllCells, Inc. (Alameda, CA).

Techniques: Flow Cytometry, Adoptive Transfer Assay, Injection, Activation Assay, Cell Culture, Negative Control, Positive Control

Journal: bioRxiv

Article Title: HA and M2 sequences alter the replication of 2013-16 H1 Live Attenuated Influenza Vaccine Infection in Human Nasal Epithelial Cell cultures

doi: 10.1101/2021.11.11.468304

Figure Lengend Snippet: M2 mutations that enhance H3N2 LAIV replication reduce H1N1 LAIV Replication in hNEC cultures. Low MOI, multistep growth curves were performed at 32oC (A,C,E,G) and 37oC (B,D,F,H) with the indicated viruses. Data are pooled from three independent replicate experiments each with n=4 wells per virus (total n = 12 wells per virus, SEM). *p < 0.05 (two-way repeated measures ANOVA with Tukey’s posttest). Dotted line indicates limit of detection. Competition growth assays were performed with the indicated viruses on hNECs at 33°C and percent of total vRNA was determined by ddPCR (I,J). Data are pooled from three independent experiments each with n=3 wells (9 wells total, SEM). (K) Ratio of determined TCID50/mL to PFU/mL for each indicated virus from MDCK grown stock viruses. Infectious virus titer was performed in duplicate and PFU/mL was averaged from 6 wells of previously shown plaque assays. M to HA segment RNA ratio was also calculated (L) using ddPCR on 13 10-fold virus dilutions. (M) These same dilutions were then used to calculate the TCID50/mL to HA RNA ratio for each virus to predict differences in infectious vs non-infectious virus production (+SEM). P< 0.05(one-way ANOVA with Tukey’s posttest).

Article Snippet: We utilized human nasal epithelial cell (hNEC) cultures collected from non-diseased patients in the Johns Hopkins Medical Institutions as a second in vitro system to assess LAIV replication, as they have previously been shown to be more sensitive to changes in LAIV replication compared to MDCK cells [ , , ].

Techniques: Virus

Journal: bioRxiv

Article Title: HA and M2 sequences alter the replication of 2013-16 H1 Live Attenuated Influenza Vaccine Infection in Human Nasal Epithelial Cell cultures

doi: 10.1101/2021.11.11.468304

Figure Lengend Snippet: Epithelial cell innate immune responses after infection with rSlov or rBol in hNEC cultures. Basolateral secretion of interferons, cytokines and chemokines was measured at 48 (A) and 96 (B) HPI during low MOI multistep growth curve experiments on hNECs at 32oC. Log fold change of rSlov relative to rBol is shown for each individual hNEC culture and as an average of all experiments. The red line indicates log fold change of 0 or no difference. Values outside red circle indicate higher expression in rSlov infected wells while values inside the red circle indicate higher expression in rBol infected wells. *p < 0.05. (two-way repeated measures ANOVA with Tukey’s posttest).

Article Snippet: We utilized human nasal epithelial cell (hNEC) cultures collected from non-diseased patients in the Johns Hopkins Medical Institutions as a second in vitro system to assess LAIV replication, as they have previously been shown to be more sensitive to changes in LAIV replication compared to MDCK cells [ , , ].

Techniques: Infection, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Triterpenoid Saponins and Flavonoid Glycosides from the Flower of Camellia flavida and Their Cytotoxic and α-Glycosidase Inhibitory Activities

doi: 10.3390/ijms252010977

Figure Lengend Snippet: Cytotoxicity (IC 50 , μM ± SD, n = 8) of compounds 1 – 10 against cell lines.

Article Snippet: The human cervical cancer cell line Hela, the human lung adenocarcinoma cell line A549, the human breast cancer cell lines MCF-7 and MDA-MB-231, the human hepatoma cell lines BEL-7402 and Hep G2, the human embryonic lung fibroblast cell line MRC-5, and the human nasal epithelial cell line HNEpC were all sourced from the China Center for Type Culture Collection (CCTCC, Wuhan University, Hubei, China).

Techniques:

Journal: Cell reports

Article Title: Regional Differences in Airway Epithelial Cells Reveal Tradeoff between Defense against Oxidative Stress and Defense against Rhinovirus

doi: 10.1016/j.celrep.2018.08.033

Figure Lengend Snippet:

Article Snippet: Primary human nasal epithelial cells , Promocell, Germany , Cat# C-12620.

Techniques: Virus, Recombinant, cDNA Synthesis, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Negative Control, Software