Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Activation of the NLRP3 Inflammasome Pathway by Uropathogenic Escherichia coli Is Virulence Factor-Dependent and Influences Colonization of Bladder Epithelial Cells

doi: 10.3389/fcimb.2018.00081

Figure Lengend Snippet: The importance of different virulence factors for IL-1β release and caspase-1 activity. The bladder epithelial cell line 5637 (A–D) and a spontaneously transformed bladder epithelial cell line HBLAK (E) were infected with CFT073, CFT073Δpap, CFT073ΔfimH, CFT073ΔhlyA, CFT073ΔhlyA/pGNH404 and CFT073 fim L-ON at MOI 10 for 3 (A,C) and 6 h (B,D,E) . IL-1β release (A,B,E) and caspase-1 activity (C,D) were measured. Caspase-1 results are presented as fold increase of mean fluorescence units (MFU) compared to unstimulated control cells. Hemolysin activity on blood agar was evaluated for CFT073, CFT073 fim L-ON, and CFT073ΔhlyA after overnight incubation (F) . Data are presented as mean ± SEM ( n = 3 independent experiments). Asterisks denote statistical significance compared to respective unstimulated control (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: The human bladder epithelial cell line HBLAK (CELLnTEC Advanced Cell Systems AG, Bern, Switzerland) has been isolated from a healthy bladder and been spontaneously transformed providing the convenience of long-term cell growth without senescence.

Techniques: Activity Assay, Transformation Assay, Infection, Fluorescence, Incubation

Journal: BMC Physiology

Article Title: Mandatory role of proteinase-activated receptor 1 in experimental bladder inflammation

doi: 10.1186/1472-6793-7-4

Figure Lengend Snippet: A-E . PAR immunohistochemistry in J82 human urothelial carcinoma cell line. Representative photomicrograph obtained in J82 cell line stained with PAR specific antibodies. J82 cells were fixed and incubated with primary polyclonal (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies: A = PAR-1 (1:20), B = PAR-2 (1:100), C = PAR-3 (1:5), and D = PAR-4 (1:50). Slides were washed and incubated with biotinylated secondary antibodies (Vector Labs), goat anti-rabbit (polyclonal antibodies). Orange dotted circles highlight some cells considered positives for the particular receptor. Original magnification was ×200. Figure 1E represents the average and SEM of number of PAR-positive cells as percent of the total cells per field.

Article Snippet: Human bladder carcinoma cell line J82 (HTB-1) were obtained from the American Tissue Culture Collection.

Techniques: Immunohistochemistry, Staining, Incubation, Plasmid Preparation

Journal: BMC Physiology

Article Title: Mandatory role of proteinase-activated receptor 1 in experimental bladder inflammation

doi: 10.1186/1472-6793-7-4

Figure Lengend Snippet: A-B . Polymerase Chain Reaction for detection of PARs message in J82 human urothelial carcinoma cell line. Figure 2A is a photomicrograph of the gel and Figure 2B represents the area under the curve as quantified using Image J software [74]. Primers used in this experiment are described in additional file (Table 1).

Article Snippet: Human bladder carcinoma cell line J82 (HTB-1) were obtained from the American Tissue Culture Collection.

Techniques: Polymerase Chain Reaction, Software

Journal: eLife

Article Title: Dynamic persistence of UPEC intracellular bacterial communities in a human bladder-chip model of urinary tract infection

doi: 10.7554/elife.66481

Figure Lengend Snippet: Figure 1. Human Bladder-chip model of UTI recapitulates the physiology of bladder filling and voiding. (A) Schematic of the human bladder-chip with co-culture of the 5637 human bladder epithelial cell line (epithelium, top) and primary human bladder microvascular endothelial cells (endothelial, bottom) on either side of the stretchable and porous membrane. Pooled human urine diluted in PBS and endothelial cell medium were perfused in the apical and vascular channels respectively to mimic bladder physiology. A negative pressure in the ‘vacuum’ channels (magenta) on either side of the main channel was applied to stretch the porous membrane to mimic stretching of the bladder. (B, C) Immunofluorescence staining of confluent epithelial and endothelial cell monolayers (anti-EpCAM (magenta) and anti-CK7 (yellow) for the epithelial cells and anti-PECAM-1 (green) for the endothelial cells) in an uninfected control chip. Some endothelial cells also stained positive for CK7. Cell nuclei were labeled with DAPI (azure). (D) Schematic of the reconstitution of the bladder filling and voiding cycle via stretching of the membrane with a duty cycle of 6 hr. The cycle consisted of a linear increase in strain through stretching of the membrane (filling bladder, 0–2 hr), maintenance of the membrane under stretch (filled bladder, 2–4 hr), a quick relaxation of applied strain over 2 min (voiding bladder, 4:02 hr) and maintenance without applied strain (voided bladder, 4:02 hr to 6 hr). (E) An overview of the timeline of the experimental protocol including infection, addition of neutrophils via the vascular channel, and two cycles of antibiotic treatment interspersed by two bacterial growth cycles. The consecutive bladder duty cycles are indicated. The online version of this article includes the following figure supplement(s) for figure 1:

Article Snippet: 5637 human bladder epithelial carcinoma cell line (procured from ATCC, HTB-9TM).

Techniques: Co-Culture Assay, Membrane, Immunofluorescence, Staining, Control, Labeling, Infection

Journal: PLoS pathogens

Article Title: Point mutations in FimH adhesin of Crohn's disease-associated adherent-invasive Escherichia coli enhance intestinal inflammatory response.

doi: 10.1371/journal.ppat.1003141

Figure Lengend Snippet: Figure 2. Adhesion ability of AIEC and non-AIEC strains with regard to fimH clade and CEACAM6 expression. (A) Cell-associated bacteria were quantified using non differentiated T84 cells after a 3 H infection period, and results were analyzed with regard to fimH clade. (B) Cell- associated AIEC bacteria belonging to the fimH S70/N78 clade were quantified using undifferentiated and differentiated T84 cells after a 3 H infection period. (C) Western blot analysis of whole protein extracts from non-differentiated and differentiated T84 cells using anti-CEACAM6 and anti-GAPDH antibodies. (D) Cell-associated bacteria (AIEC and non-AIEC bacteria belonging to the fimH S70/N78 clade) were quantified using differentiated T84 cells after a 3 H infection period. (E) Yeast agglutination titer of AIEC and non-AIEC strains belonging to the fimH S70/N78 clade. (F) Transmission electron micrograph of negatively stained AIEC and non-AIEC bacteria belonging to the fimH S70/N78 clade, magnification 625 000. doi:10.1371/journal.ppat.1003141.g002

Article Snippet: Adhesion assay of human intestinal and bladder epithelial cells T84 cells (derived from human colorectal carcinoma) and T24 cells (human bladder epithelial cells) were purchased from ATCC and maintained in an atmosphere containing 5% CO2 at 37uC in appropriate medium.

Techniques: Expressing, Bacteria, Infection, Western Blot, Agglutination, Transmission Assay, Staining

Journal: PLoS pathogens

Article Title: Point mutations in FimH adhesin of Crohn's disease-associated adherent-invasive Escherichia coli enhance intestinal inflammatory response.

doi: 10.1371/journal.ppat.1003141

Figure Lengend Snippet: Figure 4. Impact of FimH amino acid substitutions on AIEC adhesion ability. Cell-associated bacteria were quantified using undifferentiated (A) or differentiated (B) T84 cells after a 3 H infection period. WT represents the original AIEC LF82 reference strain and DfimH represents the LF82- DfimH isogenic mutant. The other constructs were made in the LF82-DfimH isogenic mutant, in which various fimH variants were reintroduced at the fimH locus. Each value is the mean 6 SEM of at least four separate experiments (* P,0.05; ** P,0.001; *** P,0.001). (C) Relative ability to bind Man1 (1 M/3 M binding ratio) with regard to undifferentiated T84 index adhesion of the various fimH chromosomal mutants constructed. (D) Bacterial binding to 1M-BSA under various shear stresses (pN/m cm2) was evaluated by measuring bacterial accumulation over 3 min. doi:10.1371/journal.ppat.1003141.g004

Article Snippet: Adhesion assay of human intestinal and bladder epithelial cells T84 cells (derived from human colorectal carcinoma) and T24 cells (human bladder epithelial cells) were purchased from ATCC and maintained in an atmosphere containing 5% CO2 at 37uC in appropriate medium.

Techniques: Bacteria, Infection, Mutagenesis, Construct, Binding Assay, Shear