17 research outputs found
Intestinal organoids model human responses to infection by commensal and Shiga toxin producing Escherichia coli
Infection with Shiga toxin (Stx) producing Escherichia coli O157:H7 can cause the potentially fatal complication hemolytic uremic syndrome, and currently only supportive therapy is available. Lack of suitable animal models has hindered study of this disease. Induced human intestinal organoids (iHIOs), generated by in vitro differentiation of pluripotent stem cells, represent differentiated human intestinal tissue. We show that iHIOs with addition of human neutrophils can model E. coli intestinal infection and innate cellular responses. Commensal and O157:H7 introduced into the iHIO lumen replicated rapidly achieving high numbers. Commensal E. coli did not cause damage, and were completely contained within the lumen, suggesting defenses, such as mucus production, can constrain non-pathogenic strains. Some O157:H7 initially co-localized with cellular actin. Loss of actin and epithelial integrity was observed after 4 hours. O157:H7 grew as filaments, consistent with activation of the bacterial SOS stress response. SOS is induced by reactive oxygen species (ROS), and O157:H7 infection increased ROS production. Transcriptional profiling (RNAseq) demonstrated that both commensal and O157:H7 upregulated genes associated with gastrointestinal maturation, while infection with O157:H7 upregulated inflammatory responses, including interleukin 8 (IL-8). IL-8 is associated with neutrophil recruitment, and infection with O157:H7 resulted in recruitment of human neutrophils into the iHIO tissue
Ocular manifestations of isolated corneal bee sting injury, management strategies, and clinical outcomes
Purpose: The purpose of this study is to describe the presenting features, management strategies, and clinical outcome following bee sting injury to the cornea. Methods: Retrospective case series involving 11 eyes of 11 patients with corneal bee sting injuries who presented over a period of 2 years. Nine of these 11 eyes had the presence of intact bee stinger in the cornea, which was removed immediately under an operating microscope and sent for microbiological and histopathological evaluation. The patients were managed as per the individual treatment protocol of the respective treating physicians. Results: Six eyes (54.5%) had a good clinical outcome (best-corrected visual acuity [BCVA] >20/40) with medical therapy alone with no need for surgical intervention. Five eyes (45.5%) had a poor clinical outcome (BCVA <20/40) with medical therapy and required surgery; of which three required a combined penetrating keratoplasty with cataract surgery, while one required isolated cataract surgery and one underwent penetrating keratoplasty. Glaucoma was present in 3/5 eyes undergoing surgery, one of which required a trabeculectomy. Five of the six eyes who had a good clinical outcome with medical therapy alone had been treated with concomitant oral steroids, along with topical antibiotic-steroid combination therapy. Conclusion: Oral corticosteroid supplementation to the topical steroid antibiotic treatment in patients with corneal bee sting injury where corneal involvement and anterior reaction is severe at presentation or inflammation not ameliorating with topical steroids alone prevents serious vision-threatening complications such as corneal decompensation, cataract, and glaucoma
Infection with pathogenic O157:H7 upregulates ROS.
<p>(A) iHIOs were injected with saline and approximately 10<sup>3</sup> commensal <i>E</i>. <i>coli</i> or pathogenic O157:H7. Mean CFU +/- standard error were determined at the indicated times from three different iHIOs for each strain. (B) ROS production was assessed 4 hours post-injection by fluorescence microscopy 30 minutes after addition of the oxidative stress detection reagent (Enzo). Bar indicates 100 μm. (C) Fluorescence intensity, pixels (mean and standard error of the mean) is plotted for three independent iHIOs. Statistical significance was assessed by using one-way ANOVA analysis of variance using Tukey’s multiple comparison posttest using GraphPad Prism 5.0. ** Very significant (<i>P</i> value 0.001 to 0.01), *** extremely significant (<i>P</i> value <i>p</i> < 0.001). Similar results were seen in an independent replicate experiment.</p
RNAseq—Venn diagram summary of upregulated genes.
<p>Venn diagram for the RNAseq analysis on iHIOs were prepared using an online tool from Bioinformatics & Evolutionary Genomics.</p
Sensitivity to LPS.
<p>Various doses of purified LPS were (A) microinjected into the iHIO lumen; or (B) introduced into the medium. Epithelial barrier function was monitored by quantifying retention of the fluorescent dye, FITC (C). Statistical significance of loss of fluorescence at 120 hours compared to control was assessed by GraphPad Prism 5 using one way ANOVA, Dunnett’s multiple comparison posttest. ***, extremely significant, P < 0.001, compared to saline control at 120 hours.</p
Influence of PMNs on bacteria and iHIOs.
<p><b>(A-D)</b> iHIOs microinjected with saline or 10<sup>3</sup> commensal bacteria or O157:H7 in the presence of the fluorescent dye FITC were incubated for approximately 4 hours to allow cytokine expression, and 5x10<sup>5</sup> fluorescently labeled human PMNs were added. Incubations were performed without antibiotics in the tissue culture medium to assess total microbial growth, or with antibiotics in the tissue culture medium to assess maintenance of the luminal barrier. Each experiment was performed in triplicate. (A) Bacterial proliferation in the absence antibiotics. In two independent experiments, CFUs were determined before and after addition of PMNs (indicated by arrow). Insert, bacteria incubated with PMNs for 4.5 hours. Full graph, bacteria incubated with PMNs for 14.5 hours. Bacterial numbers within the iHIOs and media were assessed.(B) Bacterial proliferation in the presence of antibiotics. CFUs were determined at the indicated times before and after addition of PMNs (indicated by arrow). Failure to recover O157:H7 at 23 hours is due to antibiotics in the lumen following loss of the epithelial barrier. (C) Epithelial barrier function, assessed by retention of FITC fluorescence, for experiment Part A, full graph. Significance was calculated by two-way ANOVA: *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. (D-J), <b>PMN migration into iHIOs</b>. <b>(D-F)</b>, merged bright field images of iHIOs, green FITC fluorescence (lumen), and violet PMNs, for iHIOs (from <b>Fig 7A, insert</b>) injected with saline, commensal, or pathogenic O157:H7, respectively, 8.5 hours after injection (4.5 hours after addition of PMNs). (G-I), violet fluorescent images, corresponding to D-F. (J), quantification of PMNs (violet fluorescence) within the iHIOs. Statistical analysis determined by 1-way AVOVA, Tukey’s multiple comparison posttest; ***, extremely significant, P < 0.001, comparing O157:H7 to saline and commensal (8.5 hours); **, very significant, P 0.001 to 0.01, comparing O157:H7 to saline and commensal (22.5 hours) and *, significant, P < 0.05 comparing O157:H7 (8.5 hours) to O157:H7 (22.5 hours). (K-M), merged bright field images of iHIOs (from <b>Fig 7A, full graph</b>) and sytox-orange dead-cell stain, 22.5 hours after injection (18.5 hours after addition of PMNs). (N), quantification of dead cells (red fluorescence due to sytox-orange staining). Statistical analysis determined by 1-way AVOVA, Tukey’s multiple comparison posttest: ***, extremely significant, P < 0.001: **, very significant, P 0.001 to 0.01. Red bar on micrographs indicates 100 μM.</p
Expression relative to PBS controls of select intestinal and lineage specific genes.
<p>Expression relative to PBS controls of select intestinal and lineage specific genes.</p
Bacterial growth in iHIOs.
<p><b>A, Bacterial growth</b>. iHIOs were injected with 10<sup>3</sup> commensal <i>E</i>. <i>coli</i> (SGUC183, squares), or pathogenic O157:H7 (PT29S, triangles). Open symbols, iHIOs were incubated with penicillin in the tissue culture medium to prevent bacterial growth outside of the lumen; mean CFU +/- standard deviation were determined at the indicated times from three different iHIOs for each strain. Closed symbols, in a separate experiment iHIOs were injected with 10<sup>3</sup> commensal <i>E</i>. <i>coli</i> (SGUC183, squares), or pathogenic O157:H7 (PT29S, triangles) as above, but at 18 hours, the medium was replaced with medium lacking penicillin, and bacterial counts were assessed at 27 hours post inoculation. <b>B-C</b>, Commensal <i>E</i>. <i>coli</i> (B) replicates in the lumen without damaging the iHIO, while O157:H7 (C) damages the actin layer. Cryosections of iHIOs 18 hours after injection were stained for DNA (blue), bacteria (green, anti-<i>E</i>. <i>coli</i> for commensal, anti-O157 for O157:H7), and F-actin (red). Bar indicates 20 μm. <b>D-E</b>. Cryosection 1 hour after infection with O157:H7, stained for nuclear and bacterial DNA (DAPI, blue), E-cadherin (green), and F-actin (red). White arrowheads represent bacterial nucleoids co-localized with actin. (D), Bar indicates 10 μm. (E), Magnified image of D, bar indicates 2 μm. <b>F-G</b>. Cryosections 4 hours after infection with O157:H7. White arrowheads represent bacterial co-localization with actin. (F), stained for nuclear and bacterial DNA (DAPI, blue), E-cadherin (green), and F-actin (red), bar indicates 10 μm. (G), stained for nuclear and bacterial DNA (DAPI, blue), F-actin (red), and anti-O157 (green), bar indicates 5 μm. <b>H-I</b>. Cryosections 18 hours after infection with O157:H7. Yellow arrows indicate filamentous <i>E</i>. <i>coli</i>. (H), stained for nuclear and bacterial DNA (DAPI, blue), E-cadherin (green), and F-actin (red), bar indicates 10 μm. (I), stained for nuclear and bacterial DNA (DAPI, blue), F-actin (red), anti-O157 (green), bar indicates 10 μm. Representative images of experiments performed at least four times are shown.</p
Components of iHIOs.
<p>(A) Diagram of cross section of iHIO embedded in Matrigel (extracellular matrix). (B) Histologic staining of iHIO 24 hours after microinjection with PBS; merged image stained for DNA (DAPI, blue) and antibodies to the epithelial marker E-cadherin (green), and F-actin (red). Confocal images were captured using Zeiss LSM710 Live Duo confocal microscope and merged using ImageJ software. Bar indicates 20 μm. (C) Picture of an iHIO in the process of microinjection.</p
Western blot of Stx2a.
<p>Lysed iHIOs infected with <i>E</i>. <i>coli</i> O157:H7 were harvested at the indicated time points and mechanically lysed. The cellular material was collected by centrifugation. Protein in the pellet was solubilized, and immunoblotted using polyclonal antibody to Stx2a. As control, purified Stx2a was loaded at 50 and 25 ng. The Stx2a A-subunit (33 kDa), indicated by the arrow is seen at 18 hours post-infection.</p