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1.  Persistence and Toxin Production by Clostridium difficile within Human Intestinal Organoids Result in Disruption of Epithelial Paracellular Barrier Function 
Infection and Immunity  2014;83(1):138-145.
Clostridium difficile is the leading cause of infectious nosocomial diarrhea. The pathogenesis of C. difficile infection (CDI) results from the interactions between the pathogen, intestinal epithelium, host immune system, and gastrointestinal microbiota. Previous studies of the host-pathogen interaction in CDI have utilized either simple cell monolayers or in vivo models. While much has been learned by utilizing these approaches, little is known about the direct interaction of the bacterium with a complex host epithelium. Here, we asked if human intestinal organoids (HIOs), which are derived from pluripotent stem cells and demonstrate small intestinal morphology and physiology, could be used to study the pathogenesis of the obligate anaerobe C. difficile. Vegetative C. difficile, microinjected into the lumen of HIOs, persisted in a viable state for up to 12 h. Upon colonization with C. difficile VPI 10463, the HIO epithelium is markedly disrupted, resulting in the loss of paracellular barrier function. Since similar effects were not observed when HIOs were colonized with the nontoxigenic C. difficile strain F200, we directly tested the role of toxin using TcdA and TcdB purified from VPI 10463. We show that the injection of TcdA replicates the disruption of the epithelial barrier function and structure observed in HIOs colonized with viable C. difficile.
PMCID: PMC4288864  PMID: 25312952
2.  Math5 (Atoh7) gene dosage limits retinal ganglion cell genesis 
Neuroreport  2012;23(10):631-634.
The basic helix–loop–helix factor Math5 (Atoh7) is critical for the determination of retinal ganglion cell (RGC) fate in mice. Recently, genome-wide association studies have identified the ATOH7 locus as a major determinant of variation in the human optic disc area, which is directly correlated with the RGC number. These studies suggest that the level of Math5 expression may determine the ultimate number of RGCs. To test this hypothesis, we systematically compared optic nerve area and RGC axon number in C57BL/6J congenic Math5+/– and +/+ mice at young adult and neonatal ages by transmission electron microscopy. Optic disc area and RGC abundance were not significantly different in adults, but heterozygotes had thinner optic nerves and 25–30% fewer RGCs at birth than wild-type littermates (P < 0.05). Our results suggest that Math5 dosage is important for the genesis, but not the ultimate number, of RGCs. Our findings highlight the importance of ganglion cell culling as a compensatory mechanism for retinal homeostasis, and support a quantitative role for Math5 in RGC specification.
PMCID: PMC3733793  PMID: 22660169
apoptosis; Atoh7; culling; glaucoma; genome-wide association studies; Math5; neurogenesis; optic disc area; optic nerve; retinal ganglion cell
3.  A large-scale assessment of hand hygiene quality and the effectiveness of the “WHO 6-steps” 
BMC Infectious Diseases  2013;13:249.
Hand hygiene compliance is generally assessed by observation of adherence to the “WHO five moments” using numbers of opportunities as the denominator. The quality of the activity is usually not monitored since there is no established methodology for the routine assessment of hand hygiene technique. The aim of this study was to objectively assess hand rub coverage of staff using a novel imaging technology and to look for patterns and trends in missed areas after the use of WHO’s 6 Step technique.
A hand hygiene education and assessment program targeted 5200 clinical staff over 7 days at the National University Hospital, Singapore. Participants in small groups were guided by professional trainers through 5 educational stations, which included technique-training and UV light assessment supported by digital photography of hands. Objective criteria for satisfactory hand hygiene quality were defined a priori. The database of images created during the assessment program was analyzed subsequently. Patterns of poor hand hygiene quality were identified and linked to staff demographic.
Despite the assessment taking place immediately after the training, only 72% of staff achieved satisfactory coverage. Failure to adequately clean the dorsal and palmar aspects of the hand occurred in 24% and 18% of the instances, respectively. Fingertips were missed by 3.5% of subjects. The analysis based on 4642 records showed that nurses performed best (77% pass), and women performed better than men (75% vs. 62%, p<0.001). Further risk indicators have been identified regarding age and occupation.
Ongoing education and training has a vital role in improving hand hygiene compliance and technique of clinical staff. Identification of typical sites of failure can help to develop improved training.
PMCID: PMC3689096  PMID: 23718728
4.  Evidence for STAT4 as a Common Autoimmune Gene: rs7574865 Is Associated with Colonic Crohn's Disease and Early Disease Onset 
PLoS ONE  2010;5(4):e10373.
Recent studies demonstrated an association of STAT4 variants with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), indicating that multiple autoimmune diseases share common susceptibility genes. We therefore investigated the influence of STAT4 variants on the susceptibility and phenotype of inflammatory bowel diseases (IBD) in a large patient and control cohort.
Methodology/Principal Findings
Genomic DNA from 2704 individuals of Caucasian origin including 857 patients with Crohn's disease (CD), 464 patients with ulcerative colitis (UC), and 1383 healthy, unrelated controls was analyzed for seven SNPs in the STAT4 gene (rs11889341, rs7574865, rs7568275, rs8179673, rs10181656, rs7582694, rs10174238). In addition, a detailed genotype-phenotype analysis was performed. Our analysis revealed an association of the STAT4 SNP rs7574865 with overall decreased susceptibility to CD (p = 0.047, OR 0.86 [95% CI 0.74–0.99]). However, compared to CD patients carrying the wild type genotype, the STAT4 SNP rs7574865 was significantly associated with early CD onset (p = 0.021) and colonic CD (p = 0.008; OR = 4.60, 95% CI 1.63–12.96). For two other STAT4 variants, there was a trend towards protection against CD susceptibility (rs7568275, p = 0.058, OR 0.86 [95% CI 0.74–1.00]; rs10174238, p = 0.057, OR 0.86 [95% CI 0.75–1.00]). In contrast, we did not observe any association with UC susceptibility. Evidence for weak gene-gene interaction of STAT4 with the IL23R SNP rs11209026 was lost after Bonferroni correction.
Our results identified the STAT4 SNP rs7574865 as a disease-modifying gene variant in colonic CD. However, in contrast to SLE and RA, the effect of rs7574865 on CD susceptibility is only weak.
PMCID: PMC2861592  PMID: 20454450

Results 1-4 (4)