Epithelial-mesenchymal transition, regulated by ß-catenin and Twist, leads to esophageal wall remodeling in pediatric eosinophilic esophagitis.

Eosinophilic Esophagitis (EoE) is an antigen-triggered inflammatory condition of the esophageal lining characterized by eosinophilic infiltration. EoE is associated with significant remodeling, and although this remodeling is reversed by current treatment regimens, symptoms of EoE and associated remodeling reappear upon cessation of therapies. We hypothesized that structural remodeling of cell-cell adhesion is a key factor in the pathogenesis of EoE and that epithelial to mesenchymal transition (EMT) was a viable molecular process to lead to this remodeling. Endoscopically obtained biopsy samples from 18 EoE and 18 control pediatric patients were evaluated by transmission electron microscopy to measure intercellular spaces (IS) between cells. Biopsy samples from all groups were analyzed for cellular levels of cell-cell adhesion proteins: E-cadherin, zonula occludens associated protein-1 (ZO-1), and N-cadherin. We also analyzed for cellular levels and localization two of transcription factors, Twist1 and ß-catenin, that are associated with promoting EMT. The IS was significantly increased in the EoE group compared to the control. We observed a significant decrease in E-cadherin and ZO-1 levels and a concomitant increase in N-cadherin levels in EoE samples compared to control. Further, while there was no significant change in cellular levels of ß-catenin, we observed an altered localization of the protein from the cell membrane in control tissue to a nuclear/perinuclear localization in EoE. We observed higher levels of the transcription factor Twist1 in the EoE group compared to normal which was localized mainly at the nucleus. Our results suggest that the integrity of normally sealed esophageal epithelia is compromised in the EoE patients compared to control subjects, and this is due to alterations in the expression of cell adhesion molecules at the esophageal epithelium. Our data also suggest that EMT, potentially regulated by transcription factors ß-catenin and Twist1, may be responsible for the molecular alteration which leads to the remodeling of esophageal epithelia in EoE.

Sulforaphane (SFA) protects neuronal cells from oxygen & glucose deprivation (OGD).

BACKGROUND: Perinatal brain injury results in neurodevelopmental disabilities (neuroDDs) that include cerebral palsy, autism, attention deficit disorder, epilepsy, learning disabilities and others. Commonly, injury occurs when placental circulation, that is responsible for transporting nutrients and oxygen to the fetus, is compromised. Placental insufficiency (PI) is a reduced supply of blood and oxygen to the fetus and results in a hypoxic-ischemic (HI) environment. A significant HI state in-utero leads to perinatal compromise, characterized by fetal growth restriction and brain injury. Given that over 80% of perinatal brain injuries that result in neuroDDs occur during gestation, prior to birth, preventive approaches are needed to reduce or eliminate the potential for injury and subsequent neuroDDs. Sulforaphane (SFA) derived from cruciferous vegetables such as broccoli sprouts (BrSps) is a phase-II enzyme inducer that acts via cytoplasmic Nrf2 to enhance the production of anti-oxidants in the brain through the glutathione pathway. We have previously shown a profound in vivo neuro-protective effect of BrSps/SFA as a dietary supplement in pregnant rat models of both PI and fetal inflammation. Strong evidence also points to a role for SFA as treatment for various cancers. Paradoxically, then SFA has the ability to enhance cell survival, and with conditions of cancer, enhance cell death. Given our findings of the benefit of SFA/Broccoli Sprouts as a dietary supplement during pregnancy, with improvement to the fetus, it is important to determine the beneficial and toxic dosing range of SFA. We therefore explored, in vitro, the dosing range of SFA for neuronal and glial protection and toxicity in normal and oxygen/glucose deprived (OGD) cell cultures. METHODS: OGD simulates, in vitro, the condition experienced by the fetal brain due to PI. We developed a cell culture model of primary cortical neuronal, astrocyte and combined brain cell co-cultures from newborn rodent brains. The cultures were exposed to an OGD environment for various durations of time to determine the LD50 (duration of OGD required for 50% cell death). Using the LD50 as the time point, we evaluated the efficacy of varying doses of SFA for neuroprotective and neurotoxicity effects. Control cultures were exposed to normal media without OGD, and cytotoxicity of varying doses of SFA was also evaluated. Immunofluorescence (IF) and Western blot analysis of cell specific markers were used for culture characterization, and quantification of LD50. Efficacy and toxicity effect of SFA was assessed by IF/high content microscopy and by AlamarBlue viability assay, respectively. RESULTS: We determined the LD50 to be 2 hours for neurons, 8 hours for astrocytes, and 10 hours for co-cultures. The protective effect of SFA was noticeable at 2.5 µM and 5 µM for neurons, although it was not significant. There was a significant protective effect of SFA at 2.5 µM (p<0.05) for astrocytes and co-cultures. Significant toxicity ranges were also confirmed in OGD cultures as ≥ 100 µM (p<0.05) for astrocytes, ≥ 50 µM (p<0.01) for co-cultures, but not toxic in neurons; and toxic in control cultures as ≥ 100 µM (p<0.01) for neurons, and ≥ 50 µM (p<0.01) for astrocytes and co-cultures. One Way ANOVA and Dunnett's Multiple Comparison Test were used for statistical analysis. CONCLUSIONS: Our results indicate that cell death shows a trend to reduction in neuronal and astrocyte cultures, and is significantly reduced in co-cultures treated with low doses of SFA exposed to OGD. Doses of SFA that were 10 times higher were toxic, not only under conditions of OGD, but in normal control cultures as well. The findings suggest that: 1. SFA shows promise as a preventative agent for fetal ischemic brain injury, and 2. Because the fetus is a rapidly growing organism with profound cell multiplication, dosing parameters must be established to insure safety within efficacious ranges. This study will influence the development of innovative therapies for the prevention of childhood neuroDD.

Osteosarcoma progression is associated with increased nuclear levels and transcriptional activity of activated ß-Catenin.

Osteosarcoma (OS) is an aggressive primary bone malignancy that has peak incidence in children and young adults <25 years of age. Despite current multimodal treatments, no significant change in patient outcome has been observed in two decades. Presently, there is a lack of established, reliable baseline prognostic markers for aggressive OS, other than extent and site of disease involvement. The canonical Wnt/ß-catenin pathway controls multiple cellular processes, and is known to be a critical pathway in OS progression. This pathway regulates cellular levels of ß-catenin, which is a significant player in the oncogenesis and progression of many cancers. We investigated the relationship between ß-catenin, more specifically, the transcriptionally active form of ß-catenin, Activated ß-Catenin (ABC), and OS progression. Using an in vitro model, we observed that cellular/nuclear ABC levels, but not cellular/nuclear ß-catenin levels, increase with the degree of aggressiveness in OS. Our results demonstrate a strong association between nuclear-ABC levels and aggressive OS in vitro. Furthermore, we observed significant correlation between positive nuclear-ABC and patient age and tumor stage. Our results support the potential use of ABC as a predictive marker for risk stratification in OS.

Retrospective review of tertiary and neurosyphilis cases in Alberta, 1973-2017.

OBJECTIVES: To review the notification rate and characteristics of tertiary and neurosyphilis cases in Alberta, Canada in the postantibiotic era. METHODS: A retrospective review of all neurosyphilis and tertiary syphilis cases reported in Alberta from 1973 to March 2017 was undertaken and cases classified into early neurosyphilis, late neurosyphilis and cardiovascular (CV) syphilis. Variables collected included demographics, sexual partners, HIV status, clinical parameters, symptoms and treatment and distributions were compared between early versus late neurosyphilis and asymptomatic versus symptomatic cases (stratified by early versus late stage). Data were analysed using IBM SPSS Statistics V.19.0. RESULTS: 254 cases were identified; 251 were neurosyphilis and 3 were CV. No cases of gummatous syphilis were reported. Early neurosyphilis accounted for 52.4% (n=133) and 46.1% (n=117) were late neurosyphilis cases; one (0.4%) case with unknown duration. Three outbreaks of infectious syphilis were identified during the study period and a concurrent rise in both early and late neurosyphilis was observed during the outbreak periods. The most common manifestation of symptomatic neurosyphilis was ocular involvement which was more likely in early neurosyphilis. Relative to late neurosyphilis cases, early neurosyphilis cases were more likely to be younger, Caucasian, born in Canada, HIV positive and reporting same sex partners. CONCLUSIONS: Our review of tertiary and neurosyphilis cases found that early and late neurosyphilis cases continue to occur in the context of cycling syphilis outbreaks. CV syphilis cases were extremely rare. Ongoing identification of new cases of syphilis and clinical evaluation of cases for complications continues to be important in the context of global resurgence of syphilis.