Establishment of a 3D organoid culture model for the investigation of adult slow-cycling putative intestinal stem cells.

The study of intestinal stem cells is a prerequisite for the development of therapies aimed at regenerating the gut. To enable investigation of adult slow-cycling H2B-GFP-retaining putative small intestinal (SI) stem cells in vitro, we have developed a three-dimensional (3D) SI organoid culture model based on the Tet-Op histone 2 B (H2B)-green fluorescent protein (GFP) fusion protein (Tet-Op-H2B-GFP) transgenic mouse. SI crypts were isolated from 6- to 12-week-old Tet-Op-H2B-GFP transgenic mice and cultured with appropriate growth factors and an animal-derived matrix (Matrigel). For in vitro transgene expression, doxycycline was added to the culture medium for 24 h. By pulse-chase experiments, H2B-GFP expression and retention were assessed through direct GFP fluorescence observations, both by confocal and fluorescence microscopy and by immunohistochemistry. The percentages of H2B-GFP-retaining putative SI stem cells and H2B-GFP-retaining Paneth cells persisting in organoids were determined by scoring relevant GFP-positive cells. Our results indicate that 24 h exposure to doxycycline (pulse) induced ubiquitous expression of H2B-GFP in the SI organoids. During subsequent culture, in the absence of doxycycline (chase), there was a gradual loss (due to cell division) of H2B-GFP. At 6-day chase, slow-cycling H2B-GFP-retaining putative SI stem cells and H2B-GFP-retaining Paneth cells were detected in the SI organoids. The developed culture model allows detection of slow-cycling H2B-GFP-retaining putative SI stem cells and will enable the study of self-renewal and regeneration for further characterization of these cells.

TITF1 Screening in Human Congenital Diaphragmatic Hernia (CDH).

TITF1 (Thyroid Transcription Factor-1) is a homeodomain-containing transcription factor. Previous studies showed that Titf1 null mice are characterized by failure of tracheo-oesophageal separation and impaired lung morphogenesis resulting in Pulmonary Hypoplasia (PH). In this study, we aim to evaluate the role of TITF1 in the pathogenesis of congenital diaphragmatic hernia (CDH) in humans. We investigated TITF1 expression in human trachea and lungs and performed direct mutation analysis in a CDH population. We studied 13 human fetuses at 14 to 24 weeks of gestation. Five µm sections were fixed in paraformaldehyde and incubated with anti-TITF1 primary antibody. Positive staining was visualized by biotinylated secondary antibody. We also performed TITF1 screening on genomic DNA extracted from peripheral blood of 16 patients affected by CDH and different degrees of PH, searching for mutations, insertions, and/or deletions, by sequencing the exonic regions of the gene. Histochemical studies showed positive brown staining of fetal follicular thyroid epithelium, normal fetal trachea, and normal fetal lung bronchial epithelium. Fetal esophageal wall was immunohistochemically negative. Molecular genetic analysis showed complete identity between the sequences obtained and the Wild Type (WT) form of the gene in all cases. No mutation, insertion and/or deletion was detected. Although TITF1 is expressed in the human fetal lung and has been considered to have a role in the pathogenesis of PH in CDH, the results of our study do not support the hypothesis that TITF1 mutations play a key role in the etiopathogenesis of CDH.

The Biochemical and Pharmacological Properties of Ozone: The Smell of Protection in Acute and Chronic Diseases.

Ozone therapy has been widely used in everyday clinical practice over the last few years, leading to significant clinical results in the treatment of herniated discs and pain management. Nevertheless, further studies have demonstrated its potential efficacy and safety under other clinical and experimental conditions. However, some of these studies showed controversial results regarding the safety and efficacy of ozone therapy, thus mining its potential use in an everyday clinical practice. To this regard, it should be considered that extensive literature review reported the use of ozone in a significant different dose range and with different delivery systems. The aim of the present review is to describe the various pharmacological effects of ozone in different organs and clinical conditions and to provide possible biochemical and molecular insights for ozone biological properties, thus providing a possible explanation for various controversial clinical outcomes described in the scientific literature.