Isolation of esophageal stem cells with potential for therapy

Purpose

Long-gap esophageal atresia represents a significant challenge for pediatric surgeons and current surgical approaches are associated with significant morbidity. A tissue-engineered esophagus, comprising cells seeded onto a scaffold, represents a therapeutic alternative. In this study, we aimed to determine the optimal techniques for isolation and culture of mouse esophageal epithelial cells and to isolate CD34-positive esophageal epithelial stem cells from cadaveric mouse specimens.

Methods

Primary epithelial cells were isolated from mouse esophagi by enzymatic dissociation from the mucosal layer (Dispase, Trypsin/EDTA) using three different protocols. In protocol A, isolated mucosa was minced and incubated with trypsin once. In protocol B, intact mucosal sheets underwent two trypsin incubations yielding a single-cell suspension. In protocol C, intact mucosa explants were plated epithelial side down. Epithelial cells were cultured on collagen-coated wells.

Results

Initial findings showed that Protocol B gave the best results in terms of yield, viability, and least contamination with different cell types and microbes. Esophageal epithelial cells isolated using Protocol B were stained for CD34 and sorted using fluorescence-activated cell sorting (FACS). Of the total cells sorted, 8.3 % (2–11.3) [%median (range)] were CD34 positive.

Conclusions

Our results demonstrate that mouse esophageal epithelial cells can be successfully isolated from fresh mouse esophagi using two consecutive trypsin incubations of intact mucosal sheets. Furthermore, the cells obtained using this method were successfully stained for CD34, a putative esophageal epithelial stem cell marker. Further research into the factors necessary for the successful proliferation of CD34 positive stem cell lines is needed to progress toward clinical application.     

Esophageal tissue engineering: A new approach for esophageal replacement

A number of congenital and acquired disorders require esophageal tissue replacement. Various surgical techniques, such as gastric and colonic interposition, are standards of treatment, but frequently complicated by stenosis and other problems. Regenerative medicine approaches facilitate the use of biological constructs to replace or regenerate normal tissue function. We review the literature of esophageal tissue engineering, discuss its implications, compare the methodologies that have been employed and suggest possible directions for the future. Medline, Embase, the Cochrane Library, National Research Register and ClinicalTrials.gov databases were searched with the following search terms: stem cell and esophagus, esophageal replacement, esophageal tissue engineering, esophageal substitution. Reference lists of papers identified were also examined and experts in this field contacted for further information. All full-text articles in English of all potentially relevant abstracts were reviewed. Tissue engineering has involved acellular scaffolds that were either transplanted with the aim of being repopulated by host cells or seeded prior to transplantation. When acellular scaffolds were used to replace patch and short tubular defects they allowed epithelial and partial muscular migration whereas when employed for long tubular defects the results were poor leading to an increased rate of stenosis and mortality. Stenting has been shown as an effective means to reduce stenotic changes and promote cell migration, whilst omental wrapping to induce vascularization of the construct has an uncertain benefit. Decellularized matrices have been recently suggested as the optimal choice for scaffolds, but smart polymers that will incorporate signalling to promote cell-scaffold interaction may provide a more reproducible and available solution. Results in animal models that have used seeded scaffolds strongly sug- gest that seeding of both muscle and epithelial cells on scaffolds prior to implantation is a prerequisite for complete esophageal replacement. Novel approaches need to be designed to allow for peristalsis and vascularization in the engineered esophagus. Although esophageal tissue engineering potentially offers a real alternative to conventional treatments for severe esophageal disease, important barriers remain that need to be addressed.     

Incidence of DIS/DSM-IV social phobia in adults.

OBJECTIVE: The aim of the study was to estimate the incidence of social phobia in the general population. METHOD: The Baltimore cohort of 3481 subjects, sampled during the 1981 Epidemiologic Catchment Area study, was traced. A total of 1920 subjects were re-interviewed from 1993 to 1996 using the Diagnostic Interview Schedule (DIS). A subsample of 349 subjects was interviewed by psychiatrists using the Schedules for Clinical Assessment in Neuropsychiatry. RESULTS: The estimated incidence of DIS/DSM-IV social phobia is 4-5/1000/year. New cases were found in all age groups, with the highest rates in subjects with baseline depressive and panic disorders. Psychiatric evaluations showed broad diagnostic concordance with DIS diagnoses in incident cases. However, validity indices were highly dependent on diagnostic thresholds. None of the psychiatrist-ascertained social phobics had received treatment for the disorder, although the majority were considered likely to benefit from treatment. CONCLUSION: New cases of social phobia occur in adults of all age groups, and are often secondary to other psychiatric conditions.     

Safety and efficacy of human platelet extract in skin recovery after fractional CO2 laser resurfacing of the face: A randomized,controlled, evaluator-blinded pilot study

Background

Fractional carbon dioxide (CO₂) laser resurfacing is used successfully for facial rejuvenation. Post procedure skincare is a variable that influences downtime caused by pain/tenderness, erythema, crusting, and bruising.

Aims

The primary objective of this pilot study was to demonstrate the benefits of human platelet extract (HPE) (plated)™ CALM Serum, a new topical cosmetic product, following fractionated CO₂ ablative laser resurfacing treatment to the entire face versus standard of care.

Methods

In a single-center, randomized, evaluator-blinded pilot study, a total of 18 subjects were randomized into two groups, CO₂ facial resurfacing followed by post-procedural standard of care (Stratacel silicone gel) or CO₂ facial resurfacing with the addition of HPE renewosomes in the CALM Serum.

Results

CALM Serum demonstrated statistically significant less crusting at Day 10 compared to the control group (p = 0.0193) with less downtime in the first 14 days (p = 0.03). Subjects treated with CALM Serum had statistically significant brighter appearing skin at 14 days (p = 0.007) and more youthful looking skin on Days 14 and 30 (p = 0.003 and 0.04, respectively).

Conclusions

This study demonstrates that Renewosome™ technology provides statistically significant post-laser clinical recovery over silicone gel for reducing crusting, and downtime. Subjects reported less diary days of symptoms of pain/tenderness, redness, crusting/flaking, bruising, and itching in the first 14 days compared to the control group. CALM also demonstrated statistically significant improvements in brighter and more youthful appearing skin. CALM is safe and well tolerated.     

THE AGGLUTINATION OF PLASMODIUM KNOWLESI BY IMMUNE SERUM

A specific agglutination of Plasmodium knowlesi detectable both by macroscopic and by microscopic methods is described. Agglutinins for Plasmodium knowlesi appear in the sera of monkeys between 15 and 45 days after the onset of the infection and become progressively stronger as the malarial infection gradually subsides. Agglutinins persist in the sera of chronically infected animals for a year or longer. The sera of animals which have been repeatedly superinfected agglutinate parasites at dilutions as high as 1:1,000. Sera from normal monkeys, from monkeys acutely ill with malaria, and from monkeys chronically infected with a different species of malarial parasite (Plasmodium inui) do not agglutinate Plasmodium knowlesi. Immune serum agglutinates mature intracellular or extracellular parasites but does not agglutinate unparasitized cells or cells containing immature parasites. The relation of these observations to the mechanism of active and passive immunity in monkey malaria is discussed.