Chung-Yin Lee

Cell proliferation in the small intestine and colon of intravenously fed rats: effects of urogastrone-epidermal growth factor

Abstract. There is marked intestinal hypoplasia in the intestine of intravenously fed (TPN) rats. Recombinant urogastrone‐epidermal growth factor (URO‐EGF) reversed these changes by significantly increasing the length of the intestinal crypts. Crypt diameter, however, was not affected to the same extent. Few differences in labelling indices were seen between the orally fed and TPN groups, however, this was the consequence of the concomitant changes in crypt population.

Non-steroidal anti-inflammatory drugs inhibit the processes of mucosal cell proliferation associated with duodenal ulcer healing

We demonstrate that, in patients taking non-steroidal anti-inflammatory drugs (NSAIDs), there is inhibition of the proliferation of mucosal cells that normally leads to healing of duodenal ulcers. A microdissection technique was used to quantitate mitosis in duodenal crypts at the ulcer edge, giving a regeneration index of mitotic rate at that site, as compared to nearby mucosa. In patients with duodenal ulcers occurring in the absence of NSAID therapy, there was a brisk regenerative response (median index 2.48, range 1.55-9.81, n = 8), significantly greater than in patients taking NSAIDs (median index 1.10, range 0.73-2.16, n = 10, p = 0.014). Inhibition of the process of epithelial cell division normally involved in duodenal ulcer healing could contribute to the delay in ulcer healing which may explain the higher complication rate for duodenal ulcer during NSAID therapy.

Colonic cell proliferation and growth fraction in young, adult and old rats

SummaryColonic epithelial proliferation was investigated in three groups of rats, aged 3, 60 and 121 weeks. As reported in previous work, the crypts were markedly longer in the young rats, and the number of labelled cells per crypt was significantly greater. There was an upward movement of the marker positions derived from the distribution of labelled cells within the crypt of the young rats. This was a consequence of the increased crypt length, so that the growth fraction, as expressed as a percentage of crypt length, was the same. The proliferative changes between the young rats and the other aged rats were therefore effected by altering the size of the crypts, while maintaining the kinetic organisation. There was no evidence of any proliferative changes or changes in the growth fraction when the colons of the old rats were compared with those of the 60 week old rats.

Clonality analysis of defined cell populations in paraffin-embedded tissue sections by RT-PCR amplification of X-linked G6PD gene.

This paper establishes a method of clonality analysis using the reverse transcription‐polymerase chain reaction (RT‐PCR) to amplify X‐linked G6PD transcripts on defined cell populations microdissected from archival, paraffin‐embedded tissue sections. Four known monoclonal low‐grade B‐cell lymphomas from females who were heterozygous (informative) at the 1131 exonic polymorphic locus of the G6PD gene were used to validate the method. Lymphoma and reactive lesions in each case were separated by microdissection. In order to preserve the intact RNA species in the lesion, sections were digested on the slides before microdissection. A one‐step RT‐PCR was performed with a single pair of primers, one of which contained a mismatched base adjacent to the polymorphic site, to generate a PvuI cutting site. Successful amplification and allele identification by PvuI digestion were achieved from all RNA samples studied. Three of four samples from non‐neoplastic reactive lesions showed two bands with equal intensity, representing transcription of the two alleles of the G6PD gene, while the corresponding tumour samples demonstrated a biased intensity in one allele, indicating monoclonality. To assess the method further, the clonal nature of in situ and invasive breast cancers was examined, along with adjacent normal breast tissue and hyperplastic lesions from three informative females from our archives. Apart from the clusters of normal terminal duct‐lobular units, all lesions were monoclonal. This result is in agreement with data derived from other X‐linked gene studies and loss of heterozygosity (LOH) analyses of pre‐invasive breast disease. The results suggest that the clonality analysis method presented here is simple and reliable, and is therefore potentially applicable in a wide range of pathological conditions. Copyright

Bone Marrow Cells in Murine Colitis: Multi-Signal Analysis Confirms Pericryptal Myofibroblast Engraftment without Epithelial Involvement

Background The contribution of bone marrow-derived cells to epithelial tissues in the inflamed gut remains controversial. Recent reports have suggested that cell fusion between bone marrow-derived cells and the intestinal epithelium takes place in inflammatory conditions. Methods In attempts to confirm this, we have undertaken gender mis-matched bone marrow (BM) transplants from male Swiss Webster (SWR) mice to B and T cell-deficient female Rag2 KO mice which, 4 weeks later, were given 5% dextran sodium sulphate in drinking water to induce acute colitis. A further BM-treated group of animals with a graft versus host-like condition was also studied. We developed a new method to combine up to three brightfield or fluorescent lectin- or immuno-histochemical signals with fluorescent in situ hybridisation for the Y and X chromosomes to enable us unequivocally to identify BM-derived male cells which presented as different cell types in the gastrointestinal tract. Principal Findings In rolled preparations of whole intestines we scanned around 1.5 million crypts at many tissue levels. In no instance did we see a Y chromosome-positive cell in the epithelial compartment, which was not also CD45-positive. We saw no evidence of cell fusion, based on combined X and Y chromosome analysis. Levels of CD45-positive stromal and lymphoid cells and pericryptal myfibroblasts (positive for α-smooth muscle actin) increased with time up to a plateau, which resembled the level seen in untreated control grafted animals. We saw very few Y chromosome-positive endothelial cells in intestinal stromal vessels. Conclusions We conclude that whole BM transplantation does not result in intestinal epithelial engraftment in this model. Our new methods can usefully assist in multi-signal analyses of cell phenotypes following BM transplant and in models of chimaerism and regenerative medicine.

Evaluation of a proposed technique to assess unscheduled DNA synthesis and genotoxicity.

Results from a recent, new assay suggest that omeprazole, a potent inhibitor of gastric acid secretion, is genotoxic. The principle of this assay is that the non-proliferating zone of surface gastric epithelial cells can be selectively removed by controlled digestion so that any incorporation of tritiated thymidine into these cells represents unscheduled DNA synthesis. Parietal cells (which are located below the uppermost proliferating cells) and proliferating cells in semiconservative, regular DNA synthesis could always be shown in the digested fraction, and as regular DNA synthesis takes up a thousand fold more thymidine than unscheduled DNA synthesis, any signal from unscheduled synthesis would therefore be swamped. The digestion process was also uneven, as histological analysis showed denuded patches of mucosa, and gland like structures were seen in the digest. Quantification of the number of silver grains over the nuclei showed no increase in low level labelling after omeprazole administration, indicating that there was no unscheduled DNA synthesis. The labelling index of undigested gastric tissue from omeprazole treated rats was not significantly different from that of the control group, despite an increase in the plasma gastrin value.

The ailing gut: a therapeutic role for bone marrow cells?

It is obvious that the BM does more than simply supply the GIT with cells of the innate and adaptive immune system. A growing number of studies suggest that BMCs can differentiate into ISEMFs (Lee et al., PLOS ONE 2011;6:e26082) and in the setting of inflammation can be contributors to all lineages of the neovasculature. The role of BMCs in epithelial turnover is more problematic; their contribution after transient mucosal injury seems negligible, but a number of studies in both rodents and man suggest that small numbers of BMCs can be incorporated into the epithelial compartment with more chronic injury (e.g., GvHD in man and chemically induced colitis in rodents); commonly cell fusion seems to be responsible for this. Significantly, this engraftment does not seem to occur in the stem-cell compartment, with the notable single report of the chronically infected murine gastric mucosa, where the BM origin of the stem cells can be the only rational explanation for the complete colonization of the mucosa by BMDCs. In the clinical setting, a role for MSCs in ameliorating colitis seems promising, though the mechanisms by which this is achieved remain somewhat unclear, though both immunomodulatory and regenerative effects of BMCs are likely to be important.

687 Topical Alginate Protection of Human Esophageal Epithelium for Gastro-Esophageal Reflux Disease. A Study Using Human Cell Culture and Biopsy Models

G A A b st ra ct s extra-hepatic malignancies in patients with IBD and co-existent PSC using the Explorys database. In total the Explorys database contains over 40 million unique patient records across the United States from over 400 hospitals. Methods: Explorys database from 19992014 was queried for adults with medical records of at least 4 years with a diagnosis of IBD (ICD-9: 556.xx, 555.xx). Patients were divided into 2 groups, those with ulcerative colitis (UC) and those with crohns disease (CD). Each group was further divided into patients that had PSC and those that did not. ICD-9 codes were used to identify extent and location of UC and CD, respectively. Medications were identified by generic and brand name. Analysis was completed using Chi-squared test and Odds ratio. A p-value < 0.05 was considered significant. Results: 2,170 of a total of 65,850 IBD patients had PSC. PSC was more common in UC than CD (p= 0.0001, Table 1). Caucasian males with UC were more likely to develop PSC (Table 1). While the majority of individuals with PSC were less than 65 years of age; interestingly, the data demonstrated that the incidence of PSC was highest in the age group 50-59 years (23.5%). In UC, the incidence of PSC increased with increased colonic involvement; however, disease distribution was not a factor in the CD. The current data also suggests that there is an increased risk of pancreatic cancer for both UC with PSC (p-value: 0.0001; OR: 6.53 95% (3.99-10.70)) and CD with PSC (p-value: 0.0001; OR 8.42 95% (5.14-13.80)). Conclusion: This study is the largest to date to assess the relationship of PSC and IBD. Confirming prior studies, the risk of PSC in greatest amongst Caucasian males with UC and its incidence increases with the degree of colonic involvement. The pooled data suggest a shift in the paradigm of PSC to an older patient population than previously thought, which may be reflective of the advent of potent anti-inflammatory medications limiting the degree of chronic inflammation. The relationship between CRC and PSC is well established; however a previously unknown phenomena, the increased risk of pancreatic cancer requires further exploration.