CCL25/CCR9 interactions are not essential for colitis development but are required for innate immune cell protection from chronic experimental murine colitis.

BACKGROUND: The chemokine CCL25, and its receptor CCR9, constitute a unique chemokine/receptor pair, which regulates trafficking of T lymphocytes to the small intestine under physiological conditions and is an attractive target for small bowel Crohn's disease drug development. We have previously shown that CCL25/CCR9 interactions regulate the recovery from acute dextran sulfate sodium-induced colonic inflammation. In this study, we explored whether these interactions also regulate chronic colitis development in 2 independent murine models of experimental colitis. METHODS: Histological flow cytometry and qPCR analyses were performed to evaluate the role of CL25 and CCR9 in chronic colonic inflammation induced by serial exposures to dextran sulfate sodium salts or by adoptive transfer of CD45RB(hi) CD4(+) T cell into lymphopenic mice devoid of CCL25/CCR9 interactions. RESULTS: Chronic dextran sulfate sodium exposure results in exacerbated colitis in mice deficient for either CCR9 or CCL25 when compared with wild-type control mice. Although CCR9-deficient T cells traffic to the colon and induce severe colitis similar to wild-type T cells in the CD45RB transfer model, naive wild-type T cells induce more severe disease in recipient animals devoid of CCL25 expression. CONCLUSIONS: CCL25/CCR9 interactions are required for modulating protection against large intestinal inflammation in 2 models of chronic colitis. These data may have implications for the potential effects of disrupting CCL25/CCR9 interactions in humans in the setting of intestinal disorders including inflammatory bowel disease.

MUC2 is a highly specific marker of goblet cell metaplasia in the distal esophagus and gastroesophageal junction.

Currently, the American College of Gastroenterology requires identification of goblet cells in mucosal biopsies from the esophagus to diagnose Barrett esophagus (BE). Identification of goblet cells in mucosal biopsies is fraught with limitations such as sampling and interpretation error. One previous study by our group suggested that MUC2 expression in esophageal nongoblet columnar cells represents a late biochemical reaction in the conversion of mucinous columnar cells to goblet cells in BE. We conducted this study to evaluate the prevalence, sensitivity, and specificity of MUC2 positivity in nongoblet columnar epithelium for detection of goblet cells in the distal esophagus and gastroesophageal junction (GEJ) region. We also sought to identify associations between MUC2 positivity and clinical and endoscopic risk factors for BE. This analysis utilized mucosal biopsies of the distal esophagus or GEJ from 100 patients who participated in a community clinic-based study of patients with chronic gastroesophageal reflux disease evaluated prospectively in the western part of Washington state. We randomly selected 50 patients who had columnar epithelium with goblet cells, representing the study group and 50 patients without goblet cells, representing the comparison group. Immunohistochemistry for MUC2 was performed on samples in a blinded manner without knowledge of the clinical or endoscopic features of the patients. The presence of staining was noted in both goblet and nongoblet epithelium, both close to and distant from the mucosa with goblet cells, when the latter were present. All study patients showed MUC2 positivity in goblet cells. MUC2 was present in nongoblet columnar epithelium in 78% of study patients with goblet cells, but in only 4% of controls without goblet cells (P<0.0001) (sensitivity, 78%; specificity, 96% for goblet cell metaplasia). MUC2 was significantly more common in nongoblet columnar cells close to, rather than distant from, the mucosa with goblet cells (P<0.00001). Finally, MUC2 was significantly associated with endoscopic evidence of columnar metaplasia in the distal esophagus, and with known risk factors for BE, such as older age, white race, frequent heartburn, and elevated body mass index. We conclude that goblet cells likely develop from a field of MUC2-positive mucinous columnar cells, and as such, MUC2 represents a late event in the development of goblet cells. MUC2 staining in nongoblet columnar cells is a reasonably sensitive and highly specific marker for goblet cells in the distal esophagus and GEJ, and its presence is predictive of endoscopic columnar metaplasia of the esophagus, even in patients without goblet cells.

CCL25/CCR9 interactions regulate large intestinal inflammation in a murine model of acute colitis.

BACKGROUND AIMS: CCL25/CCR9 is a non-promiscuous chemokine/receptor pair and a key regulator of leukocyte migration to the small intestine. We investigated here whether CCL25/CCR9 interactions also play a role in the regulation of inflammatory responses in the large intestine. METHODS: Acute inflammation and recovery in wild-type (WT) and CCR9(-/-) mice was studied in a model of dextran sulfate sodium (DSS)-induced colitis. Distribution studies and phenotypic characterization of dendritic cell subsets and macrophage were performed by flow cytometry. Inflammatory bowel disease (IBD) scores were assessed and expression of inflammatory cytokines was studied at the mRNA and the protein level. RESULTS: CCL25 and CCR9 are both expressed in the large intestine and are upregulated during DSS colitis. CCR9(-/-) mice are more susceptible to DSS colitis than WT littermate controls as shown by higher mortality, increased IBD score and delayed recovery. During recovery, the CCR9(-/-) colonic mucosa is characterized by the accumulation of activated macrophages and elevated levels of Th1/Th17 inflammatory cytokines. Activated plasmacytoid dendritic cells (DCs) accumulate in mesenteric lymph nodes (MLNs) of CCR9(-/-) animals, altering the local ratio of DC subsets. Upon re-stimulation, T cells isolated from these MLNs secrete significantly higher levels of TNFα, IFNγ, IL2, IL-6 and IL-17A while down modulating IL-10 production. CONCLUSIONS: Our results demonstrate that CCL25/CCR9 interactions regulate inflammatory immune responses in the large intestinal mucosa by balancing different subsets of dendritic cells. These findings have important implications for the use of CCR9-inhibitors in therapy of human IBD as they indicate a potential risk for patients with large intestinal inflammation.

Epidermal growth factor receptor gene amplification in atypical adenomatous hyperplasia of the lung.

Atypical adenomatous hyperplasia (AAH) is postulated to be the earliest morphologic precursor lesion in lung carcinogenesis. The epidermal growth factor receptor (EGFR), one of the members of the Erb-2 family of receptors, is commonly expressed in non-small cell lung carcinoma (NSCLC). A subset of the patients with NSCLC has molecular abnormalities in the EGFR gene, including missense mutations and deletions and/or abnormal gene copy numbers, and the relative importance of each of these for patient outcome is an area of great interest. Recent reports show that EGFR mutations are rare or absent in AAH and are rare in bronchioloalveolar carcinoma (BAC). However, the EGFR gene copy number status in AAH is unknown. In this study, we examined the EGFR gene copy number status in lung adenocarcinomas, synchronous AAH, and BAC in surgical pathology resection specimens. EGFR gene copy number was analyzed by chromogenic in situ hybridization (CISH) using formalin fixed paraffin embedded tissue sections and EGFR probes as recommended by the manufacturer. A known positive case of high-grade glioma was used as a positive control. The results indicate that four of eight adenocarcinomas (50%) had more than five EGFR signals per nucleus, suggesting a gain in copy number. Interestingly, in four of nine cases of AAH (44.4%) more than three EGFR signals per nucleus were noted, with scattered cells showing up to 6 signals per nucleus. In addition, in five of 12 cases of BAC (42%), more than three EGFR signals per nucleus were noted. In the remaining cases two to three intranuclear dot-like peroxidase positive signals were present consistent with non-amplification of the gene. Our study reveals an abnormal EGFR gene copy gain in several cases of AAH. In our cohort, the rate of EGFR gene copy abnormalities in AAH appears similar to BAC and lower than in lung adenocarcinomas. These findings suggest that although EGFR gene copy abnormalities may be an early event in lung carcinogenesis, they are associated with tumor progression to invasive cancer and highlight the complexity of tumor morphogenesis.