Plakophilin3 loss leads to increased adenoma formation and rectal prolapse in APCmin mice.

Plakophilin3 (PKP3) loss leads to tumor progression and metastasis of colon cancer cells. The goal of this report was to determine if PKP3 loss led to increased disease progression in mice. We generated a colonocyte-specific knockout of PKP3 in APCmin mice, which led to increased adenoma formation, the formation of rectal prolapse, and a significant decrease in survival. The observed increase in rectal prolapse formation and decrease in survival correlated with an increase in the expression of Lipocalin2 (LCN2). Increased disease progression was observed even upon treatment with 5-fluorouracil (5FU). These results suggest that an increase in LCN2 expression might lead to therapy resistance and that LCN2 might serve as a potential therapeutic target in colorectal cancer.

The Proton-activated Receptor GPR4 Modulates Intestinal Inflammation.

BACKGROUND AND AIMS: During active inflammation, intraluminal intestinal pH is decreased in patients with inflammatory bowel disease [IBD]. Acidic pH may play a role in IBD pathophysiology. Recently, proton-sensing G-protein coupled receptors were identified, including GPR4, OGR1 [GPR68], and TDAG8 [GPR65]. We investigated whether GPR4 is involved in intestinal inflammation. METHODS: The role of GPR4 was assessed in murine colitis models by chronic dextran sulphate sodium [DSS] administration and by cross-breeding into an IL-10 deficient background for development of spontaneous colitis. Colitis severity was assessed by body weight, colonoscopy, colon length, histological score, cytokine mRNA expression, and myeloperoxidase [MPO] activity. In the spontaneous Il-10-/- colitis model, the incidence of rectal prolapse and characteristics of lamina propria leukocytes [LPLs] were analysed. RESULTS: Gpr4-/- mice showed reduced body weight loss and histology score after induction of chronic DSS colitis. In Gpr4-/-/Il-10-/- double knock-outs, the onset and progression of rectal prolapse were significantly delayed and mitigated compared with Gpr4+/+/Il-10-/- mice. Double knock-out mice showed lower histology scores, MPO activity, CD4+ T helper cell infiltration, IFN-γ, iNOS, MCP-1 [CCL2], CXCL1, and CXCL2 expression compared with controls. In colon, GPR4 mRNA was detected in endothelial cells, some smooth muscle cells, and some macrophages. CONCLUSIONS: Absence of GPR4 ameliorates colitis in IBD animal models, indicating an important regulatory role in mucosal inflammation, thus providing a new link between tissue pH and the immune system. Therapeutic inhibition of GPR4 may be beneficial for the treatment of IBD.

Colonic Lesions, Cytokine Profiles, and Gut Microbiota in Plasminogen-Deficient Mice.

Plasminogen-deficient (FVB/NPan-plg(tm1Jld), plg(tm1Jld)) mice, which are widely used as a wound-healing model, are prone to spontaneous rectal prolapses. The aims of this study were 1) to evaluate the fecal microbiome of plg(tm1Jld) mice for features that might contribute to the development of rectal prolapses and colonic inflammation and 2) to assess the relevance of the inflammatory phenotype to the variability in wound healing in this model. The (plgtm1Jld) mice exhibited delayed wound healing, and they could be divided into 3 distinct groups that differed according to the time until wound closure. Colonic lesions in plg(tm1Jld) mice, which were characterized by necrotizing ulcerations and cystically dilated glands, were restricted to the intermediate and distal parts of the colon. The cytokine profile was indicative of chronic tissue damage, but the genetic modification did not change the composition of the gut microbiota, and none of the clinical or biochemical parameters correlated with the gut microbiota composition.

Expression of fibulin-5 in the skin of patients with rectal prolapse.

AIM: Components of connective tissue other than collagen have been found to be involved in patients with rectal prolapse. The organization of elastic fibres differs between controls and subsets of patients with rectal prolapse, and their importance for maintaining the structural and functional integrity of the pelvic floor has been demonstrated in transgenic mice, with animals which have a null mutation in fibulin-5 (Fbln5(i/i)) developing prolapse. This study aimed to compare fibulin-5 expression in the skin of patients with and without rectal prolapse. METHOD: Between January 2013 and February 2014, skin specimens were obtained during surgery from 20 patients with rectal prolapse and from 21 without prolapse undergoing surgery for other indications. Fibroblasts from the skin were cultured and the level of fibulin-5 expression was determined on cultured fibroblasts, isolated from these specimens by quantitative real-time polymerase chain reaction. Immunohistochemistry was performed on fixed tissue specimens to assess fibulin-5 expression. RESULTS: Fibulin-5 mRNA expression and fibulin-5 staining intensity were significantly lower in young male patients with rectal prolapse compared with age-matched controls [fibulin-5 mean ± SD mRNA relative units, 1.1 ± 0.41 vs 0.53 ± 0.22, P = 0.001; intensity score, median (range), 2 (0-3) vs 1 (0-3), P = 0.05]. There were no significant differences in the expression of fibulin-5 in women with rectal prolapse compared with controls. CONCLUSION: Fibulin-5 may be implicated in the aetiology of rectal prolapse in a subgroup of young male patients.

Impaired removal of Vß8(+) lymphocytes aggravates colitis in mice deficient for B cell lymphoma-2-interacting mediator of cell death (Bim).

We investigated the role of B cell lymphoma (BCL)-2-interacting mediator of cell death (Bim) for lymphocyte homeostasis in intestinal mucosa. Lymphocytes lacking Bim are refractory to apoptosis. Chronic colitis was induced in Bim-deficient mice (Bim(-/-) ) with dextran sulphate sodium (DSS). Weight loss and colonoscopic score were increased significantly in Bim(-/-) mice compared to wild-type mice. As Bim is induced for the killing of autoreactive cells we determined the role of Bim in the regulation of lymphocyte survival at mucosal sites. Upon chronic dextran sulphate sodium (DSS)-induced colitis, Bim(-/-) animals exhibited an increased infiltrate of lymphocytes into the mucosa compared to wild-type mice. The number of autoreactive T cell receptor (TCR) Vß8(+) lymphocytes was significantly higher in Bim(-/-) mice compared to wild-type controls. Impaired removal of autoreactive lymphocytes in Bim(-/-) mice upon chronic DSS-induced colitis may therefore contribute to aggravated mucosal inflammation.

Prolapse-related changes are a confounding factor in misdiagnosis of sessile serrated adenomas in the rectum.

The differential diagnosis of rectal serrated polyps is challenging due to its unique anatomic location, the evolving concept of serrated polyps over the past several years, and to histologic changes seen in rectal mucosal prolapse. We reclassified 95 rectal polyps diagnosed originally as "sessile serrated adenoma" (SSA), "serrated polyp," or "hyperplastic polyp (HP) with features of SSA" in a 5-year period based on World Health Organization classification criteria for colorectal serrated polyps. BRAF (V600E) mutation assay was performed to explore its value in the differential diagnosis for serrated polyps. Twenty-six originally diagnosed SSAs were reclassified as SSA (15/26, 57.7%), HP with mucosal prolapse (HP-P; 7/26, 26.9%), and HP (4/26, 15.4%). Fifty-two polyps originally diagnosed "HP with features of SSA" were reclassified as HP-P (24/52, 46.2%), HP (10/52, 19.2%), inflammatory-type polyp (5/52, 9.6%), and serrated polyp unclassifiable (13/52, 25.0%). Thirty-one of the 78 originally diagnosed SSA or HP with features of SSA were reclassified as HP-P, which accounted for 32.6% of the rectal polyps in this study. Mucosal prolapse along with chronic inflammation and tissue embedding artifact were the most common features that led to misdiagnosis in rectal serrated polyps. BRAF mutation was identified in 8 of 11 HP, 4 of 4 SSA, and 8 of 11 unclassifiable serrated polyp of the rectum, and was absent in control tissue. Thus, histopathologic changes suggesting prolapsed rectal mucosa should take precedence over BRAF results.

Compromised mitochondrial fatty acid synthesis in transgenic mice results in defective protein lipoylation and energy disequilibrium.

A mouse model with compromised mitochondrial fatty acid synthesis has been engineered in order to assess the role of this pathway in mitochondrial function and overall health. Reduction in the expression of mitochondrial malonyl CoA-acyl carrier protein transacylase, a key enzyme in the pathway encoded by the nuclear Mcat gene, was achieved to varying extents in all examined tissues employing tamoxifen-inducible Cre-lox technology. Although affected mice consumed more food than control animals, they failed to gain weight, were less physically active, suffered from loss of white adipose tissue, reduced muscle strength, kyphosis, alopecia, hypothermia and shortened lifespan. The Mcat-deficient phenotype is attributed primarily to reduced synthesis, in several tissues, of the octanoyl precursors required for the posttranslational lipoylation of pyruvate and α-ketoglutarate dehydrogenase complexes, resulting in diminished capacity of the citric acid cycle and disruption of energy metabolism. The presence of an alternative lipoylation pathway that utilizes exogenous free lipoate appears restricted to liver and alone is insufficient for preservation of normal energy metabolism. Thus, de novo synthesis of precursors for the protein lipoylation pathway plays a vital role in maintenance of mitochondrial function and overall vigor.

CCR7 deficiency causes diarrhea associated with altered ion transport in colonocytes in the absence of overt colitis.

The chemokine receptor CCR7 is a central regulator in the maintenance of cellular homeostasis of mucosal tissues. CCR7⁻/⁻ mice develop autoimmune gastritis and exocrinopathy accompanied by the formation of mucosal tertiary lymphoid follicles. Here we found that CCR7-deficient mice frequently suffered from chronic diarrhea linked with increased gastrointestinal motility and the development of severe anorectal prolapse. Enhanced formation of intestinal lymphoid follicles was associated with an elevated proportion of activated colonic T cells and increased production of the cytokine interleukin (IL)-1ß. To uncover the pathomechanisms of diarrhea in CCR7⁻/⁻ mice, colonic epithelial barrier and ion channel activities were analyzed in Ussing chambers. Although overt acute colitis was absent, CCR7 deficiency resulted in reduced electrogenic sodium absorption and colonic chloride secretion. As it is known that IL-1ß regulates epithelial sodium channel (ENaC) activity, these data imply a causal link between CCR7 expression, IL-1ß level, and Na⁺ malabsorption owing to altered ENaC expression and diarrhea.

The protein disulfide isomerase AGR2 is essential for production of intestinal mucus.

Protein disulfide isomerases (PDIs) aid protein folding and assembly by catalyzing formation and shuffling of cysteine disulfide bonds in the endoplasmic reticulum (ER). Many members of the PDI family are expressed in mammals, but the roles of specific PDIs in vivo are poorly understood. A recent homology-based search for additional PDI family members identified anterior gradient homolog 2 (AGR2), a protein originally presumed to be secreted by intestinal epithelial cells. Here, we show that AGR2 is present within the ER of intestinal secretory epithelial cells and is essential for in vivo production of the intestinal mucin MUC2, a large, cysteine-rich glycoprotein that forms the protective mucus gel lining the intestine. A cysteine residue within the AGR2 thioredoxin-like domain forms mixed disulfide bonds with MUC2, indicating a direct role for AGR2 in mucin processing. Mice lacking AGR2 were viable but were highly susceptible to colitis, indicating a critical role for AGR2 in protection from disease. We conclude that AGR2 is a unique member of the PDI family, with a specialized and nonredundant role in intestinal mucus production.

The relationship between COL3A1 exon 31 polymorphism and pelvic organ prolapse.

PURPOSE: We investigated the role of COL3A1 exon 31 polymorphism (a single base substitution from guanine to adenine at +2092), resulting in the replacement of alanine with threonine at the 698th amino acid of COL3A1, in the pathogenesis of pelvic organ prolapse. MATERIALS AND METHODS: A total of 72 postmenopausal Korean women who were not on hormonal replacement therapy and who had a history of vaginal childbirth were enrolled in this study. The patient group consisted of 36 women diagnosed with stage II or greater pelvic organ prolapse irrespective of urodynamic stress incontinence. The control group consisted of 36 healthy volunteers with pelvic organ prolapse quantification system stage 0 or I disease without urodynamic stress incontinence. After extracting the genomic DNA from peripheral blood leukocytes the polymorphism of exon 31 of COL3A1 was typed by restriction fragment length polymorphism (Alu I restriction fragment length polymorphism) and confirmed by direct sequencing. RESULTS: Frequency of the G allele was significantly higher in patients with pelvic organ prolapse than in controls (0.8 vs 0.6, p = 0.002). In women with the G allele the OR for pelvic organ prolapse was 3.2 (95% CI 1.4-7.3). CONCLUSIONS: COL3A1 exon 31 polymorphism may have a role in determining the risk of pelvic organ prolapse in women with risk factors such as aging, vaginal childbirth and hypoestrogenism.

Failure of pelvic organ support in mice deficient in fibulin-3.

Fibulin-5 is crucial for normal elastic fiber synthesis in the vaginal wall; more than 90% of fibulin-5-knockout mice develop pelvic organ prolapse by 20 weeks of age. In contrast, fibulin-1 and -2 deficiencies do not result in similar pathologies, and fibulin-4-knockout mice die shortly after birth. EFEMP1 encodes fibulin-3, an extracellular matrix protein important in the maintenance of abdominal fascia. Herein, we evaluated the role of fibulin-3 in pelvic organ support. Pelvic organ support was impaired significantly in female Efemp1 knockout mice (Fbln3(-[supi]/-)), and overt vaginal, perineal, and rectal prolapse occurred in 26.9% of animals. Prolapse severity increased with age but not parity. Fibulin-5 was up-regulated in vaginal tissues from Fbln3(-[supi]/-) mice regardless of prolapse. Despite increased expression of fibulin-5 in the vaginal wall, pelvic organ support failure occurred in Fbln3(-[supi]/-) animals, suggesting that factors related to aging led to prolapse. Elastic fiber abnormalities in vaginal tissues from young Fbln3(-[supi]/-) mice progressed to severe elastic fiber disruption with age, and vaginal matrix metalloprotease activity was increased significantly in Fbln3(-[supi]/-) animals with prolapse compared with Fbln3(-[supi]/-) mice without prolapse. Overall, these results indicate that both fibulin-3 and -5 are important in maintaining pelvic organ support in mice. We suggest that increased vaginal protease activity and abnormal elastic fibers in the vaginal wall are important components in the pathogenesis of pelvic organ prolapse.

LOXL1 deficiency negatively impacts the biomechanical properties of the mouse vagina and supportive tissues.

Mice deficient in lysyl oxidase-like1 protein (LOXL1(-/-)) develop pelvic organ prolapse (POP). We sought to determine the impact of LOXL1(-/-) on the biomechanical properties of the vagina and its supportive tissues tested as a complex. Tissues of nulliparous LOXL1(-/-) and age-matched wild type (WT) mice were tested to failure to obtain load-distension curves. Data were compared utilizing one-way analysis of variance and appropriate post hoc tests. The groups demonstrated different biomechanical behavior, with LOXL1(-/-) animals displaying a 31% decrease in ultimate load at failure (p=0.001). Experimental disruption of specific levels of support in WT mice failed to generate load-distension curves similar to the LOXL1(-/-) mice indicating a global instead of a site-specific tissue defect. The decrease in the ultimate load at failure in the LOXL1(-/-) mice suggests mechanically weaker tissues. LOXL1 mutation results in a global defect in connective tissues and correlates with altered biomechanical behavior of the vagina and supportive tissues.

Generation of a unique strain of multiple intestinal neoplasia (Apc(+/Min-FCCC)) mice with significantly increased numbers of colorectal adenomas.

The relevance of the Apc(+/Min) mouse model in the study of human colorectal cancer remains uncertain due to the predominance of small intestinal adenomas and few, if any, colorectal adenomas. A new strain of Apc(+/Min) mice (Apc(+/Min-FCCC)) with significantly greater numbers of colorectal adenomas has been generated and characterized. Male C57BL/6J-Apc(+/Min) mice (the Jackson Laboratory, Bar Harbor, ME) were crossed with wild-type (Apc(+/+)) C57BL/6J females from an independent colony at this institution (offspring=Apc(+/Min-FCCC)) and 233 animals were evaluated over 20 generations. In order to determine the contribution of genetics to the enhanced colorectal adenoma phenotype, breeding pairs (Apc(+/Min) male x wild type female C57BL/6J) were purchased from the Jackson Laboratory and offspring (Apc(+/Min-JAX)) were maintained in our facility under identical conditions (n=98). Animals were fed Purina Rodent chow (#5013) diet containing 5% fat. The entire intestinal tract was examined histopathologically in both strains. Both the Apc and Pla2g2a (candidate for Mom1) genes were sequenced and found to be identical for both the Apc(+/Min-FCCC) and Apc(+/Min-JAX) mouse strains. The multiplicity of colorectal adenomas in the Apc(+/Min-FCCC) mice was much higher than reported in the literature and significantly greater than the multiplicity of colorectal adenomas in Apc(+/Min-JAX) mice maintained in our facility (P=0.01). Apc(+/Min-FCCC) had a significantly greater incidence of rectal prolapse (P = 0.02) and small intestinal adenocarcinomes (P=0.001), and multiplicity of small intestinal adenocarcinomas (P=0.001) compared to Apc(+/Min-JAX) mice. Male Apc(+/Min-FCCC) mice had significantly greater numbers of colorectal adenomas compared to female Apc(+/Min-FCCC) mice (P=0.0002), as did male Apc(+/Min-JAX) mice vs. female Apc(+/Min-JAX) mice (P< 0.0001). These results allow us to conclude: (1) Apc(+/Min-FCCC) mice are unique in that they develop significantly greater numbers of colorectal adenomas and small intestinal cancers, and a significantly greater incidence of small intestinal cancers and rectal prolapse than Apc(+/Min-JAX) mice. (2) This study represents the first report of a significant gender difference in multiplicity of colorectal adenomas. (3) Differences between Apc(+/Min-FCCC) and Apc(+/Min-JAX) mice in currently undefined genetic modifiers may contribute to the enhanced colorectal phenotype. (4) The Apc(+/Min-FCCC) strain is highly suited for the investigation of colorectal neoplastic disease and chemoprevention studies.

Expression of IL-5 in thymocytes/T cells leads to the development of a massive eosinophilia, extramedullary eosinophilopoiesis, and unique histopathologies.

Transgenic mice were generated using regulatory elements from the CD3delta gene to drive T cell expression of IL-5. Expression of this cytokine resulted in white blood cell counts that expand virtually unabated (approximately 400,000 cells/mm3). This expansion is characterized by a profound eosinophilia (>60%) and commensurate increases in the absolute numbers of all other white blood cell types. In particular, circulating B220+ B lymphocyte populations increased >30-fold over wild-type (+/+) levels. Cell differentials and expression studies using a marker for eosinophil precursor cells (major basic protein gene expression) suggest that the peripheral eosinophilia is induced primarily through the establishment of extramedullary sites of eosinophilopoiesis. These mice display a massive peritoneal cavity cell exudate (1-2 x 10(8) cells) dominated by eosinophils (approximately 50%) and the infiltration of eosinophils in nearly all organ systems. Sudden unexplained death occurs in 70% of all transgenic animals by 12 mo of age. Surviving transgenic animals display severe inflammatory pathologies that include ulcerating skin lesions as well as lower bowel inflammation. These pathologies parallel clinical observations of patients with a profound eosinophilia and imply that IL-5 effector functions during some inflammatory responses may be contingent upon peripheral lymphohemopoietic expression.

Procidentia in identical twins. Report of cases and analysis of treatment options.

Within a six-week interval, adult identical twin brothers presented with third-degree complete rectal prolapse. Since no other predisposing factors were evident, contribution of an inherited predisposition is implied. Evaluation of alternatives supports use of presacral rectal mobilization with subtotal anterior resection and reanastamosis of sigmoid colon and rectum as the curative procedure.

Peutz-Jeghers syndrome in children: report of two cases and review of the literature.

In this article, we report two new cases of Peutz-Jeghers syndrome in children and review the literature over the past twenty years. This series of 70 cases demonstrates that the clinical pictures observed in children are similar to those of adults. Rectal prolapse or extrusion of polyps can be the clue to the diagnosis at an early age, even in the absence of pigmentation, which can appear later. Gastroduodenal polyps were strikingly frequent in the less than or equal to 16-yr-old group (62%) a circumstance that can create operative difficulties. Five out of the 70 patients (7.14%) had tumors during childhood (two gastrointestinal adenocarcinomas, two ovarian and one testicular neoplasms). A higher risk of tumor development in these patients does exist either as a result of degeneration of the polyps or of a genetic predisposition. Whenever operation becomes necessary, a very cautious approach must be advised in order to preserve as much intestinal length as possible in these patients, who have a lifelong disease which may require repeated operations.