Acquired dysfunction of CFTR underlies cystic fibrosis-like disease of the canine gallbladder.

Mucocele formation in dogs is a unique and enigmatic muco-obstructive disease of the gallbladder caused by the amassment of abnormal mucus that bears striking pathological similarity to cystic fibrosis. We investigated the role of cystic fibrosis transmembrane conductance regulatory protein (CFTR) in the pathogenesis of this disease. The location and frequency of disease-associated variants in the coding region of CFTR were compared using whole genome sequence data from 2,642 dogs representing breeds at low-risk, high-risk, or with confirmed disease. Expression, localization, and ion transport activity of CFTR were quantified in control and mucocele gallbladders by NanoString, Western blotting, immunofluorescence imaging, and studies in Ussing chambers. Our results establish a significant loss of CFTR-dependent anion secretion by mucocele gallbladder mucosa. A significantly lower quantity of CFTR protein was demonstrated relative to E-cadherin in mucocele compared with control gallbladder mucosa. Immunofluorescence identified CFTR along the apical membrane of epithelial cells in control gallbladders but not in mucocele gallbladder epithelium. Decreases in mRNA copy number for CFTR were accompanied by decreases in mRNA for the Cl-/[Formula: see text] exchanger SLC26A3, K+ channels (KCNQ1, KCNN4), and vasoactive intestinal polypeptide receptor (VIPR1), which suggest a driving force for change in secretory function of gallbladder epithelial cells in the pathogenesis of mucocele formation. There were no significant differences in CFTR gene variant frequency, type, or predicted impact comparing low-risk, high-risk, and definitively diagnosed groups of dogs. This study describes a unique, naturally occurring muco-obstructive disease of the canine gallbladder, with uncanny similarity to cystic fibrosis, and driven by the underlying failure of CFTR function.NEW & NOTEWORTHY Cystic fibrosis transmembrane conductance regulatory protein (CFTR) genomic variants and expression of mRNA, protein, and electrogenic anion secretory activity of CFTR were characterized in dog gallbladder. Acquired inhibition of CFTR expression by gallbladder epithelium was identified as underpinning a naturally occurring muco-obstructive disease of the dog gallbladder that bears striking pathological similarity to animal models of cystic fibrosis.

Molecular profiling of appendiceal epithelial tumors using massively parallel sequencing to identify somatic mutations.

BACKGROUND: Some epithelial neoplasms of the appendix, including low-grade appendiceal mucinous neoplasm and adenocarcinoma, can result in pseudomyxoma peritonei (PMP). Little is known about the mutational spectra of these tumor types and whether mutations may be of clinical significance with respect to therapeutic selection. In this study, we identified somatic mutations using the Ion Torrent AmpliSeq Cancer Hotspot Panel v2. METHODS: Specimens consisted of 3 nonneoplastic retention cysts/mucocele, 15 low-grade mucinous neoplasms (LAMNs), 8 low-grade/well-differentiated mucinous adenocarcinomas with pseudomyxoma peritonei, and 12 adenocarcinomas with/without goblet cell/signet ring cell features. Barcoded libraries were prepared from up to 10 ng of extracted DNA and multiplexed on single 318 chips for sequencing. Data analysis was performed using Golden Helix SVS. Variants that remained after the analysis pipeline were individually interrogated using the Integrative Genomics Viewer. RESULTS: A single Janus kinase 3 (JAK3) mutation was detected in the mucocele group. Eight mutations were identified in the V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and GNAS complex locus (GNAS) genes among LAMN samples. Additional gene mutations were identified in the AKT1 (v-akt murine thymoma viral oncogene homolog 1), APC (adenomatous polyposis coli), JAK3, MET (met proto-oncogene), phosphatidylinositol-4,5-bisphosphate 3-kinase (PIK3CA), RB1 (retinoblastoma 1), STK11 (serine/threonine kinase 11), and tumor protein p53 (TP53) genes. Among the PMPs, 6 mutations were detected in the KRAS gene and also in the GNAS, TP53, and RB1 genes. Appendiceal cancers showed mutations in the APC, ATM (ataxia telangiectasia mutated), KRAS, IDH1 [isocitrate dehydrogenase 1 (NADP+)], NRAS [neuroblastoma RAS viral (v-ras) oncogene homolog], PIK3CA, SMAD4 (SMAD family member 4), and TP53 genes. CONCLUSIONS: Our results suggest molecular heterogeneity among epithelial tumors of the appendix. Next generation sequencing efforts have identified mutational spectra in several subtypes of these tumors that may suggest a phenotypic heterogeneity showing mutations that are relevant for targeted therapies.

MCM3 could be a better marker than Ki-67 for evaluation of dysplastic oral lesions: an immunohistochemical study.

BACKGROUND: The aim of this study was to identify the expression of MCM3, Ki-67 and p27 in normal mucosa, leucoplakia and oral squamous cell carcinoma (OSCC) and determine whether altered expression could serve as a prognostic marker of a malignant progression of dysplastic lesions. METHODS: The samples were collected from 37 patients with oral leucoplakia (13 with mild dysplasia - MLD, 12 with moderate dysplasia - MD and 12 with severe dysplasia - SD). Eleven samples of mouth floor mucocele (M) and 50 floor mouth and tongue samples OSCC of untreated patients were included in this study. Immunohistochemical expression of MCM3, Ki-67 and p27 of all the groups was analysed. Kruskal-Wallis and Dunn's test were used to determine differences among groups, and a Pearson's correlation test was used to evaluate the correlation between the proteins. RESULTS: Ki-67 expression was higher in OSCC than M (P < 0.001) and MLD (P < 0.01) groups, and there was a lower expression in M compared with MD and SD (P < 0.05). Regarding p27, its expression was lower in OSCC compared with M, MD and SD. MCM3 expression was lower in M compared with SD and OSCC (P < 0.001), and MLD showed a lower expression when compared SD (P < 0.01) and OSCC (P < 0.001). Moreover, a better correlation was observed between the proteins MCM3 and p27 than between Ki-67 and p27 proteins when all lesions were examined together. CONCLUSIONS: This study showed that MCM3 could be a better marker than Ki-67 for evaluation of dysplastic oral lesions.

Increased lipogenesis and lipidosis of gallbladder epithelium in dogs with gallbladder mucocele formation.

BACKGROUND: Gallbladder disease in people is frequently associated with disorders of lipid metabolism and metabolic syndrome. A recently emergent gallbladder disease of dogs, referred to as mucocele formation, is characterized by secretion of abnormal mucus by the gallbladder epithelium and is similarly associated with hyperlipidemia, endocrinopathy, and metabolic dysfunction. The cause of gallbladder mucocele formation in dogs is unknown. METHODS: A prospective case-controlled study was conducted to gain insight into disease pathogenesis by characterization of plasma lipid abnormalities in 18 dogs with gallbladder mucocele formation and 18 age and breed matched control dogs using direct infusion mass spectrometry for complex plasma lipid analysis. This analysis was complemented by histochemical and ultrastructural examination of gallbladder mucosa from dogs with gallbladder mucocele formation and control dogs for evidence of altered lipid homeostasis of the gallbladder epithelium. RESULTS: Gallbladder mucocele formation in dogs carried a unique lipidomic signature of increased lipogenesis impacting 50% of lipid classes, 36% of esterified fatty acid species, and 11% of complex lipid species. Broad enrichment of complex lipids with palmitoleic acid (16:1) and decreased abundance within complex lipids of presumptive omega-3 fatty acids eicosapentaenoic (20:5) and docosahexaenoic (22:6) was significant. Severe lipidosis of gallbladder epithelium pinpoints the gallbladder as involved causally or consequently in abnormal lipid metabolism. CONCLUSION: Our study supports a primary increase in lipogenesis in dogs with mucocele formation and abnormal gallbladder lipid metabolism in disease pathogenesis.

MUC5AC and MUC5B expression in canine gallbladder mucocele epithelial cells.

Gallbladder mucocele (GBM) is one of the most common gallbladder diseases in dogs. Its pathogenesis has not yet been clarified, but excessive accumulation of a secretory gel-forming mucin, MUC5AC in the gallbladder has been reported. This study aimed to ascertain if MUC5AC overproduction resulted in mucus accumulation in the gallbladder during GBM development. Eleven dogs undergoing cholecystectomy who were pathologically diagnosed with GBM were included, and the expression level of mucins, particularly MUC5AC and MUC5B, in their gallbladder epithelial cells was compared with those in normal gallbladder epithelial cells. On reverse transcription-quantitative polymerase chain reaction screening, there was a significant difference (P<0.05) in the mRNA expression level of MUC1, but not of other mucins including MUC5AC and MUC5B, between mucocele and normal gallbladder epithelial cells. Protein expression levels were also evaluated for MUC5AC and MUC5B using immunohistochemistry. There was little immunoreactivity for MUC5AC, whereas MUC5B showed definitive staining in gallbladder epithelial cells. There was no difference in MUC5AC and MUC5B protein expression levels between mucocele and normal gallbladder epithelial cells. These data suggest that excessive production of mucin, especially MUC5AC and MUC5B, does not occur in canine GBM, and that abnormal mucus excretion, rather than excessive mucus production, may be the cause of GBM development.

Collagenous spherulosis in an oral mucous cyst.

Collagenous spherulosis is a histological pattern that has been described in both benign and malignant salivary gland tumors, proliferative lesions of breast ductal epithelium, chondroid syringomas and schwannomas. Histologic structures of similar appearance have also been reported in oral extravasation mucoceles as questionable myxoglobulosis or myxoglobulosis-like change. We report collagenous spherulosis within a mucocele removed from the lower lip of a 17-year-old female. Based upon histologic appearance, immunophenotypic data and review of the literature, we hypothesize that collagenous spherulosis and myxoglobulosis are morphologically related reaction patterns.

Expression of human beta-defensin -1, -2, and -3 in non-inflamed pseudocyst, mucoceles.

OBJECTIVES AND DESIGN: The expressions of human beta defensin-1 (HBD-1), -2 (HBD-2) and -3 (HBD-3) in non-inflamed pseudocysts such as mucoceles were investigated immunohistochemically in this study. MATERIALS AND METHODS: Mucocele specimens were obtained from 21 patients. The expression of HBDs was studied immunohistochemically by using antibodies directed against HBD-1, -2, and -3. Statistical analyses were carried out on serial sections stained with antibodies. RESULTS: Cells expressing HBDs were found in mucoceles. The expression of HBD-2 was observed in floating cells in all the specimens, whereas HBD-1 and HBD-3-expressing cells were detected in 93% and 73% of the mucoceles, respectively. The HBD-2 signal was the most intense and the HBD-3 signal intensity was weaker than that of HBD-1. HBDs were expressed in neutrophils and in other floating cells. Interestingly, the signal intensity and the population of positive cells located close to the centers of cysts were higher than those located in the peripheral areas of cysts. CONCLUSION: The expression of HBDs was found even in non-inflamed pseudocysts such as mucoceles. These results suggest that an unknown mechanism not involved in biophylaxis for the expression of HBDs may exist.

Qualitative metabolomics profiling of serum and bile from dogs with gallbladder mucocele formation.

Mucocele formation is characterized by secretion of abnormally thick mucus by the gallbladder epithelium of dogs that may cause obstruction of the bile duct or rupture of the gallbladder. The disease is increasingly recognized and is associated with a high morbidity and mortality. The cause of gallbladder mucocele formation in dogs is unknown. There is a strong breed predisposition and affected dogs have a high incidence of concurrent endocrinopathy or hyperlipidemia. These observations suggest a significant influence of both genetic and metabolic factors on disease pathogenesis. In this study, we investigated a theory that mucocele formation is associated with a syndrome of metabolic disruption. We surmised that a global, untargeted metabolomics approach could provide unique insight into the systemic pathogenesis of gallbladder mucocele formation and identify specific compounds as candidate biomarkers or treatment targets. Moreover, concurrent examination of the serum and hepatic duct bile metabolome would enable the construction of mechanism-based theories or identification of specific compounds responsible for altered function of the gallbladder epithelium. Abnormalities observed in dogs with gallbladder mucocele formation, including a 33-fold decrease in serum adenosine 5'-monophosphate (AMP), lower quantities of precursors required for synthesis of energy transporting nucleotides, and increases in citric acid cycle intermediates, suggest excess metabolic energy and a carbon surplus. Altered quantities of compounds involved in protein translation and RNA turnover, together with accumulation of gamma-glutamylated and N-acetylated amino acids in serum suggest abnormal regulation of protein and amino acid metabolism. Increases in lathosterol and 7α-hydroxycholesterol suggest a primary increase in cholesterol synthesis and diversion to bile acid formation. A number of specific biomarker compounds were identified for their ability to distinguish between control dogs and those that formed a gallbladder mucocele. Particularly noteworthy was a significant decrease in quantity of biologically active compounds that stimulate biliary ductal fluid secretion including adenosine, cAMP, taurolithocholic acid, and taurocholic acid. These findings support the presence of significant metabolic disruption in dogs with mucocele formation. A targeted, quantitative analysis of the identified serum biomarkers is warranted to determine their utility for diagnosis of this disease. Finally, repletion of compounds whose biological activity normally promotes biliary ductal secretion should be examined for any therapeutic impact for resolution or prevention of mucocele formation.

The activation of Akt/mTOR pathway by bleomycin in Epithelial-to-mesenchymal transition of human submandibular gland cells: A treatment mechanism of bleomycin for mucoceles of the salivary glands.

OBJECTIVE: Bleomycin (BLM) has been found safe and highly effective in the treatment of the mucoceles by intralesional injection in our previous study. The present research was designed to investigate whether epithelial-to-mesenchymal transition (EMT) contributes to the therapeutic effects of BLM for mucoceles of the salivary glands. MATERIAL AND METHODS: The cell proliferation and apoptosis of human submandibular gland cells (HSG cells) were examined by Cell Counting Kit-8 assay and Annexin V binding assay respectively. Epithelial and mesenchymal markers of HSG cells were measured by real-time quantitative PCR and Western blot analysis. Acinar differentiation and cell migration assays were performed to evaluate HSG cells function. RESULTS: High-dose BLM (≥0.5µg/mL) significantly inhibited the cell proliferation and induced the cell apoptosis, while the treatment with low-dose BLM (0.05 and 0.1µg/mL) for 48h induced EMT in HSG cells. Furthermore, Akt/mTOR pathway, rather than MAPK pathway, was activated through treated with 0.05 and 0.1µg/mL BLM, as well as activation of the transcription factor Slug and Zeb 1. The migration of HSG cells was also enhanced through 0.05 and 0.1µg/mL BLM, but the ability of acinar differentiation was diminished. CONCLUSION: Our results indicated that an EMT process was involved in the BLM-induced therapeutic effects on the HSG cells through the Akt/mTOR pathway. Importantly, the results indicated the potential role of this process in the BLM sclerotherapy of mucoceles of the salivary glands.

MR spectroscopy in sinus mucocele: N-acetyl mimics of brain N-acetylaspartate.

We describe MR spectroscopy in 2 patients with frontal sinus mucoceles that showed a dominant metabolite peak at 2.0-ppm chemical shift, simulating N-acetylaspartate (NAA) of normal neuronal tissue. In vitro analysis of postsurgical mucocele samples confirmed that the signal at 2.0 ppm was arising from the methyl moiety of an N-acetyl compound. This is probably caused by N-acetylgalactosamine or N-acetylglucosamine, which are glycoproteins found in normal respiratory mucus produced by the paranasal sinus epithelium.

Mammary mucocele-like lesions. Benign and malignant.

In 1986 mucocele-like lesions (MLL) were described as benign tumors; subsequent reports identified MLL associated with ductal hyperplasia or carcinoma (CA). To characterize MLL further, we studied 53 lesions from 49 patients, in whom 25 MLL were benign and 28 were malignant (14 in situ, 14 invasive). Two had bilateral benign MLL, and two had bilateral MLL with CA. Patients ranged in age from 24 to 79 years (mean, 48 years). There were no appreciable differences in age, tumor size, or laterality between patients with benign or malignant MLL, although MLL with CA had coarse calcifications more often than benign MLL and were more likely to be detected mammographically. Intraductal carcinoma was micropapillary or cribriform, and invasive carcinoma was usually mucinous. Fewer of the benign lesions were estrogen and progesterone receptor positive. HER2/neu positivity was more common in MLL with CA. Known treatment was as follows: for benign MLL, excisional biopsy was done in 22 patients (one with axillary dissection) and total mastectomy in one patient; for MLL with CA, excisional biopsy was done in 17 patients, biopsy followed by wider excision in four patients (three of whom had axillary dissection), and mastectomy and axillary dissection in five patients (one also had radiotherapy). Follow-up ranged from less than a 1 year to 15 years (mean and median, 3.7 years). Two patients had recurrences in the breast (one benign MLL and one MLL with CA). At the time of this report, all were alive without evidence of disease. We conclude that MLL with CA is a low-grade neoplasm with few clinical differences from benign MLL except for more prominent calcifications, leading to mammographic detection. Excisional biopsy is recommended for benign MLL. Breast-conserving surgery is appropriate therapy for MLL with CA. Radiotherapy is indicated if CA involves margins or if extensive intraductal carcinoma is present.

Altered mucin expression is a field change that accompanies mucinous (colloid) breast carcinoma histogenesis.

Mucinous carcinomas of the breast, so-called colloid carcinomas, exhibit better prognoses than their nonmucinous breast counterparts. This biological difference exhibited by mucinous breast carcinomas prompted us to examine the relationship of mucin expression to colloid carcinoma histogenesis. We studied 50 colloid carcinomas, 50 noncolloid cancers, and 50 normal breasts by hematoxylin-eosin (H&E) and Alcian blue staining, mucin immunohistochemistry, in situ hybridization with a battery of MUC riboprobes, and ancillary digital image analysis. We observed luminal mucin in normal ducts in 80% of colloid carcinomas compared with 10% of noncolloid carcinomas and 6% of normal breasts (P < .01). In the cases of colloid carcinoma that showed mucin-filled ducts, luminal mucin was observed in 40% of the normal ducts and acini, 40% to 75% of the ducts involved by hyperplasia, atypical ductal hyperplasia (ADH) and ductal carcinoma in situ (DCIS), respectively, and in 50% of the co-incidental areas of cysts (mucoceles), adenosis, fibroadenoma, and intraductal papilloma (P < .01). Immunohistochemistry showed that colloid carcinomas showed strong MUC2 cytoplasmic immunoreactivity and decreased MUC1 immunoreactivity compared with noncolloid carcinomas. In situ hybridization studies indicated fivefold increased MUC2 signals and twofold increased MUC5 signals within adjacent and remote normal epithelium in only the colloid carcinoma cases (P < .01; P < .05). In these cases of colloid carcinoma, these increased MUC2 and MUC5 signals were also observed in areas of hyperplasia, ADH, DCIS, and invasive carcinoma. In contrast, the noncolloid carcinomas showed fivefold increased MUC1 signals but no increases in MUC2 or MUC5. In mixed colloid/noncolloid carcinomas, the colloid areas had identical mucin expression patterns as the pure colloid carcinomas, but there was a loss of MUC2 and MUC5 expression and a gain of MUC1 expression in the noncolloid areas that was therefore identical to the pattern observed in pure noncolloid carcinoma. In this study, we conclude that the altered expression of mucin so characteristic of colloid carcinoma is also a field change present in adjacent and remote normal breast epithelium.

Lectin binding activity in appendiceal mucocele.

In order to further clarify the histologic criteria and prognostic measurement of appendiceal adenoma and well differentiated adenocarcinoma, lectin binding activity in 31 cases of appendiceal mucocele (6 hyperplasias, 16 adenomas and 9 adenocarcinomas) was studied using an immunoperoxidase staining method. An additional 45 cases of colonic lesions (15 adenomas and 30 adenocarcinomas) were also examined as the corresponding controls. Arachis hypogaea agglutinin (PNA) bound selectively to the neoplastic cells of adenocarcinomas in the appendix and colon, but in contrast, Dolichos biflorus agglutinin (DBA) binding activity was observed in both normal mucosa and adenomas, and in appendiceal hyperplasia. The fact that one case each of appendiceal adenoma and colonic villous adenoma showed a diffuse positive reactivity with PNA, which was comparable with similar binding in the adenocarcinoma cases, may suggest development of a malignant behavior prior to the histologic expression of malignant transformation in the appendiceal mucocele.

Expression of the antimicrobial peptide, human beta-defensin 1, in duct cells of minor salivary glands and detection in saliva.

The oral cavity is exposed to a variety of environmental insults. Salivary secretions play a critical role in maintaining oral health via innate host defense mechanisms and secretion of secretory IgA. Human beta-defensins (hBD) are antimicrobial peptides that are a component of the innate immune response; they are expressed in epithelia and are proposed to have a role in mucosal defense. hBD-1 mRNA is constitutively expressed in numerous mucosal tissues, including human gingiva and submandibular and parotid glands. Our objective was to detect the expression and localization of hBD-1 peptide in human salivary glands and in saliva. Minor salivary gland tissue was obtained from biopsies of patients with mucoceles (n = 20). hBD-1 peptide was detected by immunohistochemistry; expression was localized to the ductal cells and not the acinar cells of these glands. The peptide was located apically, toward the lumen in the duct cells. Further evaluation showed stronger hBD-1 expression in ducts with periductal inflammation, as indicated by the immunostaining of serial sections with anti-CD45 specific for B- and T-lymphocytes. Statistical analysis showed a strong correlation of hBD-1 staining and inflammation. Results of immunolocalization suggest that hBD-1 functions to protect salivary glands from retrograde infection, that expression of the peptide is enhanced in inflamed sites, and that post-transcriptional regulatory mechanisms may be involved in hBD-1 peptide expression. Western immunoblot analysis also detected hBD-1 peptide in unstimulated, whole, acidified saliva from normal volunteers. However, hBD-1 peptide associated with salivary mucin resulted in loss of the detection in a dot-immunoblot assay. Association of hBD-1 with salivary mucin may facilitate peptide distribution and adherence to oral surfaces and aid its function within the oral cavity.

Keratin proteins identified in epithelial fragments of salivary mucoceles.

Immunohistochemically detected keratin proteins in mucoceles of oral mucosa were used to served as markers to identify epithelial-derived cells of minor salivary glands. Normal ducts of minor salivary glands showed an intense keratin staining. Epithelial lining and or epithelial fragments in cystic walls of mucoceles displayed a strong reaction to keratin proteins too, whereas granulation tissue or connective tissue fibers of the walls were not seen. Foamy macrophages present in cystic cavities signify higher staining with the use of keratin proteins. Mucoceles in oral minor salivary glands are probably caused by ductal obstructions leading to continuous mucous secretion. Keratin proteins were used as an epithelial marker of ductal segments.

Gallium scintigraphy demonstration of an appendiceal mucocele: a proposed mechanism of uptake.

An appendiceal mucocele demonstrated intense early avidity for Ga-67, despite the lack of inflammatory cells to account for the uptake. It is proposed that the acid mucopolysaccharide component of the mucus within the lumen and lining cells accounted for the uptake of the gallium ion, in a similar manner to the uptake of its analogue, the ferric ion, as demonstrated by intense staining of mucus by the colloidal iron technique.

Oral mucoceles: a clinical, histopathological and immunohistochemical study.

The aim of study was to evaluate the clinicopathological features of oral mucoceles and the immunohistochemical expression of cellular and extracellular matrix components in these lesions. One hundred cases of oral mucoceles were examined for clinicopathological features. The expression of mast cell tryptase, CD68, MMP-1 (matrix metalloproteinase-1), MMP-9 (matrix metalloproteinase-9) and CD34 was investigated immunohistochemically in 32 cases. The lesions arose as nodules or blisters of variable color. The mean age was 23.2 years and a higher male frequency was observed. The most common locations were the lower lip (92%), followed by the floor of the mouth (7%), and palate (1%). The lesion size ranged from 0.4 to 3.0cm. Unusual histopathological findings as superficial mucoceles (n=16, 16%), pseudopapillary projections (n=3, 3%), epithelioid histiocytes (n=4, 4%), multinucleated giant cells (n=1, 1%) and myxoglobulosis (n=9, 9%) were also seen. Mast cells and CD68-positive macrophages, MMP-1, MMP-9 and CD34-positive blood vessels were seen in all cases. A significant association was seen between mast cells and MMP-1 (p=0.03) and between macrophages and MMP-1 (p=0.01). This study provided important insight into the demographic and histopathological occurrence of oral mucoceles. The tissue remodeling seen in these lesions mainly involved the migration and interaction of mast cells, macrophages and MMP-1.