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.

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.

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.

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.

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.

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.

Mucocele etmoidal e frontoetmoidal com extensão orbitária: relato de dois casos / Ethoid and front-ethmoid mucocele with orbit extension: report of two cases

As mucoceles dos seios paranasais são lesões císticas de revestimento epitelial com conteúdo mucóide, que apresentam crescimento lento com características expansivas e de reabsorção óssea. Eventualmente, podem comprometer as estruturas nobres adjacentes como aórbita e a cavidade intracraniana.Relato de caso - Caso 1: Paciente masculino, 55 anos, com tumoração na região medial da órbita esquerdacom evolução progressiva que iniciou há 10 meses. Apresentava restrição na elevação do olho em supraversão à direita. Exame tomográfico compatível com mucocele etmoidal. Submetido a etmoidectomia externa, apresentou boa evolução no pós-operatório e permaneceassintomático até o momento. Caso 2: Paciente masculino, 65 anos, com baixaacuidade visual progressiva no olho direito há 5 meses após aparecimento de tumoração indolor na região superiorda órbita direita que evoluía progressivamente há 1 ano. Apresentava acuidade visual de vultos e atrofia de nervo óptico no olho direito. Mobilidade ocular com restrição. Tomografia computadorizada confirmou o diagnóstico de mucocele fronto-etmoidal. Discussão: As mucoceles apresentam um crescimento lento e, por este motivo, em muitos casos, a lesãopode alcançar a órbita antes dos pacientes procurarem auxílio. A complicação mais grave das mucoceles periorbitárias é a perda da visão. Seu crescimento através da órbita pode produzir compressão do globo ocular caucausando lesões no nervo óptico e no pólo posterior. O tratamento das mucoceles é cirúrgico, sendo que as vias de acesso podem ser externa e endonasal.

Paranasal sinus mucoceles are cystic lesions with an epithelial revestment containing mucus, that grow slowlywith expansive characteristics, reabsorbing surrounding bones. Eventually, they can compromise important structures as the orbit and the intracranial cavity. Case report - Case 1: A 55 year-old male came to our clinic complaining of a tumor in nasal left orbit with slow progression for 10 months. Physical examination revealed supraversion of left eye. Computed tomography (CT) imaging showed ethmoid sinus mucocele. The patient underwent external ethmoidectomy with good evolution and remains asymptomatic until now. Case 2: A 65 year-old male, with progressive righteye low vision for 5 months after the appearing of a painless tumor at the superior region of the right orbit thatwas slowly growing for 1 year. Visual acuity of shadows and optic nerve atrophy in the right eye. General limitedeye movement of the right eye. CT confirmed frontal and ethmoid mucocele.Discussion: Mucoceles have slow progression and, for this reason, in many cases, they can reach the orbitbefore patient look for help. The most severe complication of the periorbital mucoceles is blindness. While theygrow inside the orbit, they can compress the eyeball and injure the posterior pole and the optic nerve. The treatmentis based on surgery, that can be performed by external or endonasal access.

Expression of IL-12 and T helper cell 1 cytokines in the fluid of paranasal sinus mucoceles.

OBJECTIVE: The objective of this study was to assess the expression of regulatory cytokines and T helper cell (Th)1/Th2 cytokines in paranasal sinus mucoceles. MATERIALS AND METHODS: Fluid samples of 12 paranasal sinus mucoceles were assessed by enzyme-linked immunosorbent assay for concentrations of regulatory cytokines (interleukin [IL]-10 and IL-12), Th1 cytokines (IL-2 and interferon gamma), and Th2 cytokines (IL-4 and IL-5). RESULTS: IL-12 was detected in all samples, whereas IL-10 was detected in only one case. The concentration of IL-12 tended to correlate with that of interferon gamma and was significantly and positively correlated with that of IL-2. CONCLUSIONS: Th1 cytokines and the Th1 regulatory cytokine IL-12, but not IL-10, potentially play a key role in the pathogenesis of paranasal sinus mucoceles. Together with our recent report showing that lipopolysaccharide is highly detected in mucocele fluid, the data from this study suggest that the Th1 response induced by lipopolysaccharide may affect the immunological inflammation in the epithelium of paranasal sinus mucoceles.

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.

Myxoglobulosis in oral extravasation mucocele: an entity?

AIMS: Myxoglobulosis is a recognized variant of mucocele of the vermiform appendix. Myxoglobulosis has recently been reported in extravasation mucocele of the oral cavity. This morphological study was carried out to identify the prevalence and possible pathogenesis of this unusual feature. MATERIALS AND METHODS: A retrospective review of archival material diagnosed as oral cavity mucocele was undertaken. This covered a period of 32 months and consisted of 76 cases. In accordance with the proposed traumatic origin of extravasation mucocele, histological changes were classified on the basis of mucin extravasation and cyst formation. Globular structures, as described in the reports of myxoglobulosis, were looked for, as were changes that could be ascribed to their formation. CONCLUSIONS: Globular structures were noted in 22 (31%) of 71 cases of extravasation mucocele. They were present within cysts (11 samples) and in extraluminal connective tissue in the rest. They appeared to originate from alterations in connective tissue collagen following mucin extravasation. Their structure is different from that described in appendicial mucocele. When observed, they are best regarded as part of the normal sequence in the formation of oral cavity extravasation mucocele, without attaching special importance to them.

IGF-1 and insulin receptor expression in the minor salivary gland tissues of Sjögren's syndrome and mucoceles--immunohistochemical study.

OBJECTIVE: To investigate the expression of IGF-1 receptors and insulin receptors on the minor salivary gland (MSG) tissues of patients diagnosed with Sjögren's syndrome (SS) and normal salivary gland tissue surrounding mucoceles. SUBJECTS AND METHODS: Five MSG tissue sections from SS and seven from mucocele patients were stained immunohistochemically using antibody to IGF-1 receptor and insulin receptor in a horse radish peroxidase and DAB system. RESULTS: The expression of the insulin receptor was increased in the SS sections compared with controls, while the insulin-like growth factor-1 receptor was more intensely expressed in the controls. CONCLUSION: The presence of differential expression of receptors for IGF and insulin might suggest a possible role of these growth factors in the pathogenesis of SS.

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.

Immunohistochemical detection of insulin-like growth factor-I in the labial salivary glands of patients with Sjögren's syndrome.

OBJECTIVE: The purpose of the present study was to investigate the presence of insulin-like growth factor-I (IGF-I) in the labial salivary glands of patients with Sjögren's syndrome and healthy controls and to determine if there are any differences between these two groups. DESIGN: An immunohistochemical study. SUBJECTS AND METHODS: Twenty-five patients with Sjögren's syndrome, 20 healthy controls and 20 patients with mucoceles of the lip were used in this study. All individuals underwent a systemic evaluation and a lip biopsy. Sections from the lip biopsies were stained with haematoxylin and eosin (H&E). Immunohistochemical staining was also performed using a three-step indirect immunoperoxidase for IGF-I. RESULTS: The light microscopic examination revealed the presence of a mononuclear infiltration in the labial salivary glands of patients with Sjögren's syndrome. Most of the infiltrates were lymphocytes. Immunohistochemically an intense staining result was apparent in the same group. In contrast sections of labial salivary glands of healthy individuals and of patients with mucoceles revealed very weak staining. CONCLUSIONS: The above findings and the fact that both lymphocytic infiltration and IGF-I were predominantly seen in ductal regions, suggest that IGF-I may be a target of autoimmunity in Sjögren's syndrome.