Mucin Staining Research Articles

Gastroprotective role of a flavonoid-rich subfraction from Fridericia chica (Bonpl.) L. G. Lohmann: a medicinal plant used in the Amazon region.

Fridericia chica is an Amazonian plant used to treat stomach disorders. However, the pharmacological activity of flavonoids in the extract has yet to be investigated. Therefore, we considered that a flavonoid-rich F. chica subfraction (FRS) has gastroprotective functions. For this, before the induction of gastric ulcers with ethanol or piroxicam, the rats received vehicle (water), omeprazole (30mg/kg), or FRS (30mg/kg), and the ulcer area was measured macro and microscopically, and the antisecretory action was investigated in pylorus-ligated rats. In addition, the roles of nitric oxide (NO) and nonprotein sulfhydryl compounds (NP-SH) in the gastroprotective effects of FRS were studied. FRS reduced ethanol- and piroxicam-induced ulcerations by 81% and 77%, respectively, as confirmed histologically. Antioxidant effects were observed for FRS through the maintenance of GSH and LPO levels, and the SOD and CAT activity similar to those found in the nonulcerated group. Moreover, FRS avoided the increase in MPO activity and TNF, IL-6, IL-4 and IL-10 levels. Moreover, mucin staining increased in ulcerated rats receiving FRS, and the pharmacological mechanism gastroprotective seems to involve the NO and NP-SH in addition to antisecretory actions. The chemical study by mass spectrometry confirmed the presence of flavonoids in FRS, and molecular docking studies have shown that these compounds interact with cyclooxygenase-1 and NO synthase. Furthermore, there was no indication that FRS had cytotoxic effects. Our results support the popular use of F. chica, and we conclude that the gastroprotection effect promoted by FRS can be attributed to the combined effect of the flavonoids.

Hydroalcoholic extract of Araucaria sp. brown propolis alleviates ulcerative colitis induced by TNBS in rats by reducing inflammatory cell infiltration and oxidative damage.

To investigate the effects of Araucaria sp. brown propolis (ABP) against trinitrobenzenesulfonic acid (TNBS)-induced colitis in rats. Animals received vehicle (1% DMSO, 1 ml/kg) or hydroalcoholic extract of ABP (hydroalcoholic extract of Araucaria sp. brown propolis (HEABP), 30, 100, and 300 mg/kg) orally, or dexamethasone (25 mg/kg, s.c.) for 5 days. On day 4, the animals received intracolonic TNBS (150 mg/kg), on day 6 they were euthanized. The weight of the animals, the macroscopic and microscopic colonic damage, reduced glutathione (GSH) and malondialdehyde (MDA) levels, and the activity of glutathione S-transferase (GST), catalase (CAT), superoxide dismutase (SOD), and myeloperoxidase (MPO) were measured in colon homogenate. The action of HEABP and two isolated compounds in neutrophil migration was recorded. HEABP (100 and 300 mg/kg), but not dexamethasone, decreased colonic lesion, and increased colonic mucin staining. In parallel, HEABP decreased MDA and restored GSH levels and the activity of SOD, CAT, and GST in the colon. A dose-dependent inhibition of MPO activity was observed (LogIC50 = 1.9). Moreover, HEBPA and the junicedric and abietic acids inhibited the neutrophil chemotaxis in vitro and HEBPA reduced neutrophil migration in vivo. HEABP may be promising in the therapies for inflammatory bowel diseases, reducing oxidative and inflammatory damage, especially mediated by neutrophils.

Insect live larvae as a new nutritional model in duck: effects on gut health

BackgroundThis study aimed to evaluate the effects of Hermetia illucens (Black soldier fly-BSF) and Tenebrio molitor (Yellow mealworm-YMW) live larvae as a new nutritional model on duck’s gut health, considering gut histomorphometry, mucin composition, cytokines transcription levels, and microbiota. A total of 126, 3-days-old, females Muscovy ducks were randomly allotted to three dietary treatments (6 replicates/treatment, 7 birds/pen): (i) C: basal diet; (ii) BSF: C + BSF live larvae; (iii) YMW: C + YMW live larvae. BSF and YMW live larvae were administered on top of the basal diet, based on the 5% of the expected daily feed intake. The live weight, average daily gain, average daily feed intake and feed conversion ratio were evaluated for the whole experimental period. On day 52, 12 ducks/treatment (2 birds/replicate) were slaughtered and samples of duodenum, jejunum, ileum, spleen, liver, thymus and bursa of Fabricius were collected for histomorphometry. Mucin composition was evaluated in the small intestine through histochemical staining while jejunal MUC-2 and cytokines transcription levels were evaluated by rt-qPCR. Cecal microbiota was also analyzed by means of 16 S rRNA gene sequencing.ResultsBirds’ growth performance and histomorphometry were not influenced by diet, with a proximo-distal decreasing gradient from duodenum to ileum (p < 0.001), respecting the physiological gut development. Mucin staining intensity and MUC-2 gene expression did not vary among dietary treatments, even though mucin intensity increased from duodenum to ileum, according to normal gut mucus physiology (p < 0.001). Regarding local immune response, IL-6 was higher in YMW group when compared to the other groups (p = 0.009). Insect live larvae did not affect cecal microbiota diversity, but BSF and YMW groups showed a higher presence of Helicobacter, Elusimicrobium, and Succinatimonas and a lower abundance of Coriobacteriaceae and Phascolarctobacterium compared to C birds (p < 0.05).ConclusionsThe use of BSF and YMW live larvae as new nutritional model did not impair gut development and mucin composition of Muscovy ducks, but slightly improved the intestinal immune status and the microbiota composition by enhancing regulatory cytokine IL-6 and by increasing minor Operational Taxonomic Units (OTUs) involved in short-chain fatty acids production.

The study of Mucin stains and Cyclin D1 expression in benign prostatic hyperplasia and prostatic adenocarcinoma (Immunohistochemical study)

The study of Mucin stains and Cyclin D1 expression in benign prostatic hyperplasia and prostatic adenocarcinoma (Immunohistochemical study)

Supported Immunohistochemical Staining of Mucins.

Immunohistochemistry (IHC) staining is the most common method to detect the distribution and localization of biomarkers in different parts of a tissue. Antibodies for tandem repeat peptide of mucins are very popular, but antibodies for glycosylation or others are also used. IHC for mucin is the same protocol as IHC for others. This description includes IHC according to ABC method for processed formalin-fixed paraffin-embedded (FFPE) tissues. Protocol of in situ hybridization is also shown.

Succinylation-Alcian Blue Staining of Mucins on Polyvinylidene Difluoride Membrane.

Mucins are often stained with the basic dye Alcian blue, but mucins with a low acidic glycan content cannot be stained with it. Succinylation-Alcian blue staining is a method that temporarily modifies glycans with succinic acid to visualize mucins with low acidic glycan content. This method can be used to stain mucins on polyvinylidene difluoride (PVDF) membranes separated via supported molecular matrix electrophoresis (SMME) and mucins blotted onto PVDF membranes from gel electrophoreses. The succinyl groups of the modified glycans can be easily and completely removed by releasing O-glycan from the stained mucin bands. Therefore, the glycans can be analyzed using the same methods as those used for mucins with a high acidic glycan content.

The Effect of Autografts from the Inferior and Superior Bulbar Conjunctiva on the Ocular Surface in Primary Pterygium Surgery: A Cytology Study.

This study aimed to evaluate the effect of using an inferior or superior conjunctival autograft in primary pterygium surgery on the postoperative ocular surface. Forty eyes of 40 patients who underwent pterygium surgery with autograft were included in the study. Cytological cell counts were performed on samples taken from the bulbar conjunctiva by impression cytology before and 1 year after the operation. Schirmer 1 test score, lissamine green conjunctival staining score, tear film break-up time (TBUT), and fluorescein corneal staining scores were evaluated. The pain levels of the patients were evaluated with visual analog scale at postoperative 1 day and 1 week. Corneal and conjunctival staining, TBUT, and Schirmer test results demonstrated significant improvement in all patient groups after surgery, but there was no difference between groups (p>0.05). In both preoperative and postoperative impression cytology, the number of goblet cells in the inferior bulbar conjunctiva was higher than in the superior bulbar conjunctiva (p<0.001), while there was no such difference in epithelial cell or mucin staining. There were no significant cytological changes postoperatively in either group (p>0.05). Pterygium surgery with autografting improved tear function tests regardless of graft location. Goblet cell count was higher in the inferior bulbar conjunctiva than in the superior bulbar conjunctiva in both postoperative and preoperative impression cytology. However, there was no significant difference in postoperative epithelial and goblet cell counts or mucin staining between the groups before and after surgery. We think that using the inferior bulbar conjunctiva is an appropriate choice in cases where the superior conjunctiva cannot be used as a graft or when future glaucoma surgery is possible.

Toxicological assessment of E-cigarette flavored E-liquids aerosols using Calu-3 cells: A 3D lung model approach

Scientific progress and ethical considerations are increasingly shifting the toxicological focus from in vivo animal models to in vitro studies utilizing physiologically relevant cell cultures. Consequently, we evaluated and validated a three-dimensional (3D) model of the human lung using Calu-3 cells cultured at an air-liquid interface (ALI) for 28 days. Assessment of seven essential genes of differentiation and transepithelial electrical resistance (TEER) measurements, in conjunction with mucin (MUC5AC) staining, validated the model. We observed a time-dependent increase in TEER, genetic markers of mucus-producing cells (muc5ac, muc5b), basal cells (trp63), ciliated cells (foxj1), and tight junctions (tjp1). A decrease in basal cell marker krt5 levels was observed. Subsequently, we utilized this validated ALI-cultured Calu-3 model to investigate the adversity of the aerosols generated from three flavored electronic cigarette (EC) e-liquids: cinnamon, vanilla tobacco, and hazelnut. These aerosols were compared against traditional cigarette smoke (3R4F) to assess their relative toxicity. The aerosols generated from PG/VG vehicle control, hazelnut and cinnamon e-liquids, but not vanilla tobacco, significantly decreased TEER and increased lactate dehydrogenase (LDH) release compared to the incubator and air-only controls. Compared to 3R4F, there were no significant differences in TEER or LDH with the tested flavored EC aerosols other than vanilla tobacco. This starkly contrasted our expectations, given the common perception of e-liquids as a safer alternative to cigarettes. Our study suggests that these results depend on flavor type. Therefore, we strongly advocate for further research, increased user awareness regarding flavors in ECs, and rigorous regulatory scrutiny to protect public health.

Discriminant Analysis Using Gabor Filter Sets for Lobular Endocervical Glandular Hyperplasia: Numerical Interpretation of Nuclear Atypia by Gabor Filter Features

Introduction: Lobular endocervical glandular hyperplasia (LEGH) is a benign lesion; however, it is considered to be the origin of gastric-type adenocarcinoma in the uterine cervix, and early diagnosis is important. At Shinshu University Hospital, screening of LEGH cells is based on the difference in color tone of cytoplasmic mucin on Papanicolaou staining and detection of gastric mucin using HIK1083-labeled latex agglutination assay. However, it is sometimes difficult to distinguish LEGH cells with subtle nuclear atypia from endocervical (EC) cells. Methods: We calculated the Gabor filter features (mean signal value, standard deviation, skewness, kurtosis) from the nuclei of cytological specimens in EC cells (37 cases) and LEGH cells (33 cases) using microscopic images, and we performed statistical analysis and discriminant analysis by linear support vector machine (LSVM) using these features. A Gabor filter is a linear filter defined as a mathematical representation of the mammalian visual system. Gabor filters with three wavelengths and eight angles were used for analysis. Results: Gabor filter features in EC cells were higher than in LEGH cells, demonstrating that the gradient of LEGH cell nuclei was milder than that of EC cell nuclei. The accuracy calculated using all Gabor filters was 91.0% and the accuracy of four Gabor filters (λ = 2/3π and θ = 0°, 45°, 90°, 135°) was 88.9%. High accuracy with low computation costs was achieved by reducing the number of features used for LSVM. Conclusion: The application of a Gabor filter with convolutional processing resulted in the edges of LEGH cells being slightly rough and thick, whereas those of EC cells were fine and thin. Thus, it is thought that the frequency of abrupt gradients of pixels was higher in EC cells than in LEGH cells, and the gradient of chromatin distribution in LEGH cell nuclei was milder than that in EC cell nuclei. It was possible to evaluate nuclear findings of EC and LEGH cells objectively by quantifying morphological features of nuclei using Gabor filters. It was possible to differentiate EC cells from LEGH cells using LSVM using Gabor filter features.

Gastric amphicrine carcinoma: A histopathological diagnostic conundrum

Amphicrine carcinoma is rarely reported in literature. It is characterised by dual positivity of neuroendocrine markers and mucin stain in the same cells depicting divergent differentiation. Recently published WorldHealth Organisation (WHO) classification of neuroendocrine neoplasm of 2022 differentiates it from adenocarcinoma and mixed neuroendocrine-nonneuroendocrine neoplasms (MiNEN) it shows varied histomorphology and on rare occasions neuroendocrine features may not be evident in poorly differentiated carcinoma. Hence, diagnosis based solely on histomorphology can be misleading. This is a case of amphicrine carcinoma in an elderly female which showed poorly differentiated morphology along with signet ring cells. Same cells demonstrated immunopositivity for synaptophysin, chromogranin A and were also positive for mucin stains (PAS, PAS-D and Alcian blue). This case report, thus, emphasises the importance of neuroendocrine markers as a part of routine immunohistochemistry in poorly differentiated gastric carcinomas to aid diagnosis of amphicrine carcinoma.

Agreement Between Mucicarmine-Stained Bronchial Brushing and Bronchial Biopsy in Subtyping Non-small Cell Lung Carcinoma.

Background Bronchial brushing and biopsy are used for the diagnosis of lung carcinoma as most of these tumors are unresectable. Recently, the subclassification of non-small cell lung carcinoma (NSCLC) into adenocarcinoma (ADC) and squamous cell carcinoma (SCC) has become mandatory due to the emergence of targeted therapies. Due to the inherent limitations of small samples, subcategorization of a tumor is not always possible. Immunohistochemical and mucin stains are being used for this purpose, especially for tumors with poorly differentiated features. In our study, we utilized mucicarmine mucin stain to refine the differentiation of SCC and ADC on bronchial brushing and determine its agreement with bronchial biopsy. This study aimed to measure the degree of agreement between mucicarmine-stained bronchial brushing and bronchial biopsy for subtyping NSCLC into SCC and ADC. Methodology This descriptive, cross-sectional study was conducted in the pathology department of Allama Iqbal Medical College. Samples were collected by the pulmonology department of Jinnah Hospital Lahore. The study was conducted for 10 months from June 2020 to April 2021. A total of 60 cases diagnosed as NSCLC, aged between 35 and 80 years, were included in this study. After cytohistological evaluation of bronchial brushing and bronchial biopsy specimens, the agreement was deduced using kappa statistics. Results The strength of agreement between mucicarmine-stained bronchial brushing and bronchial biopsy for subtyping NSCLC into SCC and ADC was found to be substantial. Conclusions As significant agreement exists between the two modalities, mucicarmine-stained bronchial brushing can be used for a reliable and rapid categorization of NSCLC.

Morphology of the esophagus of ferrets and expression profile of molecular markers

To examine the morphological characteristics and the expression profile of molecular markers of ferret esophagus and assess the feasibility of using ferrets as animal models for studying human esophageal diseases. Frozen sections and paraffin- embedded specimens of the esophageal tissues were obtained from adult ferrets (aged 6 to 8 months) and ferrets aged 1 day, 3 days, 5 days, 1 week and 2 weeks. HE staining and periodic acid-Schiff (PAS) staining were used for morphological analysis of the esophageal submucosal glands (SMGs) of adult ferrets, and the expressions of MUC5B and MUC5AC were tested using Mucin staining; The expressions of cytokeratins (CK4, CK5, CK7, CK8, CK14, CK17, CK18, CK19, and CK20) in adult ferret esophagus were examined using HE staining and immunofluorescence assay. The expressions of LEF1 in the esophageal epithelium and SMGs were detected with immunofluorescence assay. In adult ferrets, the esophageal SMGs were connective tissues below the muscularis mucosa of the esophagus with secretory functions. Cytokeratins were expressed differentially in different esophageal cells: CK4, CK8 and CK20 were expressed mainly in the mucous cells, ductal cells and epithelial cells, respectively, while the mucous cells expressed the largest variety of cytokeratins. Mucin staining showed positive MUC5B and MUC5AC expression in the cytoplasm and lumen of adult ferret esophageal glands. Lectin from DBA, ECL, GSLI, GSL Ⅱ, SBA, Tacalin bioylated, ULEX, WGA, GSL Ⅰ and GSL Ⅱ were expressed on ductal cell membrane, and ECL, PNA and WGA were detected on epithelial cell membrane. Lectin with ConA, PHA-E and PHA-L were expressed on serous cell membrane. Immunofluorescence assay showed that LEF1 in the developing glands were visible from 3 days to 1 week of age and then disappeared as the glands matured. The intensity of LEF1 expression in the esophageal glands differed significantly between ferrets aged 1 to 7 days and those aged two weeks. Ferrets and human share similar esophageal tissue structures and some common molecular markers, suggesting the possibility of using ferrets as animal models of human esophageal diseases.

Identifying the potential origin of mucin in primary cutaneous mucinoses-A retrospective study and analysis using histopathology and multiplex fluorescence staining.

Primary cutaneous mucinoses (PCM) are rare diseases characterized by dermal or follicular mucin deposits. A retrospective study characterizing PCM to compare dermal with follicular mucin to identify its potential origin on a single-cell level. Patients diagnosed with PCM between 2010 and 2020 at our department were included in this study. Biopsy specimens were stained using conventional mucin stains (Alcian blue, PAS) and MUC1 immunohistochemical staining. Multiplex fluorescence staining (MFS) was used to investigate which cells were associated with MUC1 expression in select cases. Thirty-one patients with PCM were included, 14 with follicular mucinosis (FM), 8 with reticular erythematous mucinosis, 2 with scleredema, 6 with pretibial myxedema and one patient with lichen myxedematosus. In all 31 specimens, mucin stained positive for Alcian blue and negative for PAS. In FM, mucin deposition was exclusively found in hair follicles and sebaceous glands. None of the other entities showed mucin deposits in follicular epithelial structures. Using MFS, all cases showed CD4+ and CD8+ T cells, tissue histiocytes, fibroblasts and pan-cytokeratin+ cells. These cells expressed MUC1 at different intensities. MUC1 expression in tissue histiocytes, fibroblasts, CD4+ and CD8+ T cells, and follicular epithelial cells of FM was significantly higher than the same cell types in the dermal mucinoses (p < 0.001). CD8+ T cells were significantly more involved in expression of MUC1 than all other analysed cell types in FM. This finding was also significant in comparison with dermal mucinoses. Various cell types seem to contribute to mucin production in PCM. Using MFS, we showed that CD8+ T cells seem to be more involved in the production of mucin in FM than in dermal mucinoses, which could indicate that mucin in dermal and follicular epithelial mucinoses have different origins.

A199 BIOFILMS IN THE APPENDIX AND OTHER NON-INFLAMED SECTIONS OF THE COLON IN PEDIATRIC PATIENTS WITH INFLAMMATORY BOWEL DISEASES

Abstract Background Biofilms, aggregated bacteria colonizing the extracellular polymeric substances matrix, are associated with the mucosa of inflammatory bowel diseases (IBD) patients with some studies showing a mean density of the mucosal biofilms 2-fold higher in IBD patients than in controls. The appendix, which is a highly immune organ, seems to be involved in IBD pathogenesis. Purpose In this study we aimed to evaluate biofilms in the appendix and other non-inflamed regions of the colon in pediatric IBD patients. We hypothesized that biofilms composed of pathobionts in these sections could drive inflammation in IBD patients. Method Tissues from the appendix, peri-appendicular region, cecum, and ascending colon (ASC), collected from the resected colons of 9 pediatric IBD patients and one pediatric non-IBD patient, preserved in methanol Carnoy’s solution were processed and paraffin embedded. Combined fluorescence in situ hybridization (FISH) for biofilms (probe: EUB338) and immunofluorescence (IF) mucin staining (MUC2) identified biofilms and their location. Biofilm formation capacity of culturable bacteria (identified through 16S DNA Sanger sequencing) from these sections was measured. Interleukin (IL)-8 (pro-inflammatory chemokine) and IL-10 (anti-inflammatory cytokine) expressions were assessed by tissue qPCR. Result(s) FISH demonstrated biofilms in these sections, in close proximity to epithelial cells. We used a biofilm formation assay to assess the ability of the identified bacteria from these sections to form biofilms, illustrating their potential to colonize and evade host defense and potential antibiotics. We found that Enterococcus avium has a significantly higher ability to form biofilms than the negative control. IL-8 and IL-10 both had the highest expression level in the appendix and peri-appendicular region and the lowest in the ASC among all the patients, suggesting an active immune response. Mechanistic experiments are in progress to investigate the type of bacteria involved in the biofilms and their effects on the gut barrier integrity. Conclusion(s) Biofilms adjacent to the epithelium, especially in the appendix and peri-appendix, could interact with or invade epithelial cells. The elevated chemokine transcript level in the appendix could reflect the recruitment of immune cells to this section following bacterial invasion, resulting in the activation of the immune system. Identifying the bacteria involved in the biofilms and clarifying their characteristics will aid us in developing novel microbe-altering treatment strategies or personalized medicine. Disclosure of Interest None Declared

The value of Alcian blue periodic acid Schiff staining and Ki-67 expression in diagnosing gastric reactive epithelial hyperplasia and dysplasia

Objective: To investigate the clinicopathological characteristics of reactive epithelial hyperplasia and dysplasia in the stomach, as well as the clinical value of mucin special staining and proliferating cell nuclear antigen (Ki-67) in distinguishing the two gastric lesions. Methods: The clinical pathological data of 63 patients with gastric reactive epithelial hyperplasia, 54 patients with low-grade dysplasia, and 63 patients with high-grade dysplasia diagnosed from May 2018 to May 2021 in Beijing Chaoyang Hospital, Capital Medical University, Beijing, China were analyzed. Alcian blue periodic acid Schiff (AB-PAS) and Ki-67 staining were performed to examine the mucin staining pattern, number of Ki-67 positive cells, Ki-67 staining patterns in the three groups of lesions, and histopathologic characteristics. Results: The positive rates of AB-PAS in the reactive epithelial hyperplasia and gastric dysplasia groups were 87.3%(55/63) and 10.3%(12/117), respectively. The expression of AB-PAS in the reactive epithelial hyperplasia was gradually increased from the base to the surface of the epithelium. In low-grade dysplasia and high-grade dysplasia, there was no mucin present in the dysplasia epithelium. The difference between the two groups was statistically significant (P<0.01). The positive rate of Ki-67 in the epithelial reactive hyperplasia (>10%) was 81.0% (51/63), and the positive cells were mainly located in the neck and middle parts of the mucosal glands (58/63, 92.1%). In the low-grade dysplasia group, the positive rate of Ki-67 (>10%) was 90.7%(49/54); the positive cells were mainly located in the upper mucosa (33/54, 61.1%), showing a banded distribution pattern; in the high-grade dysplasia group, the positive rate (>10%) was 95.2%(60/63), and the positive cells were mainly located in the whole mucosa (49/63, 77.8%), showing a diffuse/diffuse scattered distribution pattern. The three groups had statistically different rates and distribution patterns of Ki-67 expression (P<0.01). Conclusion: The gastric epithelial reactive hyperplasia and dysplasia can be differentiated using clinicopathological features, AB-PAS staining and Ki-67 expression pattern.

Lower cyst fluid carcinoembryonic antigencutoff is helpful in the differential diagnosis of mucinous versus non-mucinous pancreatic cysts.

Pancreatic cystic lesions (PCLs) are being diagnosed with increased frequency and have varying neoplastic potential. We conducted this multimodal, prospective study to evaluate the role of tumor cytology and molecular markers to differentiate PCL subtypes. Consecutive undiagnosed patients with PCLs (n = 100, mean age: 50.37 years; 41% males) were prospectively studied. Cyst fluid carcinoembryonic antigen (CEA), CA19.9, CA125, CA72.4, and vascular endothelial growth factor-alpha (VEGF-α) levels were measured by quantitative enzyme-linked immunosorbent assay (ELISA) method. Mutational analysis of the KRAS gene (exon 2, Codon 12 and 13) and GNAS gene (Exon 8, Codon 201) were performed by Sanger's sequencing. The mean cyst size was 4.32 ± 2.4 cm. Fluid cytology revealed definitive diagnosis in 21 (22.3%) patients. All malignant PCLs could be identified on cytology whereas 10/14 (71%) non-malignant mucinous PCLs could also be identified on cytology based on mucin staining. Among the tested tumor markers, cyst fluid CEA had the best diagnostic performance for differentiation between mucinous and non-mucinous PCLs (AUC 0.933 [95% CI 0.86-0.91]). At a cyst fluid CEA cutoff level of 45.0 ng/mL, the sensitivity, specificity, positive predictive value, and negative predictive value for differentiation between mucinous and non-mucinous cysts were 88.5%, 96.8%, 92.0%, and 95.3%, respectively (p &lt; 0.05). KRAS and GNAS mutation had no significant diagnostic benefit in comparison to fluid cytology and CEA levels. Fluid CEA at a lower cutoff of 45 ng/mL is the most accurate marker to differentiate between mucinous and non-mucinous PCL. The KRAS and GNAS mutational analysis does not improve upon the diagnostic performance of fluid cytology and tumor markers.

Optimizing Soluble Cues for Salivary Gland Tissue Mimetics Using a Design of Experiments (DoE) Approach.

The development of therapies to prevent or treat salivary gland dysfunction has been limited by a lack of functional in vitro models. Specifically, critical markers of salivary gland secretory phenotype downregulate rapidly ex vivo. Here, we utilize a salivary gland tissue chip model to conduct a design of experiments (DoE) approach to test combinations of seven soluble cues that were previously shown to maintain or improve salivary gland cell function. This approach uses statistical techniques to improve efficiency and accuracy of combinations of factors. The DoE-designed culture conditions improve markers of salivary gland function. Data show that the EGFR inhibitor, EKI-785, maintains relative mRNA expression of Mist1, a key acinar cell transcription factor, while FGF10 and neurturin promote mRNA expression of Aqp5 and Tmem16a, channel proteins involved in secretion. Mist1 mRNA expression correlates with increased secretory function, including calcium signaling and mucin (PAS-AB) staining. Overall, this study demonstrates that media conditions can be efficiently optimized to support secretory function in vitro using a DoE approach.

Role of endoscopic ultrasound and cyst fluid tumor markers in diagnosis of pancreatic cystic lesions.

BACKGROUNDPancreatic cystic lesions (PCLs) are common in clinical practice. The accurate classification and diagnosis of these lesions are crucial to avoid unnecessary treatment of benign lesions and missed opportunities for early treatment of potentially malignant lesions. AIMTo evaluate the role of cyst fluid analysis of different tumor markers such as cancer antigens [e.g., cancer antigen (CA)19-9, CA72-4], carcinoembryonic antigen (CEA), serine protease inhibitor Kazal-type 1 (SPINK1), interleukin 1 beta (IL1-β), vascular endothelial growth factor A (VEGF-A), and prostaglandin E2 (PGE2)], amylase, and mucin stain in diagnosing pancreatic cysts and differentiating malignant from benign lesions. METHODSThis study included 76 patients diagnosed with PCLs using different imaging modalities. All patients underwent endoscopic ultrasound (EUS) and EUS-fine needle aspiration (EUS-FNA) for characterization and sampling of different PCLs. RESULTSThe mean age of studied patients was 47.4 ± 11.4 years, with a slight female predominance (59.2%). Mucin stain showed high statistical significance in predicting malignancy with a sensitivity of 87.1% and specificity of 95.56%. It also showed a positive predictive value and negative predictive value of 93.1% and 91.49%, respectively (P < 0.001). We found that positive mucin stain, cyst fluid glucose, SPINK1, amylase, and CEA levels had high statistical significance (P < 0.0001). In contrast, IL-1β, CA 72-4, VEGF-A, VEGFR2, and PGE2 did not show any statistical significance. Univariate regression analysis for prediction of malignancy in PCLs showed a statistically significant positive correlation with mural nodules, lymph nodes, cyst diameter, mucin stain, and cyst fluid CEA. Meanwhile, logistic multivariable regression analysis proved that mural nodules, mucin stain, and SPINK1 were independent predictors of malignancy in cystic pancreatic lesions. CONCLUSIONEUS examination of cyst morphology with cytopathological analysis and cyst fluid analysis could improve the differentiation between malignant and benign pancreatic cysts. Also, CEA, glucose, and SPINK1 could be used as promising markers to predict malignant pancreatic cysts.

1357-P: Diabetic Hyperglycemia Impairs Intestinal Barrier Function in the Setting of Diet-Induced Obesity

Proper function of the intestinal barrier plays a crucial role to maintain normal health by selectively absorbing nutrients and keeping out pathogens. Increased gut permeability is a feature of several diseases, including metabolic syndrome, but whether it is a cause or a consequence is unknown. We therefore examined the impact on intestinal barrier function of streptozotocin (STZ) -induced diabetes in C57BL/6J mice that were fed either standard chow or high-fat high-sucrose (HFHS) diet for 13wk. Moderate hyperglycemia was induced by low-dose i.p. STZ injection and 12wk later, intestinal tissues were collected and processed for quantification of colonic mucin and tight junction proteins. In both HFHS- and chow-fed mice, STZ treatment resulted in significantly elevated blood glucose levels compared to vehicle-treated controls (HFHS blood glucose 249.1 mg/dL vs. 157.5 mg/dL, p=0.0008; chow 330.2 mg/dL vs. 139.7mg/dL, p=0.0005) . In HFHS-fed mice, STZ hyperglycemia was associated with reductions of both the colonic mucin layer, as measured by Alcian Blue (AB) staining (percent area 52.6 vs. 62.4, p&amp;lt;0.0001) , and the tight junction protein E-cadherin (percent area 34.7 vs. 55.9, p=0.0029) . In non-obese chow-fed mice, however, STZ-induced hyperglycemia had no significant impact on AB mucin staining or E-cadherin protein abundance. Similarly, levels of colonic mucins and E-cadherin were inversely correlated with blood glucose levels in HFHS-fed mice (AB R2=0.43, p=0.0116; E-cadherin R2=0.30, p=0.0439) , but not in chow-fed mice. We conclude that while the combination of hyperglycemia and diet-induced-obesity is sufficient to impair intestinal barrier function, neither obesity nor diabetes in isolation is sufficient to induce this effect. Our additional finding that the magnitude of colonic dysfunction is correlated with the degree of hyperglycemia implicates excess glucose as a potential driver of obesity-associated impairment of intestinal barrier function. Disclosure K.L.Francis: None. K.M.Alonge: None. S.J.W.Hu: None. B.N.Phan: None. T.P.Doan: None. C.A.Krutzsch: None. Z.Maat: None. J.M.Scarlett: None. M.W.Schwartz: None. Funding National Institutes of Health T32 DK007742

Azithromycin inhibits mucin secretion, mucous metaplasia, airway inflammation, and airways hyperresponsiveness in mice exposed to house dust mite extract.

Excessive production, secretion, and retention of abnormal mucus is a pathological feature of many obstructive airways diseases including asthma. Azithromycin is an antibiotic that also possesses immunomodulatory and mucoregulatory activities, which may contribute to the clinical effectiveness of azithromycin in asthma. The current study investigated these nonantibiotic activities of azithromycin in mice exposed daily to intranasal house dust mite (HDM) extract for 10 days. HDM-exposed mice exhibited airways hyperresponsiveness to aerosolized methacholine, a pronounced mixed eosinophilic and neutrophilic inflammatory response, increased airway smooth muscle (ASM) thickness, and elevated levels of epithelial mucin staining. Azithromycin (50 mg/kg sc, 2 h before each HDM exposure) attenuated HDM-induced airways hyperresponsiveness to methacholine, airways inflammation (bronchoalveolar lavage eosinophil and neutrophils numbers, and IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, and RANTES levels), and epithelial mucin staining (mucous metaplasia) by at least 50% (compared with HDM-exposed mice, P < 0.05). Isolated tracheal segments of HDM-exposed mice secreted Muc5ac and Muc5b (above baseline levels) in response to exogenous ATP. Moreover, ATP-induced secretion of mucins was attenuated in segments obtained from azithromycin-treated, HDM-exposed mice (P < 0.05). In additional ex vivo studies, ATP-induced secretion of Muc5ac (but not muc5b) from HDM-exposed tracheal segments was inhibited by in vitro exposure to azithromycin. In vitro azithromycin also inhibited ATP-induced secretion of Muc5ac and Muc5b in tracheal segments from IL-13-exposed mice. In summary, azithromycin inhibited ATP-induced mucin secretion and airways inflammation in HDM-exposed mice, both of which are likely to contribute to suppression of airways hyperresponsiveness.