Preparation of Soluble Mucin Solutions from the Salivary Glands.

Studying salivary gland mucins is important for elucidating the pathogenesis of salivary gland diseases, including tumors and xerostomia, and developing diagnostic methods for them. Classic methods for isolating mucins from salivary glands require sacrificing several animals to obtain sufficient quantities of mucin and are time-consuming. Supported molecular matrix electrophoresis (SMME) was used to characterize mucins and their glycans. With this method, mucins can be analyzed within 2 days using less than 100 mg of tissue and without using expensive equipment, such as an ultracentrifuge. This chapter describes a method for preparing mucin solutions for SMME analysis of salivary gland mucins.

Comparison of proteins with anti-influenza virus effects in parotid and submandibular-sublingual saliva in humans.

BACKGROUND: Saliva possesses antiviral activity, with submandibular-sublingual (SMSL) saliva having higher antiviral activity than parotid saliva. Various salivary proteins have inactivating effects on influenza A virus (IAV), but the detailed relationship between antiviral proteins and salivary anti-IAV activities in the parotid and SMSL glands is unknown. Here, to identify salivary proteins with anti-IAV activity, salivary proteins from parotid and SMSL glands were identified, quantified, and compared using liquid chromatography-mass spectrometry. METHODS: Twelve healthy male volunteers participated in the study. Parotid and SMSL saliva was collected by suction and collection devices. We assessed anti-IAV activities, protein concentrations, and protein-bound sialic acid concentrations in parotid and SMSL saliva. RESULTS: SMSL had significantly higher anti-IAV activity than parotid saliva. SMSL also had higher concentrations of glycoproteins, such as mucin 5B and mucin 7, protein-bound sialic acid, cystatins, and lysozyme C, compared with parotid saliva. Salivary mucin 5B and mucin 7 concentrations significantly positively correlated with the salivary protein-bound sialic acid concentration. Salivary anti-IAV activity significantly positively correlated with protein-bound sialic acid, mucin 5B, mucin 7, cystatin-C, -S, and -SN concentrations. CONCLUSION: Salivary mucins, cystatins, and lysozyme C contribute to the high anti-IAV activity of SMSL saliva.

Conjoint analysis of OPRPN and SMR3A protein expression as potential predictive biomarkers for head and neck squamous cell carcinoma after radiotherapy.

Increased submaxillary gland androgen­regulated protein 3A (SMR3A) expression was previously shown to serve as an independent risk factor for oropharyngeal squamous cell carcinoma (OPSCC) and as a surrogate biomarker for active estrogen receptor 2 signaling in radioresistant tumor cells. In the present study, it was aimed to unravel the expression and clinical significance of another member of the opiorphin family, opiorphin prepropeptide (OPRPN), in the radiotherapy for head and neck squamous cell carcinoma (HNSCC). Expression of SMR3A and OPRPN were analyzed for the prior and post fractionated irradiation (4x2 Gy) by double immunofluorescence staining in established HNSCC cell lines as well as by immunohistochemical (IHC) staining in ex vivo tumor tissues. Next, in a retrospective experimental cohort study, primary tumor samples from OPSCC patients (n=96), who received definitive surgery and adjuvant radiotherapy were reviewed, and expression levels of OPRPN protein were detected by IHC. Immunoreactivity scores (IRS) were associated with pathological and clinical risk factors by Chi­square analysis. Survival analysis was performed by using the Kaplan­Meier plot, log­rank test and Cox regression analysis. The expression levels of OPRPN and SMR3A protein were both induced by fractionated irradiation in vitro and ex vivo. In primary tumor samples, IRS of OPRPN was significantly higher than scores of SMR3A expression and positively correlated with expression patterns of SMR3A. SMR3A was confirmed to serve as an unfavorable factor, while OPRPN protein had no significant association with the clinical outcome of patients with OPSCC. A combinational analysis revealed that the subgroup with SMR3AhighOPRPNlow staining pattern had the worst clinical outcome among the various subgroups. Multivariate Cox regression analyses indicated that high expression of SMR3A serves as an independent unfavorable biomarker, while increased expression of OPRPN appears to exert protective function. In summary, the present study indicated that SMR3A and OPRPN serve as potential prognostic markers for HNSCC after radiotherapy.

The effect of long-term alcohol intoxication on the morphological structures and enzymatic activity of rat salivary glands.

BACKGROUND: To study changes in the morphological structures and enzymatic activity of the submandibular salivary gland (SMG) and parotid salivary gland (PG) in rats after prolonged alcohol intoxication. METHODS: Sexually mature male Wistar rats consumed 20% ethanol (6.9 g/kg/day) for 180 consecutive days. The PG and SMG were collected for morphometric and histochemical analyses (nonparametric Mann-Whitney U test, p < 0.05). RESULTS: After exposure to ethanol for 180 days, the PG showed a change in the shape of the acini and the secretory cells that formed them, uneven expansion of the interlobular excretory ducts, and moderate fatty infiltration in the stroma. After exposure to ethanol for 180 days, the SMG showed fatty infiltration and stromal edema, and changes in acinar cells, intercalated ducts, and striated ducts. There was a significant decrease in the relative and absolute weight of the SMG. The number of mast cells in the PG and SMG and their degranulation index increased 2-fold after exposure to ethanol. All mast cells were highly active. After ethanol exposure, the activity of alkaline phosphatase increased significantly in the myoepithelial cells of the SMG and PG; the activity of NADPH oxidase increased only in the acini SMG, and the activity of succinate dehydrogenase remained at the control level in the acini of both glands. In the ducts of these glands, the activity of other enzymes did not change. CONCLUSIONS: Changes in the morphological structures, morphometric parameters, and enzymatic activity of the rat salivary glands after 180 days of ethanol intoxication are shown for the first time. The most pronounced changes were found in the SMG.

Effect of High Fat and Fructo-Oligosaccharide Consumption on Immunoglobulin A in Saliva and Salivary Glands in Rats.

Consumption of indigestible dietary fiber increases immunoglobulin A (IgA) levels in saliva. The purpose of this study is to clarify the synergistic effect of the intake of a high amount of fats and indigestible dietary fiber on IgA levels in saliva and submandibular glands (SMG). Seven-week-old Wistar rats were fed a low-fat (60 g/kg) fiberless diet, low-fat fructo-oligosaccharide (FOS, 30 g/kg) diet, high-fat (220 g/kg) fiberless diet, or high-fat FOS diet for 70 days. The IgA flow rate of saliva (IgA FR-saliva) was higher in the low-fat FOS group than in the other groups (p < 0.05). Furthermore, the concentration of tyrosine hydroxylase (a marker of sympathetic nerve activation) in the SMG was higher in the low-fat FOS group (p < 0.05) and positively correlated with the IgA FR-saliva (rs = 0.68. p < 0.0001. n = 32) in comparison to that in the other groups. These findings suggest that during low-fat FOS intake, salivary IgA levels may increase through sympathetic nerve activation.

Improved online LC-MS/MS identification of O-glycosites by EThcD fragmentation, chemoenzymatic reaction, and SPE enrichment.

O-glycosylation is a highly diverse and complex form of protein post-translational modification. Mucin-type O-glycosylation is initiated by the transfer of N-acetyl-galactosamine (GalNAc) to the hydroxyl group of serine, threonine and tyrosine residues through catalysis by a family of glycosyltransferases, the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (E.C. 2.4.1.41) that are conserved across metazoans. In the last decade, structural characterization of glycosylation has substantially advanced due to the development of analytical methods and advances in mass spectrometry. However, O-glycosite mapping remains challenging since mucin-type O-glycans are densely packed, often protecting proteins from cleavage by proteases. Adding to the complexity is the fact that a given glycosite can be modified by different glycans, resulting in an array of glycoforms rising from one glycosite. In this study, we investigated conditions of solid phase extraction (SPE) enrichment, protease digestion, and Electron-transfer/Higher Energy Collision Dissociation (EThcD) fragmentation to optimize identification of O-glycosites in densely glycosylated proteins. Our results revealed that anion-exchange stationary phase is sufficient for glycopeptide enrichment; however, the use of a hydrophobic-containing sorbent was detrimental to the binding of polar-hydrophilic glycopeptides. Different proteases can be employed for enhancing coverage of O-glycosites, while derivatization of negatively charged amino acids or sialic acids would enhance the identification of a short O-glycopeptides. Using a longer than normal electron transfer dissociation (ETD) reaction time, we obtained enhanced coverage of peptide bonds that facilitated the localization of O-glycosites. O-glycosite mapping strategy via proteases, cut-off filtration and solid-phase chemoenzymatic processing. Glycopeptides are enriched by SPE column, followed by release of N-glycans, collection of higher MW O-glycopeptides via MW cut-off filter, O-glycopeptide release via O-protease, and finally detected by LC-MS/MS using EThcD.

Swelling of mucoadhesive electrospun chitosan/polyethylene oxide nanofibers facilitates adhesion to the sublingual mucosa.

Mucoadhesive chitosan-based electrospun nanofibers are promising candidates for overcoming challenges associated with sublingual drug delivery, yet studies focusing on evaluating the mucoadhesive properties of nanofibers for sublingual administration are limited. The aim was to elucidate the mucoadhesive properties of chitosan/polyethylene oxide (PEO) nanofibers focusing on how the degree of deacetylation (DDA, 53-96 %) of chitosan influenced their morphological and mucoadhesive properties. The mechanism of mucoadhesion was explained by the intermolecular interactions of chitosan with mucin from bovine submaxillary glands using quartz-crystal microbalance with dissipation monitoring and by adhesion of the nanofibers to ex vivo porcine sublingual mucosa. An increase in chitosan DDA improved the morphological stability of the nanofibers in water, but did not contribute to altered mucoadhesive properties. This study demonstrates excellent mucoadhesive properties of chitosan/PEO nanofibers and shows that the strong mucoadhesiveness of the nanofibers is attributed to their swelling ability.

Zn2+ stimulates salivary secretions via metabotropic zinc receptor ZnR/GPR39 in human salivary gland cells.

Zn2+ is a divalent cation that is essential for many biological activities, as it influences many ion channels and enzymatic activities. Zn2+ can evoke G-protein-coupled receptor signaling via activation of the metabotropic zinc receptor ZnR/GPR39. In spite of evidence suggesting the presence of ZnR/GPR39 in salivary gland cells, there has been no evidence of ZnR/GPR39-mediated modulation of salivary gland function. Here we characterized the role of ZnR/GPR39 in human submandibular gland cells. A 0.25% ZnCl2 solution evoked secretion of unstimulated and stimulated whole saliva in humans. We found that ZnR/GPR39 is expressed in human submandibular glands and HSG cells. Zn2+ increased cytosolic Ca2+ concentration ([Ca2+]i) in a concentration-dependent manner. Muscarinic antagonist had no effect on Zn2+-induced [Ca2+]i increase, which was completely blocked by the phospholipase C-ß inhibitor. As with muscarinic agonist, Zn2+ also induced the translocation of aquaporin-5 (AQP-5) to the plasma membrane, which was drastically decreased in ZnR/GPR39-knockdown cells. These data suggest that the metabotropic Zn2+ receptor ZnR/GPR39 can modulate salivary secretion in human submandibular gland cells independent of muscarinic or histamine receptor signaling.

Larrea divaricata Cav. aqueous extract and nordihydroguariaretic acid modulate oxidative stress in submandibular glands of diabetic rats: a buccal protective in diabetes.

BACKGROUND: Oxidative stress is an imbalance between the levels of reactive oxygen species (ROS), reactive nitrogen species (RNS) and endogenous antioxidants. The aetiology and pathogenesis of several oral diseases are attributed to this process. The antioxidant enzymes secreted in the saliva by submandibular glands maintain oral health through the scavenging of ROS. The objective of this work was to study the capacity of an aqueous extract of L. divaricata (AE), and its majority compound, nordihydroguariaretic acid (NDGA), to modulate the pro-oxidant/antioxidant status in submandibular glands in a model of oxidative stress induced by streptozotocin (STZ) in rats. METHODS: To induce oxidative stress with STZ, a group of animals was treated i.p. with 1 X PBS (control group) and other group was injected i.p. once with STZ (60 mg/kg). Ten days after the treatment, blood samples were taken from the tail vain to determine the glucose levels. Animals with glucose values ≥300 mg/ml were selected. The submandibular glands of control and STZ treated animals were incubated with either the AE (500 µg/ml) or with NDGA (1.5 µg/ml), and the content of malondialdehyde (MDA), protein carbonyl groups, ROS and RNS, and the activity and expression of peroxidase (Px), superoxide dismutase (SOD) and inducible nitric oxide synthase (iNOS) were assayed. RESULTS: AE decreased the levels of MDA (##P < 0.01) and protein carbonyl groups (#P < 0.05), and modulated the levels of ROS such as hydrogen peroxide (H2O2)(##P < 0.01), superoxide anion (O2.-) (#P < 0.05) and nitric oxide (NO) (#P < 0.05) in relation to the modulation of Px and iNOS expression. NDGA was found to be involved in these effects. CONCLUSIONS: The antioxidant activity of the AE in the submandibular glands would allow the maintenance of the antioxidant pool to prevent oral oxidative diseases.

Improved and semi-automated reductive ß-elimination workflow for higher throughput protein O-glycosylation analysis.

Protein O-glycosylation has shown to be critical for a wide range of biological processes, resulting in an increased interest in studying the alterations in O-glycosylation patterns of biological samples as disease biomarkers as well as for patient stratification and personalized medicine. Given the complexity of O-glycans, often a large number of samples have to be analysed in order to obtain conclusive results. However, most of the O-glycan analysis work done so far has been performed using glycoanalytical technologies that would not be suitable for the analysis of large sample sets, mainly due to limitations in sample throughput and affordability of the methods. Here we report a largely automated system for O-glycan analysis. We adapted reductive ß-elimination release of O-glycans to a 96-well plate system and transferred the protocol onto a liquid handling robot. The workflow includes O-glycan release, purification and derivatization through permethylation followed by MALDI-TOF-MS. The method has been validated according to the ICH Q2 (R1) guidelines for the validation of analytical procedures. The semi-automated reductive ß-elimination system enabled for the characterization and relative quantitation of O-glycans from commercially available standards. Results of the semi-automated method were in good agreement with the conventional manual in-solution method while even outperforming it in terms of repeatability. Release of O-glycans for 96 samples was achieved within 2.5 hours, and the automated data acquisition on MALDI-TOF-MS took less than 1 minute per sample. This largely automated workflow for O-glycosylation analysis showed to produce rapid, accurate and reliable data, and has the potential to be applied for O-glycan characterization of biological samples, biopharmaceuticals as well as for biomarker discovery.

Feasibility and Safety of Multicenter Tissue and Biofluid Sampling for α-Synuclein in Parkinson's Disease: The Systemic Synuclein Sampling Study (S4).

BACKGROUND: α-synuclein is a lead Parkinson's disease (PD) biomarker. There are conflicting reports regarding accuracy of α-synuclein in different tissues and biofluids as a PD biomarker, and the within-subject anatomical distribution of α-synuclein is not well described. The Systemic Synuclein Sampling Study (S4) aims to address these gaps in knowledge. The S4 is a multicenter, cross-sectional, observational study evaluating α-synuclein in multiple tissues and biofluids in PD and healthy controls (HC). OBJECTIVE: To describe the baseline characteristics of the S4 cohort and safety and feasibility of this study. METHODS: Participants underwent motor and non-motor clinical assessments, dopamine transporter SPECT, biofluid collection (cerebrospinal fluid, saliva, and blood), and tissue biopsies (skin, sigmoid colon, and submandibular gland). Biopsy adequacy was determined based on presence of adequate target tissue. Tissue sections were stained with the 5C12 monoclonal antibody against unmodified α-synuclein. All specimens were acquired and processed in a standardized manner. Adverse events were systematically recorded. RESULTS: The final cohort consists of 82 participants (61 PD, 21 HC). In 68 subjects (83%), all types of specimens were obtained but only 50 (61%) of subjects had all specimens both collected and evaluable for α-synuclein. Mild adverse events were common, especially for submandibular gland biopsy, but only 1 severe adverse event occurred. CONCLUSION: Multicenter tissue and biofluid sampling for α-synuclein is feasible and generally safe. S4 will inform understanding of the concurrent distribution of α-synuclein pathology and biomarkers in biofluids and peripheral nervous system in PD.

Genetic and scRNA-seq Analysis Reveals Distinct Cell Populations that Contribute to Salivary Gland Development and Maintenance.

Stem and progenitor cells of the submandibular salivary gland (SMG) give rise to, maintain, and regenerate the multiple lineages of mature epithelial cells including those belonging to the ductal, acinar, basal and myoepithelial subtypes. Here we have exploited single cell RNA-sequencing and in vivo genetic lineage tracing technologies to generate a detailed map of the cell fate trajectories and branch points of the basal and myoepithelial cell populations of the mouse SMG during embryonic development and in adults. Our studies show that the transcription factor p63 and alpha-smooth muscle actin (SMA) serve as faithful markers of the basal and myoepithelial cell lineages, respectively and that both cell types are endowed with progenitor cell properties. However, p63+ basal and SMA+ myoepithelial cells exhibit distinct cell fates by virtue of maintaining different cellular lineages during morphogenesis and in adults. Collectively, our results reveal the dynamic and complex nature of the diverse SMG cell populations and highlight the distinct differentiation potential of the p63 and SMA expressing subtypes in the stem and progenitor cell hierarchy. Long term these findings have profound implications towards a better understanding of the molecular mechanisms that dictate lineage commitment and differentiation programs during development and adult gland maintenance.

Differential immunohistochemical expression of type I collagen and matrix metalloproteinase 2 among major salivary glands of young and geriatric mice.

OBJECTIVE: This study aimed to demonstrate the immunohistochemical changes associated with MMP-2 and type 1 collagen separately for the first time in the major salivary glands (the parotid, submaxillary, and sublingual glands) that occur with aging in mice. MATERIAL AND METHODS: Fourteen Balb/c white mice (50-80 g) were used in this study. The animals were divided into two equal groups. Group I consisted of young animals (2-month-old) (n=7) and Group II consisted of older animals (18-month-old) (n=7). After routine histological follow-ups, Hematoxylin-eosin (H&E), Masson's Trichrome staining and immunohistochemical staining was performed for type I collagen and MMP-2. RESULTS: We observed that there were age-related decreases in the number of acinar cells, increase in eosinophilic zymogen granules in cells, collagen accumulation in fibrotic areas and dilatation in interlobular ducts. Also, while type I collagen and MMP-2 immunoreactivity were moderate in the salivary glands of the young mice, they were high in the salivary glands of the old mice (p=0.001). In the H-score assessment, MMP-2 immunoreactivity was lower at a significant level in young mice than in old mice (p=0.001). CONCLUSIONS: This study showed that anatomical, physiological and morphological abnormalities occur in all three major salivary glands as a natural consequence of aging.

Exploring gold nanoparticles interaction with mucins: A spectroscopic-based study.

The interaction between two mucin types (mucin from porcine stomach - PGM and mucin from bovine submaxillary glands - BSM) and gold nanoparticles (GNPs) of various size (5, 20 and 40 nm) and functionalization (with cysteamine or thioglycolic acid) was studied under physiological conditions, in order to investigate the affinity of the nanoparticles to the proteins. Different methods are employed to monitor the interactions: UV-vis and fluorescence spectroscopy, fluorescence lifetime, circular dichroism and transmission electron microscopy. These studies have shown the formation of a complex between GNPs and both PGM and BSM. This aspect could be of great importance for the use of gold nanoparticles for biomedical purposes in those diseases where qualitative and quantitative mucin anomalies play an essential role in mucus composition and rheology.

Evaluating the Role of Drone-Produced Chemical Signals in Mediating Social Interactions in Honey Bees (Apis mellifera).

Pheromones play a critical role in shaping societies of social insects, including honey bees, Apis mellifera. While diverse functions have been ascribed to queen- and worker-produced compounds, few studies have explored the identity and function of male-produced (drone) compounds. However, several lines of evidence suggest that drones engage in a variety of social interactions inside and outside of the colony. Here we elucidate the chemical composition of extracts of the drone mandibular gland, and test the hypothesis that compounds produced in these glands, or a synthetic blend consisting of the six main compounds, mediate drone social interactions in and out of the colony. Drone mandibular glands primarily produce a blend of saturated, unsaturated and methyl branched fatty acids ranging in chain length from nonanoic to docosanoic acids, and both gland extracts and synthetic blends of these chemicals serve to attract drones outside of the hive, but do not attract workers inside the hive. These studies shed light on the role drones and drone-produced chemicals have on mediating social interactions with other drones and highlight their potential importance in communicating with other castes.

Differential immunohistochemical expression of type I collagen and matrix metalloproteinase 2 among major salivary glands of young and geriatric mice

Abstract Objective This study aimed to demonstrate the immunohistochemical changes associated with MMP-2 and type 1 collagen separately for the first time in the major salivary glands (the parotid, submaxillary, and sublingual glands) that occur with aging in mice. Material and Methods Fourteen Balb/c white mice (50-80 g) were used in this study. The animals were divided into two equal groups. Group I consisted of young animals (2-month-old) (n=7) and Group II consisted of older animals (18-month-old) (n=7). After routine histological follow-ups, Hematoxylin-eosin (H&E), Masson's Trichrome staining and immunohistochemical staining was performed for type I collagen and MMP-2. Results We observed that there were age-related decreases in the number of acinar cells, increase in eosinophilic zymogen granules in cells, collagen accumulation in fibrotic areas and dilatation in interlobular ducts. Also, while type I collagen and MMP-2 immunoreactivity were moderate in the salivary glands of the young mice, they were high in the salivary glands of the old mice (p=0.001). In the H-score assessment, MMP-2 immunoreactivity was lower at a significant level in young mice than in old mice (p=0.001). Conclusions This study showed that anatomical, physiological and morphological abnormalities occur in all three major salivary glands as a natural consequence of aging.

Capillary isoelectric focusing with whole column imaging detection (iCIEF): A new approach to the characterization and quantification of salivary α-amylase.

Saliva is an easily collected biological fluid with potentially important diagnostic value. While gel electrophoresis is generally used for salivary analysis, we employed the capillary isoelectric focusing technique to allow for a rapid, automated mode of electrophoresis. Capillary isoelectric focusing coupled with UV whole column imaging detection (iCIEF) was used to develop a robust protocol to characterize salivary α-amylase collected from various glands. Notably, three sample preparation methods were examined: ultrafiltration, gel-filtration, and starch affinity interaction with salivary amylase. Salivary α-amylase separated into two major peaks before sample treatment; while both filtration methods and starch affinity interaction of salivary amylase enhanced the resolution of isozymes, desalting with gel-filtration displayed the best recovery and the highest resolution of isozymes. Good agreement existed between the observed isoelectric points and the values reported in the literature. In addition, a high level of precision was apparent, and the relative standard deviation for replicates was less than 0.5% for pIs (peak positions) and below 10% for peak area. Furthermore, saliva secreted from the parotid gland proved to have a higher amylase content compared to either secretions from the submandibular/sublingual complex, or whole saliva, as well as amylase enhancement under stimulation. The results suggest that the iCIEF technique can be used to accurately resolve and quantitate amylase isozymes in a rapid and automated fashion, and that gel-filtration should be applied to saliva samples beforehand to allow for optimal purification and characterization.

Immunolocalization of the TGFB1 system in submandibular gland fibrosis after experimental periodontitis in rats.

Saliva is the first barrier to entry of bacteria and viruses into the body. The submandibular glands (SMG) contribute to the maintenance of oral health and regulation of immune/ inflam matory responses. Previous studies suggest that transforming growth factor beta 1 (TGFB1) may contribute to salivary gland fibrosis but the expression of the TGFB1 system in the SMG has not been elucidated. Thus, the aim of this study was to analyze in rat SMG the immunolocalization of TGFB1 and its specific receptors ALK5 (profibrotic) and ALK1 (proproliferative) and the coreceptor endoglin (EDG) in a bilateral experimental periodontitis (EP) model (cotton thread ligature around the neck of the first lower molars) for 1 and 6 weeks. Fixed SMG were embedded in paraffin and serially cut for routine hematoxylin-eosin staining for histological analysis or immunohistochemical techniques by diaminobenzidine detection. SMG histology from animals with EP showed timedependent structural changes involving marked reduction in the height of the contoured ducts, cell destruction, loss of secretory granules, periductal congestion and excess connective tissue surrounding these ducts indicative of a fibrotic process, compared to control SMG. TGFB1, ALK5 and ALK1 receptors and the coreceptor EDG were mainly immunolocalized in ductal cells and in the fibrotic areas in EP groups. The expression of the profibrotic ALK5 receptor was increased in areas of fibrosis in SMG of animals with EP. In SMG of rats with EP, the localization of the TGFB1 specific receptors in the ducts and cells from fibrotic areas, due to the expression of TGFB1 in the surrounding areas, might indicate paracrine and autocrine actions exerted by TGFB1 via its specific receptors. The results of this study suggest that TGFB1 promotes fibrosis, inducing cell proliferation via ALK1 and EDG receptors and stimulates fibrosis relatedprocesses via ALK5 receptor, which could lead to abnormal secretor activity of the SMG during periodontal disease.

La saliva es la primera barrera para la entrada de bacterias y virus en el cuerpo. Las glándulas submandibulares (GSM) contribuyen al mantenimiento de la salud oral y a la regulación de las respuestas inmunoinflamatorias. Estudios previos sugieren que el factor de crecimiento transformante beta 1 (TGFB1) puede contribuir a la fibrosis de las glándulas salivales, pero la expresión y localización del sistema TGFB1 en las GSM no ha sido dilucidada. El objetivo del presente trabajo fue analizar por inmunohistoquímica en las GSM de ratas la expresión de TGFB1 y sus receptores específicos ALK5 (profibrótico) y ALK1 (proproliferativo) y el coreceptor endoglina (EDG) en un modelo de periodontitis bilateral experimental (PE) (hilo de algodón alrededor del cuello de los primeros molares inferiores) durante 1 y 6 semanas. Las GSM fueron fijadas y embebidas en parafina para realizar cortes seriados los cuales se tiñeron con hematoxilinaeosina para analizar la histología o se procesaron para realizar la técnica de inmunohistoquímica mediante detección con diaminobenzidine. La histología de las GSM de animales con PE reveló cambios estructurales tiempo dependientes, con una marcada reducción de la altura de los conductos, destrucción celular, pérdida de gránulos secretores, congestión periductal y exceso de tejido conectivo que rodea los conductos, indicando un proceso de fibrosis respecto de las GSM de animales control. TGFB1, ALK5 y ALK1 y el coreceptor EDG fueron principalmente inmunolocalizados en las células que forman los ductos y en las áreas de fibrosis en los grupos con PE. La expresión del receptor profibrótico ALK5 se incrementó en las áreas de fibrosis en GSM de animales con PE. En GSM de ratas con PE, la localización de los receptores específicos de TGFB1 en las células de los conductos y áreas de fibrosis, junto con la expresión de TGFB1 en las áreas circundantes, podría indicar acciones paracrinas y autocrinas ejercidas por TGFB1 a través de sus receptores específicos. Los resultados de este estudio sugieren que TGFB1 podría inducir un proceso de fibrosis promoviendo la proliferación celular a través de los receptores ALK1 y EDG, y favoreciendo procesos relacionados con la fibrosis a través de su receptor ALK5, lo que conduciría a una actividad secretora anormal de la GSM durante la enfermedad periodontal.

Human postmortem lacrimal and submandibular glands stored in RNAlater are suitable for molecular, biochemical, and cell biological studies.

PURPOSE: Gene expression and protein analysis studies require high-quality human tissue which is a challenge and difficult to obtain through live human biopsies. Human postmortem lacrimal gland (LG) and submandibular gland (SMG) tissues have the potential to provide an invaluable source for studying the mechanisms involved in LG and SMG dysfunction. Therefore, we aimed to test the suitability of post-mortem LG and SMG for molecular, biochemical, and cell biological studies. METHODS: LG and SMG tissue from healthy donors was collected and immediately placed in RNAlater solution and then shipped overnight at 4 °C. After receipt, each gland was divided into three pieces for RNA, protein, and histological analysis, respectively. Total RNA isolated from each LG and SMG was analyzed for RNA integrity using an Agilent Bioanalyzer and reverse transcription-PCR (RT-PCR). For histology, tissues were embedded in paraffin and stained with hematoxylin and eosin. For protein analysis, lysates were prepared and processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. RESULTS: When the LG and SMG samples were preserved in RNAlater, the RNA integrity number (RIN) values from the LG and SMG were >7.0 from all three donors, while the RNAs from tissue not preserved in RNAlater were of poorer quality. The gene and/or protein expression of E-cadherin, aquaporin 5, alpha-smooth muscle actin (α-SMA), ß-actin, and GAPDH was preserved in all samples. In addition, histological analyses showed normal tubuloacinar structures of all glands with serous and mucous producing acini within lobules interspersed with adipose fat. CONCLUSIONS: In this study, we determined that RNA, protein, and histological sections obtained from postmortem human LG and SMG tissue preserved in RNAlater were of high quality. This would provide a viable source of human LG and SMG tissue suitable for studies of diseases that affect these glands, such as Sjögren's syndrome.

Structural and Mechanical Properties of Thin Films of Bovine Submaxillary Mucin versus Porcine Gastric Mucin on a Hydrophobic Surface in Aqueous Solutions.

The structural and mechanical properties of thin films generated from two types of mucins, namely, bovine submaxillary mucin (BSM) and porcine gastric mucin (PGM) in aqueous environment were investigated with several bulk and surface analytical techniques. Both mucins generated hydrated films on hydrophobic polydimethylsiloxane (PDMS) surfaces from spontaneous adsorption arising from their amphiphilic characteristic. However, BSM formed more elastic films than PGM at neutral pH condition. This structural difference was manifested from the initial film formation processes to the responses to shear stresses applied to the films. Acidification of environmental pH led to strengthening the elastic character of BSM films with increased adsorbed mass, whereas an opposite trend was observed for PGM films. We propose that this contrast originates from that negatively charged motifs are present for both the central and terminal regions of BSM molecule, whereas a similar magnitude of negative charges is localized at the termini of PGM molecule. Given that hydrophobic motifs acting as an anchor are also localized in the terminal region, electrostatic repulsion between anchoring units of PGM molecules on a nonpolar PDMS surface leads to weakening of the mechanical integrity of the films.