Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles.

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many effectors with pharmaceutical properties that can diminish host immune responses and enhance pathogen transmission. One group of such effectors are microRNAs (miRNAs). miRNAs are short non-coding sequences that regulate host gene expression at the tick-host interface and within the organs of the tick. These small RNAs are transported in the tick saliva via extracellular vesicles (EVs), which serve inter-and intracellular communication. Vesicles containing miRNAs have been identified in the saliva of ticks. However, little is known about the roles and profiles of the miRNAs in tick salivary vesicles and glands. Furthermore, the study of vesicles and miRNAs in tick saliva requires tedious procedures to collect tick saliva. This protocol aims to develop and validate a method for isolating miRNAs from purified extracellular vesicles produced by ex vivo organ cultures. The materials and methodology needed to extract miRNAs from extracellular vesicles and tick salivary glands are described herein.

In situ oral lubrication and smoothness sensory perception influenced by tongue surface roughness.

BACKGROUND: The human tongue is important in the oral processing of food and in sensory perception. Tongue topography could influence delicate differences in sensory perception. It is hypothesized that tongue surface roughness could alter oral lubrication status and affect perception of smoothness. Fifteen participants with varying levels of tongue surface roughness were recruited and tested. Participants' in situ oral lubrication status without and after consumption of fluid food (milk with varying fat content and maltodextrin solutions with different shear viscosities) was measured. Participants' smoothness sensory scores were also recorded. RESULTS: The in situ friction coefficient (0.299-1.505) was significantly positively correlated with tongue-surface roughness (54.6-140.0 µm) in all types of test fluid samples across participants. Oral lubrication was significantly decreased when participants consumed the test fluid samples compared with no liquid food consumption, for all test fluid sample types (P < 0.05). No significant differences in in situ friction coefficient were found after participants consumed different test fluid samples, and this was mainly attributed to the limited quantities of fluid residuals in the oral cavity after expectoration. Participants whose tongue surface roughness differed did not exhibit significant differences in smoothness perception with different test fluid samples. CONCLUSION: Tongue surface roughness has a strong impact on in situ oral lubrication, and fluid food intake reduces in situ oral lubrication significantly. Saliva film and tongue surface roughness might play greater roles in oral lubrication and smoothness sensory perception if fluid is expectorated after consumption. The association between oral physiology and texture perception still needs further elucidation. © 2021 Society of Chemical Industry.

Oral function in amyotrophic lateral sclerosis patients: A matched case-control study.

BACKGROUND & AIMS: Patients with amyotrophic lateral sclerosis (ALS) develop swallowing difficulties with the progression of the disease. The present study aimed at comparing oral function and body composition between ALS patients and healthy controls, and at evaluating which parameters are the most discriminant between both groups. METHODS: We included ALS patients at the start of their multidisciplinary follow-up at the Geneva University Hospitals and healthy age-, gender-, and dental status-matched adults. We assessed the severity of the disease through the ALS Functional Rating Scale and the swallowing difficulties through the EAT-10 score. We performed an intraoral examination of the dental status, and measured chewing performance, bite, lip and tongue force, saliva weight, and body composition. Group comparisons were performed with t-tests or Mann-Whitney tests as appropriate. Linear discriminant analysis was used to determine the most discriminant parameters between groups. RESULTS: Twenty-six ALS patients (bulbar onset: n = 7, median (IQR) ALS Functional Rating Scale: 37 (11)) were included. The ALS patients had a significantly lower chewing performance (p < 0.001), lip force (p < 0.001), tongue force (p = 0.002), saliva weight (p < 0.004) and fat-free mass index (p < 0.001) as compared to the healthy individuals, and a higher EAT-10 score (p < 0.001). In ALS patients, a low chewing performance was correlated with a low bite (r = -0.45, p < 0.05)) and tongue force (r = -0.59, p < 0.05). The most discriminant parameters between both groups, by order of importance, were chewing performance, fat-free mass index and saliva weight and allowed the calculation of a discriminant function. CONCLUSION: Compared to healthy controls, ALS patients have significant alterations of oral function and body composition. The most discriminant parameters between both groups were chewing performance, fat-free mass index and saliva volume. It remains to be demonstrated whether oral parameters predict outcome. CLINICAL TRIAL REGISTRY: clinicaltrials.gov, identifier: NCT01772888.

Nivolumab-Related Dry Mouth and Dry Eye: Cross-Sectional Study.

To evaluate ICIs related dry eye and dry mouth in nivolumab therapy, 24 patients receiving nivolumab (group 1), 30 patients in remission without treatment for 6 months (group 2), 30 healthy participants (group 3) were cross-sectionally examined. Schirmer's 1, 2, TSH blood tests, serological analysis, salivary flow scintigraphy and minor-salivary gland biopsy were performed. Schirmer's tests were performed with anesthetic (1) and without anesthetic (2). Schirmer's scores were lower in group 1 with more frequent reduced tear production (p < 0.001). TSH levels negatively correlated with Schirmer's scores. Functional insufficiency was detected by salivary flow scintigraphy in 7 out of 10 patients with Schirmer's test positivity. In Schirmer's positive patients, lymphocytic sialadenitis was confirmed in 4 patients (focus score > 1) and CD4 T lymphocyte precipitation was observed in 6 patients. Nivolumab therapy may be associated with ICIs related immune sicca.

Effects of Oral pH Changes on Smoking Desire.

BACKGROUND: Nicotine addiction is associated with nicotine absorption from the buccal mucosa, and it is stated that the main factor that determines nicotine absorption is saliva pH. In the literature, the effects of changes in saliva pH values after eating and drinking on smoking desire in smokers were not questioned. AIM: To show the effect of saliva pH changes on smoking desire. The secondary aim was to show the impact of coffee and water drinking on saliva pH and smoking on oral-dental health (oral hygiene and gingival bleeding). STUDY DESIGN: Case-control study. METHODS: A questionnaire was administered that included "Sociodemographic Data Form" and smoking history and Fagerström Test for Nicotine Dependence (FTND). Oral and dental examinations were performed with mirror sonds and using oral hygiene standard Silness and Leöe plaque index and DMFT Index (Index of Decayed Missing or Filled Teeth). Untreated saliva samples were taken, and baseline saliva flow rate and pH values were measured. To assess pH changes, saliva pH was remeasured after sugar-free instant coffee and water consumption. Smoking desire was evaluated with the Visual Analog Scale (VAS). RESULTS: In this study, 24 (55.8%) females and 19 (44.2%) males were among the 43 smoking and 39 nonsmoking cases. Smoking was significantly associated with poor oral hygiene (in smokers 4.71 (±1.40), in non-smokers 2.30 (±1.59); P < .01). DMFT index was higher in smokers than in non-smokers (in smokers 6.45 (±3.69), in non-smokers 3.87 (±2.67); P < .01). Gingival bleeding was more prevalent in smokers (0.68 (±0.76)) than non-smokers (1.20 (±0.90); P = .009). Salivary flow rates were lower in smokers (in smokers 2.56 (±1.34), in non-smokers 3.00 (±1.22), P = .06). In both groups, pH values increased after coffee consumption and decreased after water; in smokers basal: 6.67 (±0.41), pH coffee: 6.93 (±0.36), pH water: 6.85 (±0.33); in non-smokers pH basal: 6.84 (±0.37), pH coffee: 7.02 (±0.37), pH water: 6.97 (±0.31), P < .01. The VAS values of smokers at basal 4.73 (±3.21); P < 0.01, after coffee consumption 4.91 (±3.08); P < .01, and after water 3.15 (±2.72); P < .01. CONCLUSION: The saliva pH increased after coffee consumption and decreased after drinking water. Besides, VAS values decreased significantly after drinking water. The results suggest that a simple behavior such as drinking water may be used in conjunction with behavioral and cognitive therapies to pursue smoking cessation.

Relationship among oral health status, bolus formation and food comfortability during consumption of model cheeses in elderly.

The aim of this study was to investigate the impact of oral impairment on chewing behaviour, food bolus properties and food comfortability during elderly consumption of model cheeses. Seventy-two elderly persons (aged 66 to 88) was recruited and classified into two groups according to dental status (poor vs. satisfactory). They showed a wide range of salivary flow rates whatever their dental status (stimulated: 0.2-3.8 mL min-1, resting: 0.1-0.8 mL min-1). Standardized bites of four model cheeses with an identical composition but different textures (soft, hard, processed and whipped) were tested. The time and number of chewing cycles required to form a bolus were measured. The rheological properties of the bolus were studied, as was saliva moistening. Food comfortability was assessed by means of a questionnaire composed of 5 sections (1-oral comfort, 2-bolus formation, 3-pain, 4-texture and 5-flavour perception). The chewing parameters measured were not modified by the oral health. However, elderly with poor dentition formed harder boluses than elderly with satisfactory dentition. Moreover, for elderly with poor dentition, the quantity of saliva incorporated into the bolus was correlated with the stimulated salivary flow rate, which was not the case for elderly with satisfactory dentition. General oral comfort and its different attributes were poorly associated with the oral health of the elderly. A multifactorial analysis performed on an average cheese showed that food comfortability is independent of changes in the hardness and moistening of the bolus, regardless of dental status. In particular, poor dental status increases the hardness of the bolus without modifying its comfortability.

Comprehensive histochemical and stereological study of the parotid and sublingual salivary glands in Caucasian squirrel (Sciurus anomalus).

The present investigation tries to reflect histochemical and stereological properties of the parotid gland (PG) and sublingual gland (SLG) in the Caucasian squirrel. Totally, five male Caucasian squirrels were used. The glands were examind histochemical using PAS, Alcian blue (AB, pH = 2.5) and Aldehyde fucshin (AF) stainings. Total volume of the glands, acini, ducts as well as total length of ducts were estimated using streological procedures. The PG had a unique morphology and were connected to each other with an intermediated isthmus. The SLG was oval-shaped. Serous acini of PG exhibited strong, negative and weak reaction with AF, AB and PAS, respectively. While, mucous acini of SLG showed positive staining for AB and PAS, and negative staining for AF. The ductal system was consisted of intercalated (Id), striated (SD) and excretory ducts (Ed). The Sd in PG was lined with simple cuboidal to the columnar epithelium, while it was lined with simple cuboidal epithelium in SLG. The Ed was lined with stratified cuboidal epithelium in both glands. The striated duct was longest duct in both glands, the excretory and intercalated ducts were the shortest ones in PG and SLG, respectively. The maximum total volume in both gland were blonged to the acini and straited ducts. It concluded that PG and SLG in the Caucasian squirrel have some distinct structural properties in comparison to other rodents and mammalis species.

Patch test of dental materials in Oral Lichen Planus with considering the role of saliva.

Lichen planus is the most common skin disease that affects the oral mucosa. Oral Lichen Planus is a T-cell-mediated autoimmune disorder. In the current study, for the first time, an oral cavity condition in skin patch tests with adding saliva is simulated. In addition, the patch results are compared with healthy subjects. Forty-one OLP patients and 63 healthy subjects were enrolled in the study. All participants were provided with patch tests, including allergens, in combination with saliva in chambers. Allergens from the European baseline (standard) series selected according to the most prevalent positive results in the previous study were applied. Positive results of Mercury and Cobalt tests were significantly higher in the case group. In this study, the differentiation of patients with lichen planus and lichenoid was identified according to the Van der Meij & Van der Waal criteria. The patch test was conducted for healthy individuals as well. The most important of all was the use of patients' saliva in the patch test, done for the first time in this field. In the case of OLP, a patch test can help identify positive reactions to dental materials; thus, the replacement of dental restorations may be needed.

SLI1 confers broad-spectrum resistance to phloem-feeding insects.

Resistance (R) genes usually compete in a coevolutionary arms race with reciprocal effectors to confer strain-specific resistance to pathogens or herbivorous insects. Here, we investigate the specificity of SLI1, a recently identified R gene in Arabidopsis that encodes a small heat shock-like protein involved in resistance to Myzus persicae aphids. In a panel with several aphid and whitefly species, SLI1 compromised reproductive rates of three species: the tobacco aphid M. persicae nicotianae, the cabbage aphid Brevicoryne brassicae and the cabbage whitefly Aleyrodes proletella. Electrical penetration graph recording of aphid behaviour, revealed shorter salivations and a 3-to-5-fold increase in phloem feeding on sli1 loss-of-function plants. The mustard aphid Lipaphis erysimi and Bemisia tabaci whitefly were not affected by SLI1. Unlike the other two aphid species, L. erysimi exhibited repetitive salivations preceding successful phloem feeding, indicating a role of salivary effectors in overcoming SLI1-mediated resistance. Microscopic characterization showed that SLI1 proteins localize in the sieve tubes of virtually all above- and below-ground tissues and co-localize with the aphid stylet tip after penetration of the sieve element plasma membrane. These observations reveal an unconventional R gene that escapes the paradigm of strain specificity and confers broad-spectrum quantitative resistance to phloem-feeding insects.

Smad7 attenuates TGF-ß-mediated aging-related hypofunction of submandibular glands.

Submandibular glands have essential functions in taste, mastication, swallowing, and digestion. Submandibular gland hypofunction is prevalent in the elderly, impairing the patients' quality of life. Current clinical treatment strategies have not decelerated or reversed the pathological process of submandibular gland hypofunction. Therefore, novel restoration strategies should be explored. However, studies on the mechanism of aging-related submandibular gland hypofunction remain very limited. The role of the TGF-ß/Smad pathway in fibrosis has been studied in other organs. Therefore, this study aimed to elucidate the role of TGF-ß/Smad signaling in the aging-related submandibular gland hypofunction. The results showed that Smad7 knockout in mice decreased the salivary flow rate. H&E, Masson trichrome, and immunohistochemistry staining of MCP-1 and α-SMA showed that Smad7 knockout in mice resulted in lymphocytic infiltration, acinar cell atrophy, and interstitial fibrosis. The Western blotting of collagen I and III also confirmed extensive fibrosis. We then found that Smad7 depletion resulted in the TGF-ß-mediated fibrosis via mir-21, mir-29, and np_5318, and NFκB-driven inflammation activation. This study confirmed the inhibitory role of Smad7 in the aging-related submandibular gland hypofunction. Therefore, it provided a promising treatment target for aging-related dysfunction and sialadenitis of submandibular gland.

Effects and mechanisms of histatins as novel skin wound-healing agents.

Wound healing is a complex and important physiological process that maintains the integrity of skin after various injuries. Abnormal wound healing, especially of chronic wounds, impairs normal physical function. Therefore, the search for effective and safe healing agents is one of the main concerns. Histatins are histidine-rich low molecular weight peptides that are expressed in the saliva of both humans and higher primates. Histatins have two main biological effects, cell stimulation and bacteria killing, with the former playing an important role in wound healing by promoting epithelial cell and fibroblast migration and angiogenesis and enhancing the re-epithelialization of the wounded area. Because of these biological effects, histatins have been shown to be promising agents of improved wound healing. Histatins are categorized into many subtypes, of which histatin 1 and its hydrolysates are the most effective in promoting wound healing. This review addresses the bioactivity of histatins in wound healing, such as their stimulatory effects on epithelial cells and fibroblasts, and elucidates the possible mechanisms by which histatin subtypes induce their biological effects.

Avian mud nest architecture by self-secreted saliva.

Mud nests built by swallows (Hirundinidae) and phoebes (Sayornis) are stable granular piles attached to cliffs, walls, or ceilings. Although these birds have been observed to mix saliva with incohesive mud granules, how such biopolymer solutions provide the nest with sufficient strength to support the weight of the residents as well as its own remains elusive. Here, we elucidate the mechanism of strong granular cohesion by the viscoelastic paste of bird saliva through a combination of theoretical analysis and experimental measurements in both natural and artificial nests. Our mathematical model considering the mechanics of mud nest construction allows us to explain the biological observation that all mud-nesting bird species should be lightweight.

Tick saliva-induced programmed death-1 and PD-ligand 1 and its related host immunosuppression.

The tick Rhipicephalus microplus is a harmful parasite of cattle that causes considerable economic losses to the cattle breeding industry. Although R. microplus saliva (Rm-saliva) contains several immunosuppressants, any association between Rm-saliva and the expression of immunoinhibitory molecules, such as programmed death (PD)-1 and PD-ligand 1 (PD-L1), has not been described. In this study, flow cytometric analyses revealed that Rm-saliva upregulated PD-1 expression in T cells and PD-L1 expression in CD14+ and CD11c+ cells in cattle. Additionally, Rm-saliva decreased CD69 expression in T cells and Th1 cytokine production from peripheral blood mononuclear cells. Furthermore, PD-L1 blockade increased IFN-γ production in the presence of Rm-saliva, suggesting that Rm-saliva suppresses Th1 responses via the PD-1/PD-L1 pathway. To reveal the upregulation mechanism of PD-1/PD-L1 by Rm-saliva, we analyzed the function of prostaglandin E2 (PGE2), which is known as an inducer of PD-L1 expression, in Rm-saliva. We found that Rm-saliva contained a high concentration of PGE2, and PGE2 treatment induced PD-L1 expression in CD14+ cells in vitro. Immunohistochemical analyses revealed that PGE2 and PD-L1 expression was upregulated in tick-attached skin in cattle. These data suggest that PGE2 in Rm-saliva has the potential to induce the expression of immunoinhibitory molecules in host immune cells.

The comparative inhibitory potency of salivary mucins against human immunodeficiency virus type 1.

MUC5B and MUC7 salivary mucins are reported to inhibit HIV-1 entry into target cells in vitro; however, their relative inhibitory potencies have not been quantitively compared. There is also conflicting evidence regarding whether HIV-1 infection diminishes mucins' inhibitory efficacy. We explored the effect of donor HIV-1 status upon the anti-HIV-1 potency of purified MUC5B and MUC7 while comparing their relative inhibitory potential using a pseudovirus-based neutralization assay. HIV status of sample donors had no detectable effect on HIV-1 inhibition by salivary mucins. MUC5B (median IC50 50 µg/ml, IQR 10-116 µg/ml) exhibited significantly more potent HIV-1 inhibition than MUC7 (median IC50 458 µg/ml, IQR 192->2000 µg/ml; Mann-Whitney U p < 0.0001). We suggest that larger size, gel-forming properties and extensive glycosylation of MUC5B allow more effective binding and aggregation of viral particles. MUC5B is also more abundant in the saliva and is therefore likely to make a substantially greater contribution to it's anti-HIV-1 properties.

Cell Sheets Restore Secretory Function in Wounded Mouse Submandibular Glands.

Thermoresponsive cell culture plates release cells as confluent living sheets in response to small changes in temperature, with recovered cell sheets retaining functional extracellular matrix proteins and tight junctions, both of which indicate formation of intact and functional tissue. Our recent studies demonstrated that cell sheets are highly effective in promoting mouse submandibular gland (SMG) cell differentiation and recovering tissue integrity. However, these studies were performed only at early time points and extension of the observation period is needed to investigate duration of the cell sheets. Thus, the goal of this study was to demonstrate that treatment of wounded mouse SMG with cell sheets is capable of increasing salivary epithelial integrity over extended time periods. The results indicate that cell sheets promote tissue organization as early as eight days after transplantation and that these effects endure through Day 20. Furthermore, cell sheet transplantation in wounded SMG induces a significant time-dependent enhancement of cell polarization, differentiation and ion transporter expression. Finally, this treatment restored saliva quantity to pre-wounding levels at both eight and twenty days post-surgery and significantly improved saliva quality at twenty days post-surgery. These data indicate that cell sheets engineered with thermoresponsive cell culture plates are useful for salivary gland regeneration and provide evidence for the long-term stability of cell sheets, thereby offering a potential new therapeutic strategy for treating hyposalivation.

Experimental Animal Model Systems for Understanding Salivary Secretory Disorders.

Salivary secretory disorders are life-disrupting pathologic conditions with a high prevalence, especially in the geriatric population. Both patients and clinicians frequently feel helpless and get frustrated by the currently available therapeutic strategies, which consist mainly of palliative managements. Accordingly, to unravel the underlying mechanisms and to develop effective and curative strategies, several animal models have been developed and introduced. Experimental findings from these models have contributed to answer biological and biomedical questions. This review aims to provide various methodological considerations used for the examination of pathological fundamentals in salivary disorders using animal models and to summarize the obtained findings. The information provided in this review could provide plausible solutions for overcoming salivary disorders and also suggest purpose-specific experimental animal systems.

The effect of sublingual atropine sulfate on clozapine-induced hypersalivation: a multicentre, randomised placebo-controlled trial.

BACKGROUND: Hypersalivation and drooling are commonly reported in clozapine-treated patients. Current management strategies have been evaluated using subjective measures. Many case reports describe the successful use of atropine in the treatment of the condition. AIMS: To measure the effect and safety of sublingual atropine on nocturnal unstimulated saliva secretion. Secondary aims were to evaluate the patient's satisfaction with the atropine effect on hypersalivation (or sialorrhea), drooling, and sleep. METHOD: Twenty-one clozapine-treated patients with hypersalivation, or drooling, were randomised to take a single 600-µg dose of sublingual atropine drops or a matching placebo. The saliva secretion was measured over 5 min at baseline and 2 h after the administration of the study medication. RESULTS: Sublingual atropine reduced the saliva secretion significantly more than the placebo (mean difference = - 57.21%, 95% CI: - 104.30, - 10.11, P = 0.02). A significant decrease in standing pulse rate was recorded in the participants in the atropine group (- 5.8 (- 9.54, - 2.15), P = 0.002). Subjectively, more patients in the atropine group found their pillow to have less saliva the following morning and found their sleep to be better. CONCLUSIONS: Sublingual atropine drops significantly reduces nocturnal unstimulated clozapine-induced saliva secretion. More research is required to compare the effect of sublingual atropine with other anticholinergic medications and different dosage forms. TRIAL REGISTRATION: ACTRN12618000051246.

Oral Microbiome Geography: Micron-Scale Habitat and Niche.

The mouth presents a multiplicity of local environments in communication with one another via saliva. The spatial organization of microbes within the mouth is shaped by opposing forces in dynamic equilibrium-salivary flow and adhesion, shedding and colonization-and by interactions among and between microbes and the host. Here we review recent evidence confirming that oral microbes are specialized for individual habitats within the mouth and that microbial habitats and niches are defined by micron-scale gradients in combination with short- and long-range interactions. Micron-scale structure illuminates the roles of individual taxa and provides insight into their community ecology and potential pathogenicity.