Effects of Thickening Agents on the Mucociliary Transport Function: Comparison by the Type of Thickening Agents and the Viscosity of Thickened Water.

Thickening agents effectively prevent liquid aspiration, but their impact on the ease of discharging aspirated liquids from the trachea remains unclear due to alterations in the physical properties of liquids. This study clarifies the effects of thickening agents, comprising various raw materials, on mucociliary transport function, focusing on the viscosities of thickened waters. The subjects were 23 healthy adults. Five types of saccharin solution were prepared: a solution without a thickening agent, a starch-based nectar-like solution, a starch-based honey-like solution, a xanthan-gum-based nectar-like solution, and a xanthan-gum-based honey-like solution. Using these five types of saccharin solutions randomly, each subject underwent five trials of the saccharine dye test to evaluate the mucociliary transport function of the respiratory tract. The saccharin time was defined as the time from the placement of the saccharin solution on the nasal vestibule of the subject to when the subject reported that they became aware of the sweetness. The saccharin transit times for all samples of thickened water were longer compared to those of water without a thickening agent (p < 0.01). A comparison between thickened water samples with different viscosities showed that the saccharin transit time was longer when thickened water samples with high viscosity were prepared using the same thickening agent (p < 0.01). This suggests that while thickening reduces aspiration, the use of thickening agents may increase the difficulty in discharging aspirated fluids from the trachea.

The Germinal Origin of Salivary and Lacrimal Glands and the Contributions of Neural Crest Cell-Derived Epithelium to Tissue Regeneration.

The vertebrate body comprises four distinct cell populations: cells derived from (1) ectoderm, (2) mesoderm, (3) endoderm, and (4) neural crest cells, often referred to as the fourth germ layer. Neural crest cells arise when the neural plate edges fuse to form a neural tube, which eventually develops into the brain and spinal cord. To date, the embryonic origin of exocrine glands located in the head and neck remains under debate. In this study, transgenic TRiCK mice were used to investigate the germinal origin of the salivary and lacrimal glands. TRiCK mice express fluorescent proteins under the regulatory control of Sox1, T/Brachyury, and Sox17 gene expressions. These genes are representative marker genes for neuroectoderm (Sox1), mesoderm (T), and endoderm (Sox17). Using this approach, the cellular lineages of the salivary and lacrimal glands were examined. We demonstrate that the salivary and lacrimal glands contain cells derived from all three germ layers. Notably, a subset of Sox1-driven fluorescent cells differentiated into epithelial cells, implying their neural crest origin. Also, these Sox1-driven fluorescent cells expressed high levels of stem cell markers. These cells were particularly pronounced in duct ligation and wound damage models, suggesting the involvement of neural crest-derived epithelial cells in regenerative processes following tissue injury. This study provides compelling evidence clarifying the germinal origin of exocrine glands and the contribution of neural crest-derived cells within the glandular epithelium to the regenerative response following tissue damage.

Therapeutic benefits of factors derived from stem cells from human exfoliated deciduous teeth for radiation-induced mouse xerostomia.

Radiation therapy for head and neck cancers is frequently associated with adverse effects on the surrounding normal tissue. Irreversible damage to radiation-sensitive acinar cells in the salivary gland (SG) causes severe radiation-induced xerostomia (RIX). Currently, there are no effective drugs for treating RIX. We investigated the efficacy of treatment with conditioned medium derived from stem cells from human exfoliated deciduous teeth (SHED-CM) in a mouse RIX model. Intravenous administration of SHED-CM, but not fibroblast-CM (Fibro-CM), prevented radiation-induced cutaneous ulcer formation (p < 0.0001) and maintained SG function (p < 0.0001). SHED-CM treatment enhanced the expression of multiple antioxidant genes in mouse RIX and human acinar cells and strongly suppressed radiation-induced oxidative stress. The therapeutic effects of SHED-CM were abolished by the superoxide dismutase inhibitor diethyldithiocarbamate (p < 0.0001). Notably, quantitative liquid chromatography-tandem mass spectrometry shotgun proteomics of SHED-CM and Fibro-CM identified eight proteins activating the endogenous antioxidant system, which were more abundant in SHED-CM than in Fibro-CM (p < 0.0001). Neutralizing antibodies against those activators reduced antioxidant activity of SHED-CM (anti-PDGF-D; p = 0.0001, anti-HGF; p = 0.003). Our results suggest that SHED-CM may provide substantial therapeutic benefits for RIX primarily through the activation of multiple antioxidant enzyme genes in the target tissue.

Evaluation of oral care using MA-T gel for high-risk patients: a pilot study.

BACKGROUND: Oral care with gel is a common method for preventing aspiration in high-risk patients. An oral care gel is used to clean and moisturize the oral cavity. However, the effects of gel care on the oral bacteria remain unclear. In this pilot study, we described a matching transformation system (MA-T) for elderly high-risk patients. MA-T is an on-demand aqueous chlorine dioxide solution that provides excellent safety and has various antimicrobial activities, even in the presence of abundant organic compounds. This study investigated the effects of MA-T gel in patients requiring nursing care. MATERIALS AND METHODS: Patients who were hospitalized for nursing care were included in this study. No drugs and foods were administered orally. Oral bacteria and intraoral humidity were examined by daily care using MA-T gel. Moreover, oral membranous substances were analyzed and material from the oral cavity was cultured on selective media for identifying opportunistic organisms. RESULTS: Membranous substances were present in the oral cavities of all patients. The number of bacteria decreased, and oral moisture improved, after treatment with MA-T gel. Moreover, oral humidity was also controlled with the continued use of MA-T gel. MA-T gels should be used not only for professional care but also on a daily basis for better oral care. Furthermore, the results of bacterial cultures showed that MA-T controls the propagation of opportunistic bacterial infections. CONCLUSION: Membranous substances may be observed in the oral cavity of individuals requiring nursing care for tube feeding. The results of this pilot study suggest that MA-T, a novel disinfectant, can be used for oral care in the elderly to reduce the risk of aspiration-pneumonia.

Human induced pluripotent stem cell-derived salivary gland organoids model SARS-CoV-2 infection and replication.

Salivary glands act as virus reservoirs in various infectious diseases and have been reported to be targeted by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the mechanisms underlying infection and replication in salivary glands are still enigmatic due to the lack of proper in vitro models. Here, we show that human induced salivary glands (hiSGs) generated from human induced pluripotent stem cells can be infected with SARS-CoV-2. The hiSGs exhibit properties similar to those of embryonic salivary glands and are a valuable tool for the functional analysis of genes during development. Orthotopically transplanted hiSGs can be engrafted at a recipient site in mice and show a mature phenotype. In addition, we confirm SARS-CoV-2 infection and replication in hiSGs. SARS-CoV-2 derived from saliva in asymptomatic individuals may participate in the spread of the virus. hiSGs may be a promising model for investigating the role of salivary glands as a virus reservoir.

HIF-1α regulates mTOR signaling pathway during salivary gland development.

The mammalian target of rapamycin (mTOR)-composed of multiple complexes, including mTOR complex 1/2 (mTORC1/2)-is a serine-threonine kinase that regulates embryonic development. The transcription factor, hypoxia-inducible factor-1α (HIF-1α), is also involved in embryonic development. As the relationship between mTOR and HIF-1α during embryonic development remains unclear, we investigated the relationship between the two using ex vivo submandibular salivary gland organ cultures. When the expression of HIF-1α increased under hypoxic conditions (1% O2), the expression of mTOR signaling pathway-related proteins decreased. Conversely, when the expression of HIF-1α decreased, the expression of mTOR signaling pathway-related proteins increased. These results indicate a strong relationship between HIF-1α and the mTOR signaling pathway. For the first time, we clarified that HIF-1α negatively regulates the mTOR signaling pathway and suppresses salivary gland development under 1% O2 using small molecules. Our research provides new insights into the relationship between HIF-1α and the mTOR signaling pathway in embryonic organ development.

Establishment of a Mouse Submandibular Salivary Gland Organ Culture.

The salivary glands produce saliva and are important in maintaining oral health. Saliva keeps the mouth moist, cleanses the oral cavity, aids digestion, and has antibacterial properties. Saliva also helps in swallowing and speech. Investigating the development of the salivary glands is thus relevant in the context of both health and disease. Various cell culture methods have been used to study salivary gland development, including culturing cells in two dimensions (2D). Under physiological conditions, cells constantly interact with other cells and the extracellular matrix, which controls complex biological functions such as cell migration and apoptosis, and can modulate gene expression. Since many of these functions are not accurately represented or reproduced in 2D culture, the results of in vitro experiments using such culture methods are often not reflected in vivo. The use of 3D cultures, such as organ cultures, has helped address this issue and has emerged as a model that better reflects the in vivo physiological environment. Here, we describe a protocol for establishing submandibular salivary gland organ culture that is more concise and simpler than previous methods and includes the separation and dissection of the salivary glands. We also describe the use of environmental stress (hypoxic stimulation) and inhibitors (U0126, LY294002, and rapamycin) to elucidate signaling pathways involved in salivary gland development. This protocol can provide researchers with a simpler and more robust method of salivary gland organ culture, enabling analysis of organ-based signaling pathways to advance developmental biology research. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Submandibular salivary gland organ culture Basic Protocol 2: Analysis of salivary gland development in the presence of hypoxia and signaling pathway inhibitors Basic Protocol 3: Western blotting using submandibular salivary gland organ culture.

Influence of gustatory and olfactory functions on appetite and nutritional status in older persons.

This study investigated olfactory/taste functions in older persons requiring nursing care to clarify whether these functions are associated with appetite or nutritional status. Seventy-two older persons requiring nursing care and 37 unassisted persons were surveyed for olfactory function, taste function, appetite, and nutritional status. Age-adjusted covariance analysis was conducted between the two groups. Both groups showed reduced olfactory and taste functions; these functions were more markedly reduced in participants requiring nursing care compared to those who did not. Both groups had similar appetite and nutritional status findings, suggesting that these factors are not associated with olfactory and taste functions.

Relationship between daily swallowing frequency and pneumonia in patients with severe cerebral palsy.

BACKGROUND: Aspiration pneumonia is a major complication that occurs in patients with severe cerebral palsy and is associated with their survival prognosis, necessitating appropriate assessment and response. We focused on swallowing frequency as an index of daily swallowing function due to the difficulty in evaluating the risk of pneumonia. The swallowing motion protects the airway by safely directing the food, saliva, and secretions accumulated in the pharynx into the esophagus to prevent aspiration and entry into the trachea. Thus, swallowing frequency may be correlated with the incidence of pneumonia. In this study, we aimed to investigate the relationship between swallowing frequency and history of pneumonia in patients with severe cerebral palsy. METHODS: Fifty-seven patients with cerebral palsy were included in this study. Swallowing frequency was measured three times for each patient on separate days, and the reproducibility was examined by calculating the intraclass correlation coefficient. Further, the relationship between swallowing frequency and history of pneumonia was investigated using multivariate logistic regression analysis. RESULTS: While swallowing frequency differed between participants, it was constant within individuals (intraclass correlation coefficient: 0.941). Furthermore, the swallowing frequencies per hour were 12.2 ± 12.2 and 27.0 ± 20.4 in the patient groups with and without a history of pneumonia, respectively (P < 0.001). Swallowing frequency (odds ratio: 10.489, 95% confidence interval: 2.706-40.663, P = 0.001) was significantly associated with the incidence of pneumonia in the previous year. CONCLUSIONS: Swallowing frequency could be used as an index for assessing the risk of dysphagia and pneumonia in patients with severe cerebral palsy.

Safety evaluation of MA-T after ingestion in mice.

MA-T (Matching Transformation System®) is a proprietary chemical mixture for on-demand production of aqueous chlorine dioxide that is used for the treatment of oral malodor. MA-T is also an effective disinfectant against at least 39 pathological microorganisms, including severe acute respiratory syndrome coronavirus 2, and therefore may be useful as a disinfectant mouthwash to prevent the spread of infection. Accidental ingestion is the putative worst hazard scenario associated with mouthwash use; therefore, here we investigated the safety of MA-T ingestion in mice. Mice were provided drinking water containing 0-3000 µg/ml MA-T for 7 days followed by non-spiked drinking water for an additional 14 days. At day 7, mice ingesting 1000 or 3000 µg/ml MA-T showed significantly decreased body weight and significantly increased liver, kidney, and heart tissue injury biomarkers compared with control. However, at 14 days after stopping MA-T ingestion, body weight and tissue injury biomarkers had returned to normal. Histological analysis revealed that MA-T-induced injuries in liver, kidney, spleen, stomach, duodenum, colon, and rectum had also recovered at 14 days after stopping MA-T ingestion; however, mild vascular endothelial injuries remained in heart, jejunum, and ileum in the worst-case scenario. Taken together, MA-T may be potentially safety for further development as a disinfectant mouthwash by risk management, such as placing a caution of the label and adding a distinctive flavor.

The Bactericidal Effect of MA-T for Factitiously Contaminated and Used Masks.

Matching transformation system (MA-T), an on-demand aqueous chlorine dioxide solution, is an excellent safety disinfectant, because chlorine dioxide is not detected during storage or before use. The production of chlorine dioxide in MA-T is induced by a catalytic reaction in the presence of target microorganisms. In this study, we investigated MA-T disinfection of masks as a reuse method to eliminate mask shortages. After spraying Escherichia coli on sterilized surgical mask, samples (factitiously contaminated masks) were treated with MA-T spraying or immersion, and the bactericidal efficacy was assessed by culturing. Used surgical masks were also sprayed with MA-T or were immersed in MA-T, and then were cultured to verify the bactericidal effect. The performance of N95 masks was assessed before and after application of MA-T. After spraying with MA-T, the numbers of bacteria of factitiously contaminated masks and used masks were drastically reduced compared with control samples (not applicable and p = 0.002, respectively). After MA-T immersion, the bacterial counts of both masks (factitiously contaminated masks and used masks) were significantly reduced (both p = 0.002). Taken together, the disinfection test on factitiously contaminated with E. coli and used surgical masks showed that masks can be disinfected by MA-T spray and sterilized by immersion, respectively. The N95 mask performance test after 30 min of immersion in MA-T showed that MA-T disinfected the mask without degrading the performance of the mask. In conclusion, MA-T is useful for the reuse of masks because of its decontamination effect and safety while maintaining the function of the mask.

Analysis of medication-induced xerostomia in elderly Japanese patients.

OBJECTIVES: To determine the general condition of elderly xerostomia patients, we collected their background and medication data in order to potentially treat their xerostomia. It is critical to identify the drugs causing xerostomia in elderly patients. A total of 521 patients who were examined at the Xerostomia Clinic of Osaka University Dental Hospital were included in the study. We obtained patients' data on age, sex, number of primary illnesses, Saxon test scores, oral moisture test, subjective symptoms, and drug types from their clinical records. RESULTS: The mean age of the patients was 65.2 ± 13.3 years. Although all patients exhibited xerostomia symptoms, there were a lot of patients without hyposalivation. With respect to medication, each elderly xerostomia patient took an average of 6.8 ± 4.4 medicines. A total of 26.1% of patients in their 70 s took more than ten number of drugs. In addition, the number of frequently used medication medicine was different between elderly and young patients. Most of the medicines had xerostomia as a side effect in medical package inserts. Moreover, the quantity of salivation significantly decreased in patients who took more than seven drugs in comparison with the patients who did not take medicine. CONCLUSIONS: As patients age, the number of medications they take tends to increase, subsequently increasing their risk of xerostomia. For the health of the patients, it is critical that an accurate diagnosis is made. CLINICAL RELEVANCE: To establish therapeutic strategies for treatment of xerostomia, this study provides new and important information that will help in the development of xerostomia medical treatment.

The HIF-1α pathway plays a critical role in salivary gland development in ex vivo organ cultures.

The transcription factor, hypoxia-inducible factor-1α (HIF-1α), has previously been shown to upregulate the expression of hypoxia-related genes, including erythropoietin (EPO). However, the role of hypoxia-inducible factor-1α in morphogenesis during salivary gland development is unclear. We investigated the function of HIF-1α in submandibular gland (SMG) organ cultures obtained from embryonic day 13.5 embryos from ICR female mice. Expression of HIF-1α, glucose transporter 1, and vascular endothelial growth factor was induced under hypoxia (5% O2 ). We further showed that BAY 87-2243-mediated inhibition of HIF-1α suppressed salivary gland development. Under severe hypoxia (1% O2 ), HIF-1α did not promote salivary gland development; this was due to suppression of cell proliferation and inhibition of the cell cycle and not because of autophagy and apoptosis. Additionally, using the inhibitor U0126, we verified that the ERK1/2 pathway is upstream of HIF-1α. Overall, we found that the HIF-1α signaling pathway plays a critical role in salivary gland development in ex vivo SMG organ cultures.

The impact of different medical complications on the feasibility of removing the feeding tube from infants with dysphagia.

PURPOSE: This study examined whether certain medical complications influence the feasibility of tube removal. METHODS: 42 subjects with dysphagia who were under the age of 2.5 years were nourished entirely through feeding tubes. Additionally, they were judged to have no aspiration. The following data about the infants were collected through a retroactive survey: age at which oral feeding training commenced, gender, and whether certain medical complications (cardiovascular, respiratory, digestive, neurological, or oral) had been present at birth. The data were analyzed to determine which type of medical complication affected the likelihood of removing the feeding tube from the infant at 3 years of age. RESULTS: Of the five medical complications examined, cardiovascular complications significantly affected the feasibility of tube removal (p = 0.049). CONCLUSION: Pediatric dysphagia patients with cardiac complications, compared to those with other complications, may take longer to transition off tube feeding.

Brief communication: Immunohistochemical detection of ACE2 in human salivary gland.

The novel, severe acute respiratory syndrome coronavirus (SARS-CoV-2) was firstly reported in late December of 2019 and subsequently caused a global outbreak. It has been shown that SARS-CoV-2 uses ACE2 (Angiotensin Converting Enzyme 2) as a cellular receptor for host cell entry through the surface unit of SARS-CoV spike glycoprotein. In this brief report, we analyze ACE2 protein expression and localization in human salivary gland, and propose a possible role of saliva in the pathogenesis of Coronavirus disease 2019 (COVID-19).

Evaluation of the Saxon test for patients with hyposalivation without Sjögren's syndrome.

BACKGROUND: Dry mouth is associated with salivary gland hypofunction, which may result from several conditions such as Sjögren's syndrome (SS), head and neck cancers, and side effects of medications. The Saxon test is a useful diagnostic method for hyposalivation in clinical settings. However, previous reports indicate that the test has mostly been used for patients with SS. OBJECTIVE(S): In the present study, we focused on patients with dry mouth who were not diagnosed with SS (patients without SS). METHODS: For patients without SS (n = 302), we examined the factors affecting Saxon test scores using multiple regression analysis. Additionally, we performed a correlation analysis comparing the Saxon test with other diagnostic methods. RESULTS: In 57.6% patients, the Saxon test score was more than 2.00 g/2 min, which is considered negative for hyposalivation. Multiple regression analysis revealed that the age and sex of patients significantly influenced test scores. The mean Saxon test score was less than 2.00 g/2 min in older patients and women. Moreover, the test showed a significant correlation with other methods used to measure salivary flow. CONCLUSION: The Saxon test is useful not only for patients with SS but also for patients without SS.

ΔNp63 is upregulated during salivary gland regeneration following duct ligation and irradiation in mice.

The transcription factor p63, a component of the p53 family, has important functions in development, homeostasis, and regeneration of epithelial tissues. However, the role of p63 in the regeneration of exocrine glands, including the salivary glands (SGs), has not been fully investigated. We investigated p63 expression in SG regeneration induced by duct ligation and irradiation. The expression of ΔNp63, a p63 isoform, increased and was colocalized with keratin 5 positive cells were myoepithelial cells. Furthermore, ΔNp63 expression was regulated by FGF7 stimulation via p38 MAPK phosphorylation and affected SG morphogenesis. These results suggest that ΔNp63 is essential for SG regeneration and may be a new target for regenerative treatment.

Transforming Growth Factor-Beta and Sonic Hedgehog Signaling in Palatal Epithelium Regulate Tenascin-C Expression in Palatal Mesenchyme During Soft Palate Development.

During palatogenesis, the palatal shelves first grow vertically on either side of the tongue before changing their direction of growth to horizontal. The extracellular matrix (ECM) plays an important role in these dynamic changes in palatal shelf morphology. Tenascin-C (TNC) is an ECM glycoprotein that shows unique expression in the posterior part of the palatal shelf, but little is known about the regulation of TNC expression. Since transforming growth factor-beta-3 (TGF-ß3) and sonic hedgehog (SHH) signaling are known to play important roles in palatogenesis, we investigated whether TGF-ß3 and SHH are involved in the regulation of TNC expression in the developing palate. TGF-ß3 increased the expression of TNC mRNA and protein in primary mouse embryonic palatal mesenchymal cells (MEPM) obtained from palatal mesenchyme dissected at embryonic day 13.5-14.0. Interestingly, immunohistochemistry experiments revealed that TNC expression was diminished in K14-cre;Tgfbr2 fl/fl mice that lack the TGF-ß type II receptor in palatal epithelial cells and exhibit cleft soft palate, whereas TNC expression was maintained in Wnt1-cre;Tgfbr2 fl/fl mice that lack the TGF-ß type II receptor in palatal mesenchymal cells and exhibit a complete cleft palate. SHH also increased the expression of TNC mRNA and protein in MEPM cells. However, although TGF-ß3 up-regulated TNC mRNA and protein expression in O9-1 cells (a cranial neural crest cell line), SHH did not. Furthermore, TGF-ß inhibited the expression of osteoblastic differentiation markers (osterix and alkaline phosphatase) and induced the expression of fibroblastic markers (fibronectin and periostin) in O9-1 cells, whereas SHH did not affect the expression of osteoblastic and fibroblastic markers in O9-1 cells. However, immunohistochemistry experiments showed that TNC expression was diminished in the posterior palatal shelves of Shh-/+ ;MFCS4 +/- mice, which have deficient SHH signaling in the posterior palatal epithelium. Taken together, our findings support the proposal that TGF-ß and SHH signaling in palatal epithelium co-ordinate the expression of TNC in the posterior palatal mesenchyme through a paracrine mechanism. This signal cascade may work in the later stage of palatogenesis when cranial neural crest cells have differentiated into fibroblast-like cells. The spatiotemporal regulation of ECM-related proteins by TGF-ß and SHH signaling may contribute not only to tissue construction but also to cell differentiation or determination along the anterior-posterior axis of the palatal shelves.

A survey on medications received by elderly persons with dysphagia living at home or in a nursing home.

Polypharmacy in elderly persons living at home or in a nursing home is an issue. In the field of dentistry, strategies to reduce polypharmacy must be promoted; however, there is insufficient basic data on medications received by elderly persons with dysphagia living at home or in a nursing home. The subjects were 106 elderly persons with dysphagia living at home or in a nursing home. Based on their medical records, the presence of disease and number/type of drugs being administered were investigated. Stroke, dementia, and hypertension were common. The mean number of drugs per person was 6.3 (minimum: 0, maximum: 15). Drugs for digestive ulcers were the most frequently prescribed medication, followed by hypotensive drugs, anti-parkinsonism drugs, and other central nervous drugs. Fifty-nine patients (52.8%) had taken drugs that may cause dysphagia, and 19 (17.9%) had taken drugs that may cause aspiration. Of the subjects, 68.9% had taken ≥5 drugs, demonstrating polypharmacy in elderly persons with dysphagia living at home or in a nursing home. Many drugs that may cause dysphagia or aspiration had been prescribed, suggesting the importance of dentists' reducing polypharmacy from the viewpoint of swallowing.