Histamine deficiency inhibits lymphocyte infiltration in the submandibular gland of aged mice via increased anti-aging factor Klotho

ObjectivesHistidine decarboxylase (HDC), a histamine synthase, is expressed in various tissues and is induced by proinflammatory cytokines such as TNFα. As they age, C57BL/6 mice show auto-antibody deposition and lymphocyte infiltration into various tissues, including salivary glands. However, the mechanism underlying cell infiltration and the change in HDC expression in salivary glands with aging remain unclear. Thus, we aimed to elucidate the relationship between histamine and inflammaging.MethodsWe investigated the change in histology and HDC expression in the major salivary glands (parotid, submandibular, and sublingual) of 6-week- and 9-month-old wild-type mice. We also determined the histological changes, cytokine expression, and anti-aging factor Klotho in the salivary glands of 9-month-old wild-type and HDC-deficient (HDC-KO) mice.ResultsCell infiltration was observed in the submandibular gland of 9-month-old wild-type mice. Although most cells infiltrating the submandibular glands were CD3-positive and B220-positive lymphocytes, CD11c-positive and F4/80-positive monocyte lineages were also detected. HDC, TNFα, and IL-1β mRNA expression increased in the submandibular gland of 9-month-old wild-type mice. The expression of PPARγ, an anti-inflammatory protein, declined in 9-month-old wild-type mice, and Klotho expression increased in 9-month-old HDC-KO mice. Immunohistochemistry showed that Klotho-positive cells disappeared in the submandibular gland of 9-month-old wild-type mice, while Klotho was detected in all salivary glands in HDC-KO mice of the same age.ConclusionOur findings demonstrate the multifunctionality of histamine and can aid in the development of novel therapeutic methods for inflammatory diseases such as Sjogren's syndrome and age-related dysfunctions.     

Adipose-derived mesenchymal stem cells promote salivary duct regeneration via a paracrine effect

ObjectivesThe self-regeneration of exocrine tissues, including salivary glands, is limited and their regeneration mechanism has not yet been fully elucidated. Here we identify the role of adipose-derived mesenchymal stem cells (AMSCs) in salivary gland regeneration.MethodsAMSCs expressing mesenchymal stem cell markers were applied to a submandibular gland injury model and the mechanism of salivary gland repair and regeneration was analyzed.ResultsTransplanted green fluorescent protein (GFP)-labeled AMSCs grew tightly together and promoted ductal regeneration in the regenerative nodule, with slight infiltration of nonspecific immune cells. A comprehensive gene analysis through RNA-sequencing revealed increased expression of bone morphogenetic protein (BMP), transforming growth factor (TGF), and Wnt in AMSC-transplanted regenerative nodules. The factors released from AMSCs scavenge hydrogen peroxidase-induced reactive oxygen species (ROS) through Wnt promoter activity in vitro. Furthermore, AMSC-conditioned medium recovered the growth of the hydrogen peroxidase-damaged primordium of the submandibular gland culture ex vivo.ConclusionsThese results suggest that AMSC-released factors scavenge ROS and maintain salivary gland repair and regeneration via paracrine effects. Thus, AMSCs could be a practical and applicable tool for use in salivary gland regeneration.     

A Ser252Trp substitution in mouse FGFR2 results in hyperplasia of embryonic salivary gland parenchyma

ObjectivesMutations in the fibroblast growth factor receptor 2 (FGFR2) gene are responsible for several severe forms of craniosynostotic disorders, such as Apert and Crouzon syndromes. Patients with craniosynostotic disorders caused by a mutation in Fgfr2 present with several clinical symptoms, including hypersalivation. Here we used a transgenic mouse model of Apert syndrome (Fgfr2^+/S252W mice) to evaluate the morphology of the submandibular glands at embryonic day 15.5 (E15.5), the time point reported to mark the start of lumen formation.MethodsFgfr2^+/S252W mice were generated by crossing ACTB-Cre^+/+ and Fgfr2^+/Neo-S252W mice. After measuring body weight, the submandibular glands were collected at E15.5. H&E staining, immunostaining, and RT-qPCR were performed to investigate the development of the submandibular gland.ResultsThe number of ducts and acini in Fgfr2^+/S252W mice was significantly higher than in control littermates; however, lumen formation was not affected. The mRNA expression of Fgf1, Fgfr1, Mmp2, Bmp4, Bmp7, Dusp6, and Etv5 in Fgfr2^+/S252W mice was significantly higher compared to control littermates. Immunoreactivity for FGF3, FGF1, BMP4, and F4/80 was detected in the parenchyma of Fgfr2^+/S252W mice. The area of apoptotic cells stained with TUNEL in Fgfr2^+/S252W mice was significantly larger than that of the control littermates.ConclusionsThese results suggested that increased FGFR1 signaling and apoptosis in the submandibular glands of Fgfr2^+/S252W mice occurred at E15.5, leading to parenchymal hyperplasia. This study demonstrated that a Ser252Trp substitution in mouse FGFR2 resulted in hyperplasia of the submandibular gland parenchyma during development.     

Effect of Myd88 deficiency on gene expression profiling in salivary glands of female non-obese diabetic (NOD) mice

ObjectivesSjögren's syndrome (SS) is a chronic autoimmune disease characterized by inflammatory lesions in the salivary and lacrimal glands, which are caused by distinct lymphocytic infiltrates. Female non-obese diabetic (NOD) mice spontaneously develop inflammatory lesions of the salivary glands with SS-like pathological features. Previous studies have shown that MyD88, a crucial adaptor protein that activates innate immune signaling, affects lymphocytic infiltration, but its detailed role remains unclear. In this study, we investigated the role of MyD88 through gene expression profiling in the early phase of pathogenesis in the salivary glands of female NOD mice.MethodsSubmandibular glands collected from 10-week-old female wild-type and Myd88-deficient NOD mice were used for RNA preparation, followed by microarray analysis. The microarray dataset was analyzed to identify Myd88-dependent differentially expressed genes (DEGs). Data generated were used for GO enrichment, KEGG pathway, STRING database, and INTERFEROME database analyses.ResultsMyd88 deficiency was found to affect 230 DEGs, including SS-associated genes, such as Cxcl9 and Bpifa2. Most of the DEGs were identified as being involved in immunological processes. KEGG pathway analysis indicated that the DEGs were putatively involved in autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis. Furthermore, the DEGs included 149 interferon (IFN)-regulated genes.ConclusionsMyD88 is involved in the expression of specific genes associated with IFN-associated immunopathological processes in the salivary glands of NOD mice. Our findings are important for understanding the role of MyD88-dependent innate immune signaling in SS manifestation.     

Assessment of the possible ameliorative effect of curcumin nanoformulation on the submandibular salivary gland of alloxan-induced diabetes in a rat model (Light microscopic and ultrastructural study)

BackgroundNowadays, attention is directed to herbal treatments in an attempt to lessen the adverse effects of diabetes. Nanoformulation of curcumin (NC) was shown to enhance stability and water solubility compared to native curcumin.ObjectiveTo examine the effect of different NC concentrations on the histopathological structure of the submandibular salivary gland of diabetic rats.Methods28 rats were divided equally into 4 groups. Group I: Control group, Group II (diabetic), III (diabetic + nanocurcumin low dose), and IV (diabetic + nanocurcumin high dose): Rats of groups II, III and IV were injected with a single dose of alloxan (140 mg/kg) to induce diabetes. After 7 days, groups III and IV were treated for 6 weeks with NC (100 mg/kg/day) for group III, and (200 mg/kg/day) for group IV. Submandibular salivary glands were assessed histologically, immunohistochemically using α smooth muscle actin (α SMA) and ultrastructurally.ResultsDiabetic samples showed destruction of parenchymal elements of the gland, with thick fiber bundles encircling the excretory ducts and minimal reaction for α SMA. Amelioration of the gland's architecture was detected in groups III and IV with reduction of collagen deposition and elevation of positive immunoreactivity to α SMA.ConclusionNC profoundly repaired the induced diabetic histopathological and ultrastructural alterations of the gland in a dose dependent manner.     

Saliva diagnostics: Salivaomics,saliva exosomics,and saliva liquid biopsy

BackgroundEach day, humans produce approximately 0.5 through 1.5 liters of saliva, a biofluid that is rich in biological omic constituents. Our lack of understanding how omic biomarkers migrate from diseased tissue to the saliva has impeded the clinical translation of saliva testing. Although such biomarkers must be conveyed via the vascular and lymphatic systems to the salivary glands, the molecular mechanisms that underlie this transport remain unclear. Although COVID-19 highlighted the need for rapid and reliable testing for infectious diseases, it represents only one of the many health conditions that potentially can be diagnosed using a saliva sample.Types of Studies ReviewedThe authors discuss salivaomics, saliva exosomics, and the mechanisms on which saliva diagnostics are based and introduce a novel electrochemical sensing technology that may be exploited for saliva liquid biopsy.ResultsThe utility of saliva for screening for lung cancer is under investigation. Saliva testing may be used to stratify patients, monitor treatment response, and detect disease recurrence. The authors also highlight the landscapes of saliva-based SARS-CoV-2 testing and ultrashort cell-free DNA and outline how these fields are likely to evolve in the near future.Practical ImplicationsBreakthroughs in the study of saliva research, therefore, will facilitate clinical deployment of saliva-based testing.     

Localization of phospholipid-related signal molecules in salivary glands of rodents: A review

BackgroundIn the 1950s, Hokin conducted initial studies on phosphoinositide turnover/cycle in salivary glandular cells. From these studies, the idea emerged that receptor-mediated changes in intramembranous levels of phosphoinositides represent an early step in the stimulus-response pathway. Based on this idea and the general view that knowledge of the exact localization of a given endogenous molecule in cells in situ is important for understanding its functional significance, we have reviewed available information about the localization of several representative phosphoinositide-signaling molecules in the salivary glands in situ in mice.HighlightWe focused on phosphatidylinositol 4-kinase, phosphatidylinositol 4 phosphate 5-kinase α, β, γ, phospholipase Cβ, muscarinic cholinoceptors 1 and 3, diacylglycerol kinase ζ, phospholipase D1 and 2, ADP-ribosylation factor 6 and its exchange factors for Arf6, and cannabinoid receptors. These molecules individually exhibit differential localization in a spatiotemporal manner in the exocrine glands, making it possible to deduce their functional significance, such as their involvement in secretion and cell differentiation.ConclusionAlthough phosphoinositide-signaling molecules whose in situ localization in glandular cells has been clarified are still limited, the obtained information on their localization suggests that their functional significance is more valuable in glandular ducts than in acini. It thus suggests the necessity of greater attention to the ducts in their physio-pharmacological analyses. The purpose of this review is to encourage more in situ localization studies of phosphoinositide-signaling molecules with an aim to further understand their possible involvement in the pathogenesis of salivary gland diseases.     

Review of genes potentially related to hyposecretion in male non-obese diabetic (NOD) mice,a Sjögren's syndrome model

BackgroundSjögren's syndrome (SS) is known to cause dry eyes and mouth due to inflammation of the lacrimal and salivary glands. However, some reports imply that other factors trigger dry eyes and mouth. We previously investigated various factors using RNA-sequencing analysis of lacrimal glands from male non-obese diabetic (NOD) mice, an SS model. In this review, we described (1) the exocrine features of male and female NOD mice, (2) the up- and down-regulated genes in the lacrimal glands of male NOD mice as revealed by our RNA-sequencing data, and (3) comparisons between these genes and data in the Salivary Gland Gene Expression Atlas.HighlightsMale NOD mice exhibit a steady worsening of lacrimal hyposecretion and dacryoadenitis, whereas females exhibit a complex pathophysiological condition that includes diabetic disease, salivary hyposecretion, and sialadenitis. Ctss, an up-regulated gene, is a potential inducer of lacrimal hyposecretion and is also expressed in salivary glands. Two other up-regulated genes, Ccl5 and Cxcl13, may worsen the inflammation of SS in both the lacrimal and salivary glands. The genes Esp23, Obp1a, and Spc25 were detected as down-regulated, but judging the relationship between these genes and hyposecretion is difficult as only limited information is available. Another down-regulated gene, Arg1, is involved in lacrimal hyposecretion, and it also has the potential to cause salivary hyposecretion in NOD mice.ConclusionIn NOD mice, males may be better than females at evaluating the pathophysiology of SS. Some regulated genes revealed by our RNA-sequencing data might be potential therapeutic targets for SS.     

Salivary factors related to caries in pregnancy: A systematic review and meta-analysis

BackgroundThe authors of this meta-analysis aimed to assess saliva-related caries risk factors, including calcium and phosphate, hydrogen ion concentration, buffer capacity, Streptococcus mutans and Lactobacillus counts, flow rate, and decayed, missing and filled teeth index in each trimester during pregnancy.Types of Studies ReviewedThe authors searched electronic databases up to July 1, 2019. Eligible observational studies were included. The authors assessed the quality of the included studies by using the Joanna Briggs Institute scale. To estimate the effects of pregnancy, standardized mean differences with 95% confidence intervals were pooled using the random-effects model. Subgroup analysis and meta-regression were used to explore heterogeneity. Publication bias was assessed using Begg and Egger tests.ResultsTwenty-nine studies were included in the meta-analysis, representing 1,230 pregnant women in the case groups and 715 in the control groups (nonpregnant women). The results showed that salivary calcium concentration decreased in the third trimester, salivary phosphate decreased in the second and third trimesters, saliva hydrogen ion concentration decreased in the first and third trimesters, stimulated saliva flow rate increased in the third trimester, and salivary S mutans count increased in the second and third trimesters. In addition, the results showed that saliva calcium, phosphate, S mutans, and buffer capacity amounts had changed from the first trimester to the third.Conclusions and Practical ImplicationsIn the third trimester, most salivary factors related to caries change and can increase the risk of developing caries in the future. Interventions and screening for caries prevention in pregnancy should start in the first or second trimesters.     

The influence of periodontal status and serum biomarkers on salivary leptin levels in systemic lupus erythematosus patients

ObjectiveThis study aimed to investigate the influence of periodontal status, clinical data, and serum markers on salivary leptin levels in patients with systemic lupus erythematosus (SLE).MethodsA case–control study was conducted with 38 patients with SLE and 29 healthy controls. Periodontal data included periodontal probing depth (PPD), clinical attachment level (CAL), and gingival bleeding on probing (BOP). Stimulated saliva samples were collected to analyze salivary leptin levels. Clinical and serum data were collected from the SLE group. Statistical analysis included the t-test, Mann–Whitney test, Spearman correlation coefficient, and a structural equation model.ResultsThe SLE group had a lower salivary leptin level than the control group (P = 0.002). The model revealed that SLE had an inverse and independent effect on salivary leptin (standardized estimate =  ? 0.289, P = 0.023). Moreover, salivary leptin level negatively correlated with the serum levels of triglyceride, creatinine, and leukocytes, positively correlated with the serum total cholesterol, but was not significantly correlated with the periodontal status.ConclusionThese findings suggest that patients with SLE have a lower salivary leptin level. In addition, the level of salivary leptin does not appear to be related to periodontal status in patients with SLE.     

Responses of salivary glands to intake of soft diet

BackgroundModernization has made individuals prefer processed and cooked foods (soft food), but this eating habit may have negative effects on the oral cavity. However, laboratory animals fed with soft diet are commonly used in an attempt to clarify this issue, and various oral tissues, including the salivary glands have been examined. In this review, we summarize the findings of previous studies concerning the responses of salivary glands to daily intake of soft diet.HighlightThe weight of the parotid glands decreased in rodents fed with soft diet (liquid or powder). In atrophic parotid glands, acinar cell shrinkage is histologically observed and the DNA content is reduced, showing that the atrophy is caused by a decrease in the size and number of acinar cells. Immunohistochemical examinations showed that the decrease in the acinar cell number was induced by suppression of acinar cell proliferation and acceleration of apoptosis. The atrophic parotid glands recovered following a change from soft to pellet diet. Other salivary glands, such as the submandibular, sublingual, and palatine glands, responded only slightly to the soft diet feeding.ConclusionAccumulated research data showed that a soft diet negatively affects the parotid glands much more than other salivary glands and that atrophic parotid glands are able to recover by switching to a hard diet. Therefore, it should be emphasized that good eating habits are important for not only digestion but also the health of oral tissues, including the salivary glands.     

The impact of salivary lactoperoxidase and histatin-5 on early childhood caries severity in relation to nutritional status

IntroductionEarly childhood caries is a multifactorial disease. Saliva plays an important role in initiation and protection against caries, and its composition is greatly affected by nutritional status. This study was conducted to determine the impact of salivary lactoperoxidase and histatin-5 on the severity of ECC in relation to nutritional status.Materials and methodsThe sample consisted of 120 children aged 5 years, classified into eight groups: mild ECC in underweight children, mild ECC in normalweight children, moderate ECC in underweight children, moderate in ECC normal weight children, severe ECC in underweight children, severe ECC in normalweight, caries-free (control) underweight children and caries-free normalweight children. Each group consisted of 15 children. Stimulated saliva was collected. Salivary lactoperoxidase was analysed using Human LPO/ Lactoperoxidase ELISA Kit (CLIA)-LS-F29892, and salivary histatin-5 was analysed using Human Histatin-5 ELISA Kit MBS705083_48T.ResultsLactoperoxidase and histatin-5 concentrations were significantly higher in caries-free children than in children with ECC, and they were higher in children with mild ECC than in children with moderate ECC or in children with severe ECC. They were significantly higher among children with normal weight than among those who were underweight (p < 0.01). ECC and nutritional status recorded non-significant interactions with both LPO and HST-5 (p > 0.01), but there was significant interaction between these two variables and LPO and HST-5 together (p < 0.01). The Pearson's correlation coefficient test recorded significant negative correlations between ECC severity and both salivary lactoperoxidase and histatin-5 among the eight study groups, whereas significant positive correlations were recorded between BMI values and both salivary lactoperoxidase and histatin-5 among the eight study groups.ConclusionSalivary lactoperoxidase and histatin-5 may be affected by nutritional status, and these two parameters may play an important role in caries prevention at high concentrations. There is interaction between these two parameters and ECC severity and nutrition.     

Impact of SARS-CoV-2 on saliva: TNF-⍺, IL-6, IL-10, lactoferrin,lysozyme, IgG,IgA, and IgM

ObjectivesUnderstanding the role of certain salivary components, such as TNF-?, IL-6, IL-10, lactoferrin, lysozyme, IgG, IgA, and IgM, in airway defense during the ongoing SARS-CoV-2 pandemic is essential. The salivary immune barrier of patients with COVID-19 may play a role in their prognosis. The present study aims to evaluate the impact of SARS-CoV-2 on saliva composition.MethodsA longitudinal study was carried out with male and female firefighters aged 24–48 years. The study sample (n = 34) was divided into 3 groups: asymptomatic volunteers with a negative polymerase chain reaction (PCR) test for SARS-CoV-2 (group 1, Control, n = 21); patients with symptoms of COVID-19 of less than 7 days’ duration and a diagnosis of SARS-CoV-2 infection by PCR (group 2, COVID-19, n = 13); and recovered patients from group 2 who were free of COVID-19 symptoms for at least 2 months (group 3, post-COVID-19 recovery, n = 13). All groups underwent real-time PCR to detect the presence of SARS-CoV-2, as well as analysis of the salivary concentrations of TNF-?, IL-6, IL-10, lactoferrin, lysozyme, IgG, IgA, and IgM by the ELISA method.ResultsLactoferrin concentrations were significantly decreased in the infected group (COVID-19) when compared to those not infected by SARS-CoV-2 (control) (p = 0.032). IgA concentrations were decreased in the COVID-19 and post-COVID-19 groups compared to the control group (p = 0.005 and p = 0.016, respectively). Comparison of the COVID-19 and post-COVID-19 groups also revealed an increase in IgM concentrations during acute SARS-CoV-2 infection (p = 0.010).ConclusionSARS-CoV-2 alters the composition of the salivary immune barrier.     

Conditioned-medium of stem cells from human exfoliated deciduous teeth prevent apoptosis of neural progenitors

PurposeThis study aimed to evaluate the neuroprotective ability of the conditioned medium of stem cells from human exfoliated deciduous teeth (CM-SHED) to prevent glutamate-induced apoptosis of neural progenitors.Materials and methodsNeural progenitors were isolated from two-day-old rat brains, and the conditioned medium was obtained from a mesenchymal stem cell SHED. Four groups were examined: neural progenitor cells cultured in neurobasal medium with (N + ) and without (N-) glutamate and glycine, and neural progenitor cells cultured in CM-SHED with (K + ) and without (K-) glutamate and glycine.ResultsThe expression of GABA A1 receptor (GABAAR1) messenger RNA (mRNA) in neural progenitor measured by real-time quantitative PCR. GABA contents were measured by enzyme-linked immunosorbent assay, whereas the apoptosis markers caspase-3 and 7-aminoactinomycin D were analysed with a Muse® cell analyzer. The viability of neural progenitor cells in the K + group (78.05 %) was higher than the control group N- (73.22 %) and lower in the N + group (68.90 %) than in the control group. The K + group showed the highest GABA content, which significantly differed from that in the other groups, whereas the lowest content was observed in the N + group. The expression level of GABAAR1 mRNA in the K + group was the highest compared to that in the other groups. CM-SHED potently protected the neural progenitors from apoptosis.ConclusionsCM-SHED may effectively prevent glutamate-induced apoptosis of neural progenitors.     

Identification of salivary volatile organic compounds as potential markers of stomach and colorectal cancer: A pilot study

ObjectivesThe purpose of the pilot study was to determine the potential diagnostic capabilities for the analysis of oxygen-containing salivary volatile organic compounds (VOCs) in stomach and colorectal cancer.MethodsSaliva samples of 11 patients with stomach cancer, 18 patients with colorectal cancer, and 16 healthy volunteers were analyzed through capillary gas chromatography. The levels of lipid peroxidation products and catalase activity were determined in all samples. To assess saliva diagnostic potential, we constructed a Classification and Regression Tree (CART).ResultsIt was shown that the use of a combination of saliva VOCs (acetaldehyde, acetone, propanol-2, and ethanol) allowed classification into Cancer/Control groups with a sensitivity and specificity of 95.7 and 90.9%, respectively. To clarify the location of the tumor, it was necessary to add a methanol level; in this case, the sensitivity for detecting stomach and colorectal cancer was 80.0% and 92.3%, respectively, while the specificity in both cases was 100%. When the lipid peroxidation product content was added to the VOC indicators, they were selected as the main factors for constructing the decision tree. For classification into Cancer/Control groups, only the triene conjugate and Schiff base content in saliva was sufficient. The combination of VOCs in saliva and lipid peroxidation indices improved the sensitivity and specificity for classification to 100%.ConclusionPreliminary data were obtained on the sensitivity and specificity of the diagnosis of stomach and colorectal cancer, which confirmed the promise of further studies on saliva VOCs for the purpose of clinical laboratory diagnostics.     

In silico exploration of enzymes involved in sialic acid biosynthesis and their possible role in SARS-CoV-2 infection

Salivary glands are considered important targets of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Recent evidence suggests that along with angiotensin converting enzyme 2, certain cell surface sialic acids (Sia) may function as receptors for binding SARS-CoV-2 spike protein. Over 50 forms of Sia have been identified in nature, with N-acetylneuraminic acid (Neu5Ac) being the most abundant. We explored the Human Protein Atlas repository to analyze important enzymes in Neu5Ac biosynthesis and propose a hypothesis that further highlights the significance of salivary glands in coronavirus disease 19 (COVID-19). This work may facilitate research into targeted drug therapies for COVID-19.