Identification of citrullinated α1-antitrypsin (A1AT) in saliva in a mouse model of rheumatoid arthritis.

OBJECTIVES: Rheumatoid arthritis (RA) is an autoimmune disease characterized by progressive joint destruction. Early diagnosis and treatment, before joint deformation or destruction occurs, are crucial. Identifying novel biomarkers for RA in saliva could potentially enable early detection of the disease, prior to its onset. METHODS: We conducted a comprehensive proteomic analysis of salivary proteins in a mouse model of RA. Proteins were identified using western blotting and enzyme-linked immunosorbent assay in the serum, saliva, and ankle joints of DBA/1JJmsSlc mice, a model of RA. Ankle joints and submandibular glands were stained with hematoxylin and eosin and immunostained, and the results were compared with those of control mice. RESULTS: Citrullinated alpha-1 antitrypsin (A1AT, 46 kDa) was commonly detected in the saliva, serum, and ankle joints of mice with severe RA and was confirmed through proteomic analysis. Western blotting showed a band corresponding to 46 kDa in the serum, saliva, and ankle joints. Immunostaining of the ankle joints with the A1AT antibody showed a strong positive signal in the synovium. CONCLUSIONS: In DBA/1JJmsSlc mice, cyclic citrullinated peptide antibodies and A1AT may be involved in citrullination and contribute to the development and severity of RA, making them valuable treatment targets requiring further study.

Association between salivary proteases and protease inhibitors linked with viral infections and oral inflammatory diseases.

INTRODUCTION: Despite the role of transmembrane protease, serine 2 (TMPRSS2) in facilitating the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the primary cause of the global COVID-19 pandemic, the interaction of extracellular and intracellular proteases in this process remains poorly elucidated. Thus, we monitored the salivary expression concentration (SEC) of TMPRSS2 and its inhibitor, alpha-1 antitrypsin (A1AT), and investigated whether oral inflammatory diseases affected the SEC of both proteins. MATERIALS AND METHODS: We collected saliva samples before and after surgical treatment of inflammatory cystic diseases (radicular and inflammatory dentigerous cysts) in 25 patients. The SEC of TMPRSS2 and A1AT was measured using enzyme-linked immunosorbent assay. SEC in multiple patient status groups and subgroups of each status were investigated. Finally, the correlation between TMPRSS2 and A1AT SEC was analyzed. RESULTS: The TMPRSS2 and A1AT SEC did not significantly change pre- or post-treatment. The TMPRSS2 SEC was significantly higher before and after treatment in patients aged >50 years, patients with radicular cysts, and patients with the basic disease. A1AT SEC was significantly decreased after treatment in the acute inflammation, large-sized, and patients without basic disease groups. No significant correlation was observed between the SEC of either protein before and after treatment. DISCUSSION: Individual-specific SEC for TMPRSS2 may be influenced by age, lesion type, and basic disease; however, oral inflammatory diseases may not have a direct effect. Moreover, the extent of oral inflammatory diseases and the presence of basic diseases may be associated with A1AT SEC. Furthermore, the SEC between the two proteins may be independent.

Detection of SARS-CoV-2 and Its Related Factors on the Mucosal Epithelium of the Tongue.

SARS-CoV-2 infects a variety of tissues, including the oral cavity. However, there are few reports examining the association of SARS-CoV-2 with tongue mucosal tissues with sticky tongue debris. This study investigated the presence of SARS-CoV-2 and its associated molecules by dissecting tongue tissue from autopsy specimens of 23 patients who died of COVID-19-related illness (pneumonia). Immunohistochemical staining, electron microscopy, and PCR analysis were performed on the tongue tissue specimens. The mucosal epithelium of the tongue formed a very thick keratinized with well-developed filiform papillae in all cases. ACE2 and TMPRSS2 were consistently co-expressed in all samples in the epithelium. The S-protein was strongly expressed in basal cells and the epithelial surface. S-protein-positive viral particles were detected in the tongue's stratified squamous epithelium via an immunoelectron microscope. Based on PCR amplification of the N1 and N2 regions, the SARS-CoV-2 gene was detected on the tongue epithelium, tongue submucosa, and in tongue debris. This suggests that tongue debris, including the squamous epithelial tissue, could be a source of SARS-CoV-2 in saliva. Furthermore, removing tongue debris may decrease the amount of SARS-CoV-2 in the oral cavity.

Effects of Diabetes and Voluntary Exercise on IgA Concentration and Polymeric Immunoglobulin Receptor Expression in the Submandibular Gland of Rats.

Background and Objectives: Patients with diabetes are more susceptible to upper respiratory tract infections (URTIs) because they are easily infected. Salivary IgA (sali-IgA) levels play a major role in transmitting URTIs. Sali-IgA levels are determined by salivary gland IgA production and polymeric immunoglobulin receptor (poly-IgR) expression. However, it is unknown whether salivary gland IgA production and poly-IgR expression are decreased in patients with diabetes. While exercise is reported to increase or decrease the sali-IgA levels, it is unclear how exercise affects the salivary glands of patients with diabetes. This study aimed to determine the effects of diabetes and voluntary exercise on IgA production and poly-IgR expression in the salivary glands of diabetic rats. Materials and Methods: Ten spontaneously diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats (eight-week-old) were divided into two groups of five rats each: a non-exercise group (OLETF-C) and a voluntary wheel-running group (OLETF-E). Five Long-Evans Tokushima Otsuka (LETO) rats without diabetes were bred under the same conditions as the OLETF-C. Sixteen weeks after the study began, the submandibular glands (SGs) were collected and analyzed for IgA and poly-IgR expression levels. Results: IgA concentrations and poly-IgR expression levels in SGs were lower in OLETF-C and OLETF-E than in LETO (p < 0.05). These values did not differ between the OLETF-C and OLETF-E. Conclusions: Diabetes decreases IgA production and poly-IgR expression in the salivary glands of rats. Moreover, voluntary exercise increases sali-IgA levels but does not increase IgA production and poly-IgR expression in the salivary glands of diabetic rats. Increasing IgA production and poly-IgR expression in the salivary glands, which is reduced in diabetes, might require slightly higher-intensity exercise than voluntary exercise under the supervision of a doctor.

Prevalence of saliva immunoglobulin A antibodies reactive with severe acute respiratory syndrome coronavirus 2 among Japanese people unexposed to the virus.

While the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a threat to public health as the number of cases and COVID-19-related deaths are increasing worldwide, the incidence of the virus infection is extremely low in Japan compared with many other countries. To explain this uncommon phenomenon, we investigated the prevalence of naturally occurring ("natural") antibodies, focusing on those of the secretory immunoglobulin A (sIgA) form, reactive with SARS-CoV-2 among Japanese people. One hundred and eighty healthy Japanese volunteers of a wide range of age who had been considered to be unexposed to SARS-CoV-2 participated in this study. Saliva samples and blood samples were collected from all of the 180 participants and 139 adults (aged ≥ 20 years) included therein, respectively. The determination of saliva IgA antibodies, mostly comprising sIgA antibodies, as well as serum IgA and immunoglobulin G antibodies, reactive with the receptor binding domain of the SARS-CoV-2 spike-1 subunit proteins was conducted using an enzyme-linked immunosorbent assay. The major findings were that 52.78% (95% confidence interval, 45.21%-60.25%) of the individuals who had not been exposed to SARS-CoV-2 were positive for saliva IgA antibodies with a wide range of levels between 0.002 and 3.272 ng/mL, and that there may be a negative trend in positivity for the antibodies according to age. As we had expected, a frequent occurrence of assumable "natural" sIgA antibodies reactive with SARS-CoV-2 among the studied Japanese participant population was observed.

Analysis of false-negatives in exfoliative cytology in oral potentially malignant disorders: A retrospective cohort study.

INTRODUCTION: Keratinized lesions have been a conceivable false-negative (FN) factor in oral exfoliative cytology (OEC); however, other factors are poorly analyzed. In this study, we aimed to identify the factors influencing the accuracy of OEC and FNs focusing on the lesion characteristics, patient background, and surgeon factors in oral potentially malignant disorders (OPMD). MATERIAL AND METHODS: We retrospectively studied 44 patients who underwent both OEC and histopathological diagnosis. Sensitivity, specificity, FN rate, false-positive (FP) rate, and prevalence of both methods were compared. Similarly, accuracy indices were compared among clinical diagnosis groups (leukoplakia vs. other diagnosis). The association between patient and surgeon-related factors influencing FN OEC results were investigated using Fisher's exact test and a multiple logistic regression analysis. RESULTS: Overall, the sensitivity; specificity; and FN, FP, and prevalence rates of OEC were 31.8%, 82.1%, and 68.8%, 17.9%, and 36.4%, respectively. Leukoplakia was significantly more common in clinical diagnosis (P = 0.007) with sensitivity, specificity, and FN rates of 20.0%, 95.2%, and 80.0%, respectively. Contrarily, non-keratinized lesions had sensitivity, specificity, and FN of 83.3%, 85.7%, and 16.7%, respectively. In the prevalent group, leukoplakia and anucleate squamous cells were significantly associated with FN cases (P = 0.013, P = 0.050). On multivariate analysis in OEC negative patients, age ≤64 (P = 0.050) and location on the tongue (P = 0.047) was independently associated with FNs. CONCLUSION: FN of OEC was conceivable to be due to poor deep-seated cell sampling, which was associated with leukoplakia, age, and location. Therefore, these factors may be considered in the evaluation of OEC results.

Histopathological analysis of the association between mucosal epithelial changes and the lamina propria vascular network in irritation fibroma.

OBJECTIVES: Irritation fibroma in the oral cavity causes atrophy or squamous epithelium thickening with respect to external injury-associated factors. However, ulcers do not occur in most cases. This study aimed to elucidate the mechanism by which ulcers do not form, focusing on the vascular network in the mucosal epithelium of irritation fibroma. METHODS: Immunostaining was performed using an enzyme antibody method with primary antibodies against CD31 and Ki-67 in 17 cases of irritation fibroma in the buccal mucosa. One section was taken at three points from the margin and three points from just above the lesion for measurement. The number of blood vessels in the superficial and deep lamina propria at the measurement site were determined, and the area per blood vessel was measured. RESULTS: The number and area of blood vessels in the superficial lamina propria just below the lesion epithelium were smaller than those in the margin. No difference was observed in the number and area of blood vessels in the deep lamina propria between the margins and lesions. CONCLUSIONS: Our results suggest that the vascular network in the deep lamina propria is maintained and compensates for the nutrient supply to the covering epithelium.

Cognitive Dysfunction in a Mouse Model of Cerebral Ischemia Influences Salivary Metabolomics.

Vascular dementia, caused by cerebrovascular disease, is associated with cognitive impairment and reduced hippocampal metabolite levels. Specifically, cognitive impairment can be induced by decreased hippocampal brain-derived neurotrophic factor (BDNF) expression. The development of low or non-invasive biomarkers to characterize these diseases is an urgent task. Disturbance of metabolic pathways has been frequently observed in cognitive impairment, and salivary molecules also showed the potentials to reflect cognitive impairment. Therefore, we evaluated salivary metabolic profiles associated with altered hippocampal BDNF expression levels in a cerebral ischemia mouse model using metabolomic analyses. The effect of tacrine (a cholinesterase inhibitor) administration was also examined. The arteries of ICR mice were occluded with aneurysm clips to generate the cerebral ischemia model. Learning and memory performance was assessed using the elevated plus maze (EPM) test. Hippocampal and blood BDNF levels were quantified using an enzyme-linked immunosorbent assay. Glutamate decarboxylase 1 (GAD1) mRNA expression, is associated with cognitive impairment, was quantified by a real-time polymerase chain reaction. The EPM test revealed impaired spatial working memory in the cerebral ischemia mouse model; tacrine administration ameliorated this memory impairment. Cerebral ischemia suppressed GAD1 expression by decreasing hippocampal BDNF expression. In total, seven salivary metabolites, such as trimethylamine N-oxide and putrescine, were changed by cognitive impairment and tacrine administration. Our data suggest that salivary metabolite patterns were associated with cognitive function.

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.

Hypertriglyceridemia-induced brain-derived neurotrophic factor in rat submandibular glands.

OBJECTIVES: Salivary glands produce brain-derived neurotrophic factor (BDNF), which increases plasma BDNF content. Salivary BDNF influences the hippocampus and enhances anxiety-like behaviors. Dyslipidemia affects the brain, promoting depression and anxiety-like behaviors. This study was performed to investigate whether hypertriglyceridemia influences salivary BDNF expression. METHODS: Hypertriglyceridemia was induced in rats by high-fat diet intake for 10 weeks. BDNF protein levels in the saliva and submandibular glands were measured using enzyme-linked immunosorbent assay (ELISA). Bdnf mRNA levels in the submandibular gland were determined using real-time polymerase chain reaction. RESULTS: A hypertriglyceridemia rat model was established. Body weight did not differ between the control and hypertriglyceridemia groups. Bdnf mRNA and protein expression was increased in the submandibular gland in the hypertriglyceridemia group compared to the control group. BDNF expression was also significantly increased in the saliva of the hypertriglyceridemia group. CONCLUSIONS: This is first study to show that hypertriglyceridemia induces BDNF expression in the rat submandibular gland and suggests that salivary BDNF is associated with lipid metabolism.

Existence of SARS-CoV-2 Entry Molecules in the Oral Cavity.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor, angiotensin-converting enzyme 2 (ACE2), transmembrane protease serine 2 (TMPRSS2), and furin, which promote entry of the virus into the host cell, have been identified as determinants of SARS-CoV-2 infection. Dorsal tongue and gingiva, saliva, and tongue coating samples were examined to determine the presence of these molecules in the oral cavity. Immunohistochemical analyses showed that ACE2 was expressed in the stratified squamous epithelium of the dorsal tongue and gingiva. TMPRSS2 was strongly expressed in stratified squamous epithelium in the keratinized surface layer and detected in the saliva and tongue coating samples via Western blot. Furin was localized mainly in the lower layer of stratified squamous epithelium and detected in the saliva but not tongue coating. ACE2, TMPRSS2, and furin mRNA expression was observed in taste bud-derived cultured cells, which was similar to the immunofluorescence observations. These data showed that essential molecules for SARS-CoV-2 infection were abundant in the oral cavity. However, the database analysis showed that saliva also contains many protease inhibitors. Therefore, although the oral cavity may be the entry route for SARS-CoV-2, other factors including protease inhibitors in the saliva that inhibit viral entry should be considered.

Faster Short-Chain Fatty Acid Absorption from the Cecum Following Polydextrose Ingestion Increases the Salivary Immunoglobulin A Flow Rate in Rats.

Salivary immunoglobulin A (IgA) plays a vital role in preventing upper respiratory tract infections (URTI). In our previous study, we showed that the intake of carbohydrates increases the intestinal levels of short-chain fatty acids (SCFAs), which in turn increase salivary IgA levels. However, the mechanism underlying this phenomenon has not been fully elucidated. In this study, we investigated in rats the effect of polydextrose (PDX) ingestion on salivary IgA level and SCFA concentration in cecal digesta and the portal vein. Five-week-old rats were fed with a fiber-free diet (control) or with 40 g/kg of PDX for 28 days. Compared to the control, ingestion of PDX led to a higher salivary IgA flow rate (p = 0.0013) and a higher concentration of SCFAs in the portal vein (p = 0.004). These two data were positively correlated (rs = 0.88, p = 0.0002, n = 12). In contrast, the concentration of SCFAs in cecal digesta and cecal digesta viscosity were significantly lower following PDX ingestion, compared to the control (p = 0.008 and 0.05, respectively). These findings suggest that the ingestion of PDX increases the absorption rate of SCFAs in the intestine through PDX-induced fermentation, which is accompanied by an increase in SCFA levels in the blood, and ultimately leads to increased salivary IgA levels.

Comparison of CD34 expression in fibrous reactive hyperplasia and healthy oral mucosa.

OBJECTIVES: Fibrous reactive hyperplasia (FRH) is a common fibrous lesion in the oral cavity. The disease characteristics of FRH, including the expression patterns of CD34, which is a well-known fibroblast marker, have not been investigated in detail. Therefore, in this study, we aimed to investigate the characteristics of FRH compared to those of the healthy mucosa, based on CD34 expression profiles. METHODS: CD34 expression was analyzed at the protein and mRNA levels using immunohistochemistry, quantitative polymerase chain reaction, and in situ hybridization (ISH). RESULTS: CD34 was not expressed in the lamina propria of the oral mucosa, but was commonly observed in submucosal fibroblasts. CD34-positive fibroblasts were commonly observed in FRH. A total of 17 out of 19 cases (89.5%) were CD34-positive. Furthermore, we identified a significant difference in the ratio of CD34-positive cells between the healthy and FRH tissues. Quantitative polymerase chain reaction showed that CD34 mRNA was expressed in all cases of FRH, and CD34 mRNA expression in FRH samples was found to be localized to spindle-shaped fibroblasts, as determined by ISH. A positive correlation was also found between the CD34 mRNA levels and the proportion of the CD34-positive cells. CONCLUSIONS: These findings suggest that the increase in collagen synthesis in CD34-positive fibroblasts in the submucosa leads to the development of FRH. To our knowledge, this is the first report confirming the mRNA expression patterns of CD34 in FRH.

Brain-derived neurotrophic factor is related to stress and chewing in saliva and salivary glands.

Chewing is one of the most important orofacial functions. During this process, food is reduced in size, while saliva moistens the food and binds it into a bolus that can be easily swallowed. Characteristics of the oral system, including the number of teeth, bite force, and salivary flow, influence the masticatory process. In addition, salivary glands produce several cell growth factors and play an important role in human health. The nerve growth factor (NGF) family consists of NGF, brain-derived neurotrophic factor (BDNF), and neurotrophins-3 to 7. BDNF is a well-studied neurotrophin involved in the neurogenesis, differentiation, and maintenance of select peripheral and central neuronal cell populations during development and adulthood. However, there has been no detailed description of the expression of neurotrophins other than NGF in the salivary gland. We previously studied the effect of immobilization stress + chewing on BDNF secretion and its receptor, tyrosine receptor kinase B, in rat submandibular glands and found increased BDNF expression in duct cells under these conditions. In this review, we describe recent advances in understanding the role of stress and chewing-related BDNF in the saliva and salivary glands.

Histopathological analysis of the differential diagnosis of peripheral odontogenic fibroma from fibrous epulis.

OBJECTIVES: Peripheral odontogenic fibroma (POF) is a relatively rare odontogenic tumor of the gingiva. Although its histological differential diagnosis from fibrous epulis (FE) is important, no study has reported the differences in their expression of immunohistochemical markers. Here, we compared the expression of tumor markers that are frequently used for the differential diagnosis of fibroproliferative lesions between POF and FE. METHODS: Forty cases were selected, including 20 POF and 20 FE cases. CD34, B cell lymphoma (Bcl)-2, and Ki-67 were used as markers for immunohistochemical examination. The positive cell ratio was calculated, and Mann-Whitney U test was performed for statistical analysis. RESULTS: POF and FE were negative for CD34 expression but showed Bcl-2 and Ki-67 expression. The ratio of Bcl-2- and Ki-67-positive cells was significantly higher in POF than in FE (p < 0.001). CONCLUSIONS: POF is CD34 negative, and Bcl-2 and Ki-67 positive-cell ratio differs between POF and FE, suggesting that these proteins may serve as immunohistochemical markers for the differential diagnosis of POF.

Detection of anti-citrullinated protein antibody (ACPA) in saliva for rheumatoid arthritis using DBA mice infected with Porphyromonas gingivalis.

OBJECTIVE: The anti-citrullinated protein antibody (ACPA), an autoantibody of rheumatoid arthritis (RA), is very specific in the diagnosis of RA and has been detected in early cases and several years before the onset of the disease. In this study, we focused on ACPA and examined whether it could be detected in saliva whether it is associated with periodontal disease. DESIGN: Porphyromonas gingivalis (Pg) or Escherichia coli (Ec) was administered into the oral cavity of DBA/1JJmsSlc mice. The arthritis index was measured in foot bones, and collected saliva and serum. The amount of ACPA in serum and saliva was measured using ELISA, and antibodies in serum, saliva, and foot bones were detected and analysed by western blotting. RESULT: Histopathological analysis of foot bones of the Pg/RA group detected greater inflammatory cell infiltration than in the RA group, and bone resorption was evident. Furthermore, ELISA results show that the amount of ACPA in serum was significantly higher in the Pg/RA group (P < 0.05), with a tendency to also increase in the saliva. In addition, western blotting results show a 55 kDa citrullinated protein in the serum and saliva of the RA and Pg/RA groups. CONCLUSIONS: We conclude that Pg infection increases ACPA in the serum and is reflected in the saliva, and may be involved in the inflammatory progression of RA.