Salivary Gland Cells Research Articles

Super-resolution microscopy reveals stochastic initiation of replication in Drosophila polytene chromosomes

Studying the probability distribution of replication initiation along a chromosome is a huge challenge. Drosophila polytene chromosomes in combination with super-resolution microscopy provide a unique opportunity for analyzing the probabilistic nature of replication initiation at the ultrastructural level. Here, we developed a method for synchronizing S-phase induction among salivary gland cells. An analysis of the replication label distribution in the first minutes of S phase and in the following hours after the induction revealed the dynamics of replication initiation. Spatial super-resolution structured illumination microscopy allowed identifying multiple discrete replication signals and to investigate the behavior of replication signals in the first minutes of the S phase at the ultrastructural level. We identified replication initiation zones where initiation occurs stochastically. These zones differ significantly in the probability of replication initiation per time unit. There are zones in which initiation occurs on most strands of the polytene chromosome in a few minutes. In other zones, the initiation on all strands takes several hours. Compact bands are free of replication initiation events, and the replication runs from outer edges to the middle, where band shapes may alter.

Apigenin, a Single Active Component of Herbal Extract, Alleviates Xerostomia via ERα-Mediated Upregulation of AQP5 Activation.

Xerostomia is a common symptom in menopausal women, suggesting the role of sex steroids in disease development. Shreds of literature had reported the potential use of herbal extracts to relieve xerostomia. However, a cocktail of multiple components in herbal extract makes it difficult to understand the exact mechanism of action. Aquaporin5 (AQP5), the specific aquaporin expressed in salivary glands, plays an important role in salivary secretion as a downstream of estrogen signaling. In this study, we aimed to unravel a single active herbal component as a therapeutic for xerostomia and investigate its mechanism of action. The effects of apigenin (flavonoid), dauricine (alkaloids), protopine (alkaloids), and lentinan (polysaccharides) on AQP5 transcription were screened in vitro. Only apigenin robustly induced AQP5 transcription and expression, and this effect was even robust compared to the effect of estradiol (E2, a positive control). Overexpression of estrogen receptor α (ERα) in the human salivary gland cell line (HSG) upregulated the AQP5 transcription and expression and the knockdown ERα reversed this effect, suggesting the role of ERα signaling on AQP5 activation in HSG cells. Docking results showed apigenin-specific binding sites in ERα. We further analyzed the therapeutic effect of apigenin on ovariectomized mice as a xerostomia model. The saliva secretion in the xerostomia group was reduced to one-third of the sham group, whereas the apigenin or E2 treatment for 12 weeks reversed this effect. Meanwhile, the water consumption in the xerostomia group was augmented obviously compared to the sham group, whereas the water consumption in the apigenin and E2 group was declined to the level of the sham group. Immunohistochemistry of submandibular glands revealed the downregulation of AQP5 expression in xerostomia mice compared to control. Apigenin, or E2 treatment, upregulated AQP5 expression in xerostomia mice. In conclusion, apigenin, a single active component of herbal extract, upregulated AQP5 expression in HSG cells via activation of ERα signaling and restored saliva flow rates in OVX mice. These results revealed apigenin as a single active component of herbal extract with the potential to treat xerostomia.

Fine needle aspiration cytology diagnosed pleomorphic adenoma of soft palate- A rare case report

Pleomorphic adenoma (PA) is benign tumor of salivary gland. Pleomorphic adenoma contains both epithelial and myoepithelial elements and it arises from myoepithelial cells of salivary gland. The index study to describe rare case of pleomorphic adenoma of soft palate diagnosed by FNAC (fine needle aspiration cytology).

CCR6 activation links innate immune responses to mucosa-associated lymphoid tissue lymphoma development.

The genesis of extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) is driven by oncogenic co-operation among immunological stimulations and acquired genetic changes. We previously identified recurrent CCR6 mutations in MALT lymphoma, with majority predicted to result in truncated proteins lacking the phosphorylation motif important for receptor desensitization. Functional consequences of these mutational changes, the molecular mechanisms of CCR6 activation and how this receptor signaling contributes to MALT lymphoma development remain to be investigated. In the present study, we demonstrated that these mutations impaired CCR6 receptor internalization and were activating changes, being more potent in apoptosis resistance, malignant transformation, migration and intracellular signaling, particularly in the presence of the ligands CCL20, HBD2 (human b defensin 2) and HD5 (human a defensin 5). CCR6 was highly expressed in malignant B cells irrespective of the lymphoma sites. HBD2 and CCL20 were constitutively expressed by the duct epithelial cells of salivary glands, and also those involved in lymphoepithelial lesions (LEL) in salivary gland MALT lymphoma. While in the gastric setting, HBD2, and HD5, to a less extent CCL20, were highly expressed in epithelial cells of pyloric and intestinal metaplasia respectively including those involved in LEL, which are adaptive responses to chronic Helicobacter pylori infection. These findings suggest that CCR6 signaling is most likely active in MALT lymphoma, independent of its mutation status. The observations explain why the emergence of malignant B cells and their clonal expansion in MALT lymphoma are typically around LEL, linking the innate immune responses to lymphoma genesis.

The LRRC family of BK channel regulatory subunits: potential roles in health and disease.

Large conductance K+ channels, termed BK channels, regulate a variety of cellular and physiological functions. Although universally activated by changes in voltage or [Ca2+ ]i , the threshold for BK channel activation varies among loci of expression, often arising from cell-specific regulatory subunits including a family of leucine rich repeat-containing (LRRC) γ subunits (LRRC26, LRRC52, LRRC55 and LRRC38). The 'founding' member of this family, LRRC26, was originally identified as a tumour suppressor in various cancers. An LRRC26 knockout (KO) mouse model recently revealed that LRRC26 is also highly expressed in secretory epithelial cells and partners with BK channels in the salivary gland and colonic goblet cells to promote sustained K+ fluxes likely essential for normal secretory function. To accomplish this, LRRC26 negatively shifts the range of BK channel activation such that channels contribute to K+ flux near typical epithelial cell resting conditions. In colon, the absence of LRRC26 increases vulnerability to colitis. LRRC26-containing BK channels are also likely important regulators of epithelial function in other loci, including airways, female reproductive tract and mammary gland. Based on an LRRC52 KO mouse model, LRRC52 regulation of large conductance K+ channels plays a role both in sperm function and in cochlear inner hair cells. Although our understanding of LRRC-containing BK channels remains rudimentary, KO mouse models may help define other organs in which LRRC-containing channels support normal function. A key topic for future work concerns identification of endogenous mechanisms, whether post-translational or via gene regulation, that may impact LRRC-dependent pathologies.

ACE2 and TMPRSS2 immunolocalization and oral manifestations of COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry into the host cells depends on the expression of angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2). We investigated the distribution of ACE2- and TMPRSS2-expressing cells in various oral tissues to identify the underlying mechanism of oral manifestations in patients with coronavirus disease 2019. We analyzed the expression patterns of ACE2 and TMPRSS2 in the oral mucosa (tongue, palate, and buccal mucosa), trigeminal ganglion, vessels, and salivary glands of 9 Sprague-Dawley rats using immunohistochemistry and immunofluorescence. ACE2 and TMPRSS2 were strongly expressed in the intermediate layer of the squamous epithelia of tongue papillae and buccal mucosa. ACE2- and TMPRSS2-positive cells were observed in the taste buds of the tongue. Additionally, ACE2 and TMPRSS2 were co-expressed in the ductal epithelium and acinar cells of salivary glands. Furthermore, both ACE2 and TMPRSS2 were stained in the neuronal cell body of trigeminal ganglia, but not in Schwann cells. Moreover, ACE2 and TMPRSS2 were expressed in capillaries, but not in venules/arterioles. SARS-CoV-2 can spread the suprabasal area of squamous epithelia of the oral mucosa, invades taste bud, trigeminal nerve, parotid gland, and microvessel, resulting in oral manifestations.

Encapsulation of Primary Salivary Gland Acinar Cell Clusters and Intercalated Ducts (AIDUCs) within Matrix Metalloproteinase (MMP)-Degradable Hydrogels to Maintain Tissue Structure and Function.

Progress in the development of salivary gland regenerative strategies is limited by poor maintenance of the secretory function of salivary gland cells (SGCs) in vitro. To reduce the precipitous loss of secretory function, a modified approach to isolate intact acinar cell clusters and intercalated ducts (AIDUCs), rather than commonly used single cell suspension, is investigated. This isolation approach yields AIDUCs that maintain many of the cell-cell and cell-matrix interactions of intact glands. Encapsulation of AIDUCs in matrix metalloproteinase (MMP)-degradable PEG hydrogels promotes self-assembly into salivary gland mimetics (SGm) with acinar-like structure. Expression of Mist1, a transcription factor associated with secretory function, is detectable throughout the in vitro culture period up to 14 days. Immunohistochemistry also confirms expression of acinar cell markers (NKCC1, PIP and AQP5), duct cell markers (K7 and K5), and myoepithelial cell markers (SMA). Robust carbachol and ATP-stimulated calcium flux is observed within the SGm for up to 14 days after encapsulation, indicating that secretory function is maintained. Though some acinar-to-ductal metaplasia is observed within SGm, it is reduced compared to previous reports. In conclusion, cell-cell interactions maintained within AIDUCs together with the hydrogel microenvironment may be a promising platform for salivary gland regenerative strategies.

Alginate Hydrogel Microtubes for Salivary Gland Cell Organization and Cavitation.

Understanding the different regulatory functions of epithelial and mesenchymal cell types in salivary gland development and cellular organization is essential for proper organoid formation and salivary gland tissue regeneration. Here, we demonstrate a biocompatible platform using pre-formed alginate hydrogel microtubes to facilitate direct epithelial–mesenchymal cell interaction for 3D salivary gland cell organization, which allows for monitoring cellular organization while providing a protective barrier from cell-cluster loss during medium changes. Using mouse salivary gland ductal epithelial SIMS cells as the epithelial model cell type and NIH 3T3 fibroblasts or primary E16 salivary mesenchyme cells as the stromal model cell types, self-organization from epithelial–mesenchymal interaction was examined. We observed that epithelial and mesenchymal cells undergo aggregation on day 1, cavitation by day 4, and generation of an EpCAM-expressing epithelial cell layer as early as day 7 of the co-culture in hydrogel microtubes, demonstrating the utility of hydrogel microtubes to facilitate heterotypic cell–cell interactions to form cavitated organoids. Thus, pre-formed alginate microtubes are a promising co-culture method for further understanding epithelial and mesenchymal interaction during tissue morphogenesis and for future practical applications in regenerative medicine.

Using co-culture system to differentiate periodontal ligament-derived stem cell to ductal salivary gland cells

Using co-culture system to differentiate periodontal ligament-derived stem cell to ductal salivary gland cells

Two Sjogren syndrome-associated oral bacteria, <i>Prevotella melaninogenica</i> and Rothia mucilaginosa, induce the upregulation of major histocompatibility complex class I and hypoxia-associated cell death, respectively, in human salivary gland cells

Two Sjogren syndrome-associated oral bacteria, <i>Prevotella melaninogenica</i> and Rothia mucilaginosa, induce the upregulation of major histocompatibility complex class I and hypoxia-associated cell death, respectively, in human salivary gland cells

Egg White Alginate as a Novel Scaffold Biomaterial for 3D Salivary Cell Culturing.

Saliva production by salivary glands play a crucial role in oral health. The loss of salivary gland function could lead to xerostomia, a condition also known as dry mouth. Significant reduction in saliva production could lead to further complications such as difficulty in speech, mastication, and increased susceptibility to dental caries and oral infections and diseases. While some palliative treatments are available for xerostomia, there are no curative treatments to date. This study explores the use of Egg White Alginate (EWA), as an alternative scaffold to Matrigel® for culturing 3D salivary gland cells. A protocol for an optimized EWA was established by comparing cell viability using 1%, 2%, and 3% alginate solution. The normal salivary simian virus 40-immortalized acinar cell (NS-SV-AC) and the submandibular gland-human-1 (SMG-hu-1) cell lines were also used to compare the spheroid formation and cell viability properties of both scaffold biomaterials; cell viability was observed over 10 days using a Live–Dead Cell Assay. Cell viability and spheroid size in 2% EWA was significantly greater than 1% and 3%. It is evident that EWA can support salivary cell survivability as well as form larger spheroids when compared to cells grown in Matrigel®. However, further investigations are necessary as it is unclear if cultured cells were proliferating or aggregating.

Multiple stimulation with spheroids comprising salivary gland and adipose-derived stem cells enhances regeneration of radiation-damaged salivary glands

Tissue engineering approaches using a single stem cell type have been unsuccessful in achieving regeneration of radiation-damaged salivary glands (SGs). Therefore, we combined adult SG stem cells (SGSCs) and adipose-derived stem cells (ASCs) in optimized spheroids to enhance the regeneration of damaged SGs in mice. SGSC/ASC spheroids survived longer and secreted more vascular endothelial growth factor (VEGF) than did SGSC spheroids. To enhance cell viability and differentiation, SGSC/ASC spheroids were stimulated with the Wnt/β-catenin signaling activator CHIR99021 and retinoic acid inhibitor BMS431 to maintain stemness. FGF7/10 induced the differentiation of multiple stimulated SGSC/ASC spheroids. SGSC/ASC spheroids exhibited improved viability, calcium channel function, differentiation, and VEGF secretion after multiple, sequential stimulation. The 21-day survival rate of multiply stimulated SGSC/ASC spheroids was two-fold that of non-treated SGSC/ASC spheroids. Optimized SGSC/ASC spheroids enhanced the in vivo regeneration and functional recovery of SG tissue in mice by promoting neovascularization. Genes related to VEGF, such as Vegfa and Vegfb, were highly expressed 2∼6 times for in vitro and 1.5 times for in vivo experiment in the multiply stimulated SGSC/ASC spheroids. Therefore, SGSC/ASC spheroids that have undergone multiple stimulation may be useful for healing radiation-damaged SGs.

Hobit confers tissue-dependent programs to type 1 innate lymphoid cells

Identification of type 1 innate lymphoid cells (ILC1s) has been problematic. The transcription factor Hobit encoded by Zfp683 has been proposed as a major driver of ILC1 programs. Using Zfp683 reporter mice, we showed that correlation of Hobit expression with ILC1s is tissue- and context-dependent. In liver and intestinal mucosa, Zfp683 expression correlated well with ILC1s; in salivary glands, Zfp683 was coexpressed with the natural killer (NK) master transcription factors Eomes and TCF1 in a unique cell population, which we call ILC1-like NK cells; during viral infection, Zfp683 was induced in conventional NK cells of spleen and liver. The impact of Zfp683 deletion on ILC1s and NK cells was also multifaceted, including a marked decrease in granzyme- and interferon-gamma (IFNγ)-producing ILC1s in the liver, slightly fewer ILC1s and more Eomes+ TCF1+ ILC1-like NK cells in salivary glands, and only reduced production of granzyme B by ILC1 in the intestinal mucosa. NK cell-mediated control of viral infection was unaffected. We conclude that Hobit has two major impacts on ILC1s: It sustains liver ILC1 numbers, while promoting ILC1 functional maturation in other tissues by controlling TCF1, Eomes, and granzyme expression.

Protective role of resveratrol and apigenin against toxic effects of bisphenol a in rat salivary gland

Objectives The aim of this study was to investigate the cellular changes caused by Bisphenol A (BPA) exposure in salivary gland cells and to examine the protective role of resveratrol (RSV) and apigenin (APG) molecules against the negative effects of BPA. Materials and Methods Forty-two rats were randomly divided into 6 groups as; (i) control, (ii) BPA (130 mg/kg), (iii) BPA + RSV100 (100 mg/kg), (iv) BPA + RSV200 (200 mg/kg), (v) BPA + APG100 (100 mg/kg), and (vi) BPA + APG200 (200 mg/kg). In all experimental groups, the chemicals were given by gavage every day for a total of 28 days. Results The BPA administration caused a significant increase in tissue oxidative stress parameters as opposed to a significant decrease in tissue antioxidant levels (p < 0.05). On the other hand, it was observed that RSV and APG treatment reversed this situation (p < 0.05). The BPA administration did not cause a significant change in tissue prostaglandin E2 (PGE2) and nitric oxide levels, whereas low-dose RSV significantly reduced the tissue PGE2 levels compared to BPA (p < 0.05). BPA caused cytopathological changes and apoptosis in salivary gland cells. In the BPA group, edema, nuclear pleomorphism, and pyknotic nuclei were observed. Moreover, both RSV and APG were found to provide protection against BPA-induced cellular damage, while RSV provided better cellular protection than APG. The control group had a normal histological structure. Conclusion BPA caused cytopathological changes and apoptosis in salivary gland cells. As a result, it was observed that these phytochemicals probably have cytoprotective effects in BPA intoxication.

A Synopsis of Signaling Crosstalk of Pericytes and Endothelial Cells in Salivary Gland.

The salivary gland (SG) microvasculature constitutes a dynamic cellular organization instrumental to preserving tissue stability and homeostasis. The interplay between pericytes (PCs) and endothelial cells (ECs) culminates as a key ingredient that coordinates the development, maturation, and integrity of vessel building blocks. PCs, as a variety of mesenchymal stem cells, enthrall in the field of regenerative medicine, supporting the notion of regeneration and repair. PC-EC interconnections are pivotal in the kinetic and intricate process of angiogenesis during both embryological and post-natal development. The disruption of this complex interlinkage corresponds to SG pathogenesis, including inflammation, autoimmune disorders (Sjögren’s syndrome), and tumorigenesis. Here, we provided a global portrayal of major signaling pathways between PCs and ECs that cooperate to enhance vascular steadiness through the synergistic interchange. Additionally, we delineated how the crosstalk among molecular networks affiliate to contribute to a malignant context. Additionally, within SG microarchitecture, telocytes and myoepithelial cells assemble a labyrinthine companionship, which together with PCs appear to synchronize the regenerative potential of parenchymal constituents. By underscoring the intricacy of signaling cascades within cellular latticework, this review sketched a perceptive basis for target-selective drugs to safeguard SG function.

코로나바이러스감염증-19 환자의 구강증상과 병리학적 기전

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, is rapidly spreading worldwide. Oral manifestations of COVID-19, including sore throat, loss of taste, dry mouth, and ulceration have been widely reported. SARS-CoV-2 enters host cells using angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2). Therefore, high levels of expression of ACE2 and TMPRSS2 may enhance SARS-CoV-2 invasion. ACE2 and TMPRSS2 were expressed in the taste bud and intermediate layer of the squamous epithelia of tongue papillae. Additionally, ACE2 and TMPRSS2 were co-expressed in the ductal epithelium and acinar cells of salivary glands and in the neuronal cell body of trigeminal ganglia. SARS-CoV-2 directly invades the oral taste buds, causing loss of taste. Additionally, the possibility of neuroinvasion innervation of taste bud cells cannot be excluded. SARS-CoV-2 spreads around the suprabasal areas of the oral squamous epithelia, and invades the trigeminal nerve, resulting in oral manifestations. SARS-CoV-2 can directly invade the parotid and submandibular glands, as well as minor salivary glands, through the duct orifice. This may lead to sialadenitis and salivary contamination. (J Clinical Otolaryngol 2021;32:163-169)

LAMP3 inhibits autophagy and contributes to cell death by lysosomal membrane permeabilization

ABSTRACT Sjögren syndrome (SS) is a chronic and progressive autoimmune disease characterized by dry mouth and dry eyes, and characteristic autoantibodies. Evidence of altered macroautophagy/autophagy and apoptosis has been associated with SS, but a mechanistic understanding of the gene expression changes associated with these abnormal processes has not been realized. Recently, increased LAMP3 (lysosomal associated membrane protein 3) expression was found in a subset of SS patients and was associated with increased apoptosis and autoantigen accumulation and release. To better understand how LAMP3 expression might modulate apoptosis, cell biology, and biochemical studies were used to examine the effect of LAMP3 expression in minor salivary gland cells. LAMP3 expression resulted in degradation of LAMP1 increasing lysosomal membrane permeabilization and relocalization of cathepsins to the cytoplasm, resulting in destabilizing autophagic flux and caspase activation. These findings highlight the central role of LAMP3 expression in the pathogenesis of SS.

SARS-COV2 Associated Oral Lesions- A Review

Introduction: COVID-19 disease is caused by SARS-CoV-2 virus and it was declared pandemic by World Health Organization (WHO) on March 11,2020. The coronavirus infection has an affinity for ACE2 receptors and by attaching to them, the virus enters the host cells. Along with many body organs like lungs, kidney, liver, upper respiratory tract, nervous system, skeletal muscles, ACE2 concentration is also found in abundance in epithelial cells of tongue and salivary glands.&#x0D; Materials and Methods: Recent studies, researches, documents and case reports published in the world medical literature in the year 2020-2021 were searched and documented in our study. The search engines used were PUBMED, google scholar, WEB OF SCIENCE etc.&#x0D; Results: Dysgeusia, xerostomia, sore throat, aphthous and herpetiform ulcers, candidiasis, enanthema, Kawasaki like lesions were the most common among various oral manifestations. Others includes plaque like changes, gingival inflammation, necrotizing gingivitis, erythema -multiforme, angina-bullosa like lesions, Melkersson-Rosenthal Syndrome, Oral mucormycosis etc.The sites of infection mainly include tongue, gingiva, hard and soft palate, buccal and labial mucosa etc.&#x0D; Conclusion: The etiopathogenesis of such lesions cannot be directly corelated with COVID-19 and factors such as stress, immunosuppression, co-infections, secondary lesions, opportunistic infections, systemic diseases, poor oral hygiene etc. must be considered. Management of stress is an important factor. In this review article various oral lesions are discussed in COVID-19 infection states in detail. The importance of earliest diagnosis of oral lesions is to be kept in mind to prevent further complications.

Aging-associated stem/progenitor cell dysfunction in the salivary glands of mice

Although stem cell aging leads to a decline in tissue homeostasis and regenerative capacity, it remains unclear whether salivary gland stem cell function changes during this process. However, the salivary glands are gradually replaced by connective tissue during aging. Here, we show a decline in the stem cell ability of CD133-positive stem/progenitor cells in the salivary glands of aged mice. The CD133-positive cells were isolated from young, adult, and aged mice. The number of CD133-positive cells was significantly decreased in aged mice. They also showed a lower sphere formation capacity compared to young and adult mice. RNA sequencing revealed that CD133-positive cells in aged mice exhibited lower gene expression of several aging-related genes, including FoxO3a, than those in young and adult mice. Salivary gland cells infected with a recombinant lentivirus encoding the FoxO3a gene showed a reduction in oxidative stress induced by hydrogen peroxide compared with those infected with a control virus. Thus, FoxO3a may inhibit stem cell aging via oxidative stress.

Dissection and Immunostaining of Larval Salivary Glands from Anopheles gambiae Mosquitoes.

Mosquito salivary glands (SGs) are a requisite gateway organ for the transmission of insect-borne pathogens. Disease-causing agents, including viruses and the Plasmodium parasites that cause malaria, accumulate in the secretory cavities of SG cells. Here, they are poised for transmission to their vertebrate hosts during a subsequent blood meal. As adult glands form as an elaboration of larval SG duct bud remnants that persist beyond early pupal SG histolysis, the larval SG is an ideal target for interventions that limit disease transmission. Understanding larval SG development can help develop a better understanding of its morphology and functional adaptations and aid in the assessment of new interventions that target this organ. This video protocol demonstrates an efficient technique for isolating, fixing, and staining larval SGs from Anopheles gambiae mosquitoes. Glands dissected from larvae in a 25% ethanol solution are fixed in a methanol-glacial acetic acid mixture, followed by a cold acetone wash. After a few rinses in phosphate-buffered saline (PBS), SGs can be stained with a broad array of marker dyes and/or antisera against SG-expressed proteins. This method for larval SG isolation could also be used to collect tissue for in situ hybridization analysis, other transcriptomic applications, and proteomic studies.