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.

The relationships of lifetime physical activity and diet with salivary cell telomere length in current ultra-endurance exercisers

BACKGROUND: Physical activity and a healthy diet may delay the aging process and ultra-endurance exercise is an extreme form of physical activity. Telomeres are protective DNA sequences located at the ends of eukaryotic chromosomes which shorten as we age. OBJECTIVE: The aim of this study was to investigate the relationships of lifetime physical activity and diet with salivary cell telomere length in current ultra-endurance exercisers (n = 49; %female = 37, age range 26–74 years). METHODS: Physical activity and dietary intake were measured using the Lifetime Physical Activity and Diet Questionnaire (LPADQ) and salivary cell telomere length was measured using quantitative polymerase chain reaction. RESULTS: In this group of current ultra-endurance exercisers there was no relationship between lifetime physical activity or diet (according to food category scores) and telomere length. In contrast to the expected age-related decrease in telomere length, there was no relationship between age and telomere length (95%confidence interval [CI]: –38.86, 14.54, p = 0.359) in this group of current ultra-endurance exercisers. CONCLUSIONS: The relationships of lifetime physical activity and diet with telomere length remain uncertain. It is possible that lifetime physical activity (including ultra-endurance exercise) and lifetime diet may independently, or in combination, contribute to a decrease in the rate of age-related telomere shortening in current ultra-endurance exercisers. ultra-endurance exercisers.

Draft genome sequences of Hirudo medicinalis and salivary transcriptome of three closely related medicinal leeches

Background Salivary cell secretion (SCS) plays a critical role in blood feeding by medicinal leeches, making them of use for certain medical purposes even today. Results We annotated the Hirudo medicinalis genome and performed RNA-seq on salivary cells isolated from three closely related leech species, H. medicinalis, Hirudo orientalis, and Hirudo verbana. Differential expression analysis verified by proteomics identified salivary cell-specific gene expression, many of which encode previously unknown salivary components. However, the genes encoding known anticoagulants have been found to be expressed not only in salivary cells. The function-related analysis of the unique salivary cell genes enabled an update of the concept of interactions between salivary proteins and components of haemostasis. Conclusions Here we report a genome draft of Hirudo medicinalis and describe identification of novel salivary proteins and new homologs of genes encoding known anticoagulants in transcriptomes of three medicinal leech species. Our data provide new insights in genetics of blood-feeding lifestyle in leeches.

Draft genome sequences ofHirudo medicinalisand salivary transcriptome of three closely related medicinal leeches

Salivary cell secretion (SCS) plays a critical role in blood feeding by medicinal leeches, making them of use for certain medical purposes even today. We annotated theHirudo medicinalisgenome and performed RNA-seq on salivary cells isolated from three closely related leech species,H. medicinalis, Hirudo orientalis,andHirudo verbana.Differential expression analysis verified by proteomics identified salivary cell-specific genes, many of which encode previously unknown salivary components. However, the genes encoding known anticoagulants were not differentially expressed in the salivary cells. The function-related analysis of the unique salivary cell genes enabled an update of the concept of interactions between salivary proteins and components of haemostasis. Thus, our study provides one of the most comprehensive knowledge of the genetic fundamentals of the blood-sucking lifestyle in leeches.

Use of the most recent reagent (CuF) for stimulation of NO synthesis by the medicinal leech salivary cell secretion in the cultures of human endothelium cells (HUVEC) and in rat cardiomiocytes

The medicinal leech salivary cell secretion (SCS) may stimulate NO-production in cultures of human endothelium cells (HUVEC) and rat cardiomiocytes (RCM). This effect was detected using a NO specific reagent, - the complex Cu2+ with a fluorescein derivative (Cu-Fl). NO had also been detected in the cells by fluorescent electronic microscopy and determined quantitatively in the cells and in culture fluid by the fluorescence method. SCS stimulated NO synthesis in HUVEC cells (but not in RCM) is accompanied by NO release into intercellular space. Localization of NO synthesis сenters is presented and it is shown that the increase in NO levels during the SCS action on HUVEC and RCM is associated with the increase in the activity of eNOS/nNOS, but not iNOS. In endothelial cells SCS activates nitrosylation processes, assessed by the increase of nitrite-ions in the culture medium. It is therefore important to use Cu-Fl, other than Griss-reagent, during the first hour of analysis of NO synthesis. The NO-depended mechanism of SCS action on endothelial cells might be a factor in providing of its positive action in hirudotheraphy.

Viruses and Salivary Gland Disease (SGD)

Viral infections are often associated with salivary gland pathology. Here we review the pathogenesis of HIV-associated salivary gland disease (HIV-SGD), a hallmark of diffuse infiltrative lymphocytosis syndrome. We investigate the presence and contributions of viral diseases to the pathogenesis of salivary gland diseases, particularly HIV-SGD. We have detected BK viral shedding in the saliva of HIV-SGD patients consistent with viral infection and replication, suggesting a role for oral transmission. For further investigation of BKV pathogenesis in salivary glands, an in vitro model of BKV infection is described. Submandibular (HSG) and parotid (HSY) gland salivary cell lines were capable of permissive BKV infection, as determined by BKV gene expression and replication. Analysis of these data collectively suggests the potential for a BKV oral route of transmission and salivary gland pathogenesis within HIV-SGD.

Distinct pathways of ERK activation by the muscarinic agonists pilocarpine and carbachol in a human salivary cell line

Cholinergic-muscarinic receptor agonists are used to alleviate mouth dryness, although the cellular signals mediating the actions of these agents on salivary glands have not been identified. We examined the activation of ERK1/2 by two muscarinic agonists, pilocarpine and carbachol, in a human salivary cell line (HSY). Immunoblot analysis revealed that both agonists induced transient activation of ERK1/2. Whereas pilocarpine induced phosphorylation of the epidermal growth factor (EGF) receptor, carbachol did not. Moreover, ERK activation by pilocarpine, but not carbachol, was abolished by the EGF receptor inhibitor AG-1478. Downregulation of PKC by prolonged treatment of cells with the phorbol ester PMA diminished carbachol-induced ERK phosphorylation but had no effect on pilocarpine responsiveness. Depletion of intracellular Ca2+([Ca2+]i) by EGTA did not affect ERK activation by either agent. In contrast to carbachol, pilocarpine did not elicit [Ca2+]imobilization in HSY cells. Treatment of cells with the muscarinic receptor subtype 3 (M3) antagonist N-(3-chloropropyl)-4-piperidnyl diphenylacetate decreased ERK responsiveness to both agents, whereas the subtype 1 (M1) antagonist pirenzepine reduced only the carbachol response. Stimulation of ERKs by pilocarpine was also decreased by M3, but not M1, receptor small interfering RNA. The Src inhibitor PP2 blocked pilocarpine-induced ERK activation and EGF receptor phosphorylation, without affecting ERK activation by carbachol. Our results demonstrate that the actions of pilocarpine and carbachol in salivary cells are mediated through two distinct signaling mechanisms—pilocarpine acting via M3receptors and Src-dependent transactivation of EGF receptors, and carbachol via M1/M3receptors and PKC—converging on the ERK pathway.

Epidermal growth factor upregulates β-adrenergic receptor signaling in a human salivary cell line

The effects of epidermal growth factor (EGF) on the β-adrenergic receptor-coupled adenylyl cyclase system were studied in a human salivary cell line (HSY). The β-adrenergic agonist isoproterenol (10−5 M) stimulated adenylyl cyclase activity by ∼2-fold, and the isoproterenol response was increased 1.8-fold after prolonged (48 h) exposure to EGF (5 × 10−10 M). In contrast, enzyme activation via stimulatory prostaglandin receptors and by agents acting on nonreceptor components of the adenylyl cyclase system was not enhanced by EGF. β-Adrenergic receptor density, assessed by binding of the β-adrenergic receptor antagonist (−)-[125I]iodopindolol, was increased threefold after EGF treatment. Competition binding studies with unlabeled antagonists selective for β1- and β2-adrenergic receptor subtypes indicated that the increase in (−)-[125I]iodopindolol binding sites induced by EGF reflected an increased number of β2-adrenergic receptors. Likewise, Northern blot analysis of RNA from EGF-treated cells revealed selective induction of β2-adrenergic receptor mRNA, which was blocked by the RNA synthesis inhibitor actinomycin D. The increase in β-adrenergic receptor density produced by EGF was unaltered after phorbol ester-induced downregulation of protein kinase C (PKC). Enhancement of isoproterenol-responsive adenylyl cyclase activity and phosphorylation of mitogen-activated protein kinase (MAPK) by EGF were both blocked by the MAPK pathway inhibitor PD-98059. The results suggest that in HSY cells EGF enhances β-adrenergic responsiveness by upregulating β2-adrenergic receptor expression at the transcriptional level. Moreover, the stimulatory effect of EGF on β2-adrenergic receptor signaling appears to be mediated by the MAPK pathway and independent of PKC activation.