Salivary gland tissue chip screening identifies candidate radioprotective drugs

During head and neck cancer treatment, off-target ionizing radiation damage to the salivary glands commonly causes a permanent loss of secretory function. Due to the resulting decrease in saliva production, patients have trouble eating, speaking and are predisposed to oral infections and tooth decay. While the radioprotective antioxidant drug Amifostine is approved to prevent radiation-induced hyposalivation, it has intolerable side effects that limit its use, motivating the discovery of alternative therapeutics. To address this issue, we previously developed a salivary gland mimetic (SGm) tissue chip platform. Here, we leverage this SGm tissue chip for high-content drug discovery. First, we developed in-chip assays to quantify glutathione and cellular senescence (β-galactosidase), which are biomarkers of radiation damage, and we validated radioprotection using WR-1065, the active form of Amifostine. Following validation, we tested other reported radioprotective drugs, including, Edaravone, Tempol, N-acetylcysteine (NAC), Rapamycin, Ex-Rad, and Palifermin, confirming that all drugs but NAC and Ex-Rad exhibited robust radioprotection. Next, a Selleck Chemicals library of 438 FDA-approved drugs was screened for radioprotection. We discovered 25 hits, with most of the drugs identified with mechanisms of action other than antioxidant activity. Hits were down-selected using EC 50 values and pharmacokinetics and pharmacodynamics data from the PubChem database leading to testing of Phenylbutazone (anti-inflammatory), Enoxacin (antibiotic), and Doripenem (antibiotic) for in vivo radioprotection in mice using retroductal injections. Results confirm that Phenylbutazone and Enoxacin exhibited equivalent radioprotection to Amifostine. This body of work demonstrates the development and validation of assays using a SGm tissue chip platform for high-content drug screening and the successful in vitro discovery and in vivo validation of novel radioprotective drugs with nonantioxidant primary indications pointing to possible, yet unknown novel mechanisms of radioprotection.

During head and neck cancer treatment, off-target ionizing radiation damage to the salivary glands commonly causes a permanent loss of secretory function. Due to the resulting decrease in saliva production, patients have trouble eating, speaking and are predisposed to oral infections and tooth decay. While the radioprotective antioxidant drug Amifostine is FDA approved to prevent radiation-induced hyposalivation, it has intolerable side effects that limit its use, motivating the discovery of alternative therapeutics. To address this issue, we previously developed a salivary gland mimetic (SGm) tissue chip platform. Here, we leverage this SGm tissue chip for high-content drug discovery. First, we developed in-chip assays to quantify glutathione and cellular senescence (β-galactosidase), which are biomarkers of radiation damage, and we validated radioprotection using WR-1065, the active form of Amifostine. Other reported radioprotective drugs including Edaravone, Tempol, N-acetylcysteine (NAC), Rapamycin, Ex-Rad, and Palifermin were also tested to validate the ability of the assays to detect cell damage and radioprotection. All of the drugs except NAC and Ex-Rad exhibited robust radioprotection. Next, a Selleck Chemicals library of 438 FDA-approved drugs was screened for radioprotection. We discovered 25 hits, with most of the drugs identified exhibiting mechanisms of action other than antioxidant activity. Hits were down-selected using EC50 values and pharmacokinetic and pharmacodynamic data from the PubChem database. This led us to test Phenylbutazone (anti-inflammatory), Enoxacin (antibiotic), and Doripenem (antibiotic) for in vivo radioprotection in mice using retroductal injections. Results confirm that Phenylbutazone and Enoxacin exhibited radioprotection equivalent to Amifostine. This body of work demonstrates the development and validation of assays using a SGm tissue chip platform for high-content drug screening and the successful in vitro discovery and in vivo validation of novel radioprotective drugs with non-antioxidant primary indications pointing to possible, yet unknown novel mechanisms of radioprotection.

Although α-synucleinopathy has been confirmed in the submandibular gland (SMG) tissue of Parkinson's disease (PD) patients, in-depth disease-related molecular research, such as tissue-specific transcriptional signals, has not been performed. In the present study, disease-relevant tissue-specific transcriptional signals in SMG tissue from PD patients were investigated to identify potential diagnostic, prognostic, and pathophysiologic biomarkers. Here, seven de novo drug-naïve PD patients and six age- and sex-matched individuals without neurological or psychological diseases were enrolled. Total RNA sequencing (RNA-seq) and total small RNA-seq (smRNA-seq) were performed on SMG tissue and blood samples, with 26 RNA-seq and 26 smRNA-seq samples used for the final analysis. Differentially expressed genes (DEGs) and microRNAs in SMG tissue and blood from PD patients were obtained and their functional integration and interaction network were analyzed. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the DEGs interacted with cytokine-, inflammation-, and immune-related pathways. Synphilin-1 expression was significantly downregulated in SMG tissue of PD patients, and α-synuclein expression did not significantly differ between PD patients and controls in either SMG tissue or blood. Fifteen tissue-specific miRNA signals in SMG tissue were identified that showed better diagnostic ability compared with those in blood samples. The correlation between DEGs and environmental factors appeared altered in PD patients. The results indicated the DEGs and microRNA signatures identified in SMG tissue may be promising diagnostic and prognostic biomarkers. These molecular insights offer potential avenues for the development of novel therapeutic strategies targeting the underlying disease mechanisms in PD patients.

The simple and safety method for removing mesenchymal tissue from the extracted submandibular gland (SMG) tissue is required for the in vitro study of SMG tissue development. For this purpose, we fabricated polydimethylsiloxane (PDMS) tissue culture substrate having micropillar structure. By applying O 2 plasma treatment and fibronectin coating, the surface affinity of PDMS substrate for cell and tissue was improved. Moreover, the pillar structure showed more effective to remove the mesenchymal tissue from isolated SMG tissue. Thus, the micro-fabricated surface with suitable chemical and physical modification is effective to remove mesenchymal tissue from extracted SMG tissue.

Tight junctions (TJs) are necessary for salivary gland function and may serve as indicators of salivary gland epithelial dysfunction. IgG4-related disease (IgG4-RD) is a newly recognized fibro-inflammatory condition which disrupts the TJ associated epithelial barrier. The salivary glands are one of the most frequently involved organs in IgG4-RD, however, changes of the TJ associated epithelial barrier in salivary gland duct epithelium is poorly understood. Here, we investigated the regulation and function of TJs in human submandibular gland ductal epithelial cells (HSDECs) in normal and IgG4-RD. We examined submandibular gland (SMG) tissue from eight control individuals and 22 patients with IgG4-RD and established an HSDEC culture system. Immunohistochemistry, immunocytochemistry, western blotting, and measurement of transepithelial electrical resistance (TER) were performed. Claudin-4, claudin-7, occludin, and JAM-A were expressed at the apical side of the duct epithelium in submandibular gland (SMG) tissue and at the cell borders in HSDECs of normal and IgG4-RD. The expression and distribution of TJs in SMG tissue were not different in control individuals and patients with IgG4-RD in vivo and in vitro. Although interferon-gamma (IFNγ) generally disrupts the integrity and function of TJs, as manifested by decreased epithelial barrier function, IFNγ markedly increased the epithelial barrier function of HSDECs via upregulation of claudin-7 expression in HSDECs from patients with IgG4-RD. This is the first report showing an IFNγ-dependent increase in epithelial barrier function in the salivary gland duct epithelium. Our results provide insights into the functional significance of TJs in salivary gland duct epithelium in physiological and pathological conditions, including IgG4-RD.

Objective: To compare the general biological characteristics and the expressions of proteins involved in secretion in stem cells from the pulp of human exfoliated deciduous teeth (SHED) and dental pulp stem cells (DPSC). Methods: SHED and DPSC were cultured and collected at passage 4 (P4) and P7. The submandibular gland epithelial and interstitial cells were cultured with tissue culture method. The cell morphology was observed using a phase contrast microscope. Flow cytometry was used to detect stem cell surface markers. Cell counting kit-8 (CCK-8) and IncuCyte ZOOM were used to evaluate cell proliferation. Quantitative real-time PCR (qPCR) was performed to examine the mRNA expressions of proteins involved in fluid and protein secretion. Results: P4 and P7 SHED and DPSC were spindle-shaped. There was no difference in cell morphology among the 4 group cells. P4 and P7 SHED and DPSC expressed CD29, CD44, CD73, and CD90, the mesenchymal stem cell markers, while, CD49f and CD117, the epithelium markers were undetected. There was no difference in cell proliferation among the 4 group cells. Compared with P4 SHED, the expressions of muscarinic cholinergic receptor 1 (MR1), MR3, aquaporin 5 (AQP5), β1-adrenoceptor (β1-AR), α-amylase, and mucin 5B in SHED were not different, while β2-AR expression was decreased (P<0.05). Compared with P4 DPSC, the expressions of MR3, β2-AR, and α-amylase in P7 DPSC were not different, while, the expressions of MR1, AQP5, β1-AR, and mucin 5B were decreased (P<0.05). Compared with primary cultured submandibular gland epithelial cells and gland tissues from a child, the expressions of proteins involved in secretion were all decreased. Compared with submandibular epithelial cells from adults, the expression of AQP5 in P4 DPSC was decreased (P<0.05), while other proteins were not different. The expressions of AQP5, β1-AR, α-amylase and mucin 5B in P7 DPSC were increased (P<0.05), while other proteins were not different. In P4 and P7 DPSC, all the protein expression levels were decreased, compared with those in submandibular gland tissues (P<0.01). Conclusions: Compared with DPSC, SHED have stable growth and the expressions of protein involved fluid and protein secretion are low. Based on its extensive sources and easy separation, SHED can be used as the ideal seed cell for salivary gland tissue engineering and the treatment of salivary gland hypofunction, and the P4 to P7 SHED can be used for experimental study.

Parkinson's disease (PD), dementia with Lewy bodies (DLB) and other related adult‐onset neurodegenerative disorders are characterized by accumulation of the pathological form of aggregated α‐synuclein (αSynagg) and are collectively known as α‐synucleinopathies. The identification of α‐synucleinopathies requires the detection of αSynagg, typically postmortem, as a molecular hallmark in various brain regions. Currently, no early diagnostic biomarkers exist for PD or related α‐synucleinopathies. Detection of αSynagg in living patients has promising diagnostic biomarker potential for α‐synucleinopathies. Therefore, we strategically adopted the real‐time quaking‐induced conversion (RT‐QuIC) assay to detect ultra‐low levels of αSynagg. First, we purified monomeric recombinant human wild‐type αSyn and evaluated the validity of the αSyn RT‐QuIC assay for detecting αSynagg as a biomarker in brain homogenates from human subjects with PD, DLB, Alzheimer's disease (AD) along with age‐matched controls. Next, we tested cerebrospinal fluid (CSF) samples from clinically confirmed PD and progressive supranuclear palsy (PSP) subjects. Additionally, we quantified the protein aggregation rate (PAR) for αSynagg based on RT‐QuIC assay kinetics by calculating the time to cross threshold fluorescence. The PD and DLB BH samples showed significantly higher αSynagg PAR compared to age‐matched healthy controls and AD. Importantly, the RT‐QuIC assay was highly reproducible with 100% specificity and 94% sensitivity. Correspondingly, CSF samples from PD subjects demonstrated significantly higher αSynagg PAR compared to age‐matched healthy controls, with 100% sensitivity and specificity. In addition to brain and biofluids, we also tested peripherally accessible submandibular gland (SMG) tissues from PD and controls with 100% sensitivity and 94% specificity using the αSyn RT‐QuIC assay. In addition to demonstrating bonafide α‐synucleinopathies, we tested metal‐induced neurotoxicity in human subjects because exposure to metals such as Mn and other transition metals has been implicated in environmentally linked Parkinsonism. Therefore, we examined the serum and plasma exosomes of welders occupationally exposed to welding fumes and subsequent metal‐induced neurotoxicity. We were able to differentiate the welders from age‐matched controls based on the αSyn RT‐QuIC assay with a high degree of sensitivity and specificity. Collectively, our results demonstrate the potential diagnostic utility of the αSyn RT‐QuIC assay on clinically useful and easily accessible samples, such as CSF, SMG tissues as well as exosomes isolated from serum and plasma, thereby demonstrating its immediate translational potential in biomarker discovery efforts for early diagnosis of neurodegenerative diseases.Support or Funding InformationGrant support‐NIH ES026892, NS100090, NS088206; DoD PD170118; Lloyd and Armbrust endowments to AGK.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The tight junction–based paracellular pathway plays an important role in saliva secretion. Zonula occludens (ZO) proteins are submembranous proteins of tight junction complex; however, their function in salivary epithelium is poorly understood. Here, we found that activation of transient receptor potential vanilloid subtype 1 (TRPV1) by capsaicin increased rat saliva secretion both in vivo and ex vivo. Meanwhile, TRPV1 activation enlarged the width of tight junctions between neighboring acinar cells, increased the paracellular flux of 4-kDa fluorescein isothiocyanate (FITC)-dextran in submandibular gland (SMG) tissues, and decreased transepithelial electric resistance (TER) in SMG-C6 cells. ZO-1, -2, and -3 were distributed principally to the apical lateral region of acinar cells in SMG tissues and continuously encircled the peripheries of SMG-C6 cells in the untreated condition. TRPV1 activation obviously diminished ZO-1 and -2 staining, but not ZO-3 or β-catenin, at the cell-cell contacts ex vivo and in vitro. Moreover, in untreated SMG-C6 cells, ZO-1 and -2 single or double knockdown by small interfering RNA (siRNA) increased the paracellular flux of 4-kDa FITC-dextran. In capsaicin-treated cells, ZO-1 and -2 single or double knockdown abolished, whereas their re-expression restored, the capsaicin-induced increase in paracellular permeability. Furthermore, TRPV1 activation increased RhoA activity, and inhibition of either RhoA or Rho kinase (ROCK) abolished the capsaicin-induced TER decrease as well as ZO-1 and -2 redistribution. These results indicate that ZO-1 and -2 play crucial roles in both basal salivary epithelial barrier function and TRPV1-modulated paracellular transport. RhoA-ROCK signaling pathway is responsible for TRPV1-modulated paracellular permeability as well as ZO-1 and -2 redistribution.

Primary Sjogren's syndrome (pSS) is a systemic autoimmune disease that causes dysfunction of secretory glands and the specific pathogenesis is still unknown. The CXCL9, 10, 11/CXCR3 axis and G protein-coupled receptor kinase 2 (GRK2) involved in many inflammation and immunity processes. We used NOD/Ltj mice, a spontaneous SS animal model, to elucidate the pathological mechanism of CXCL9, 10, 11/CXCR3 axis promoting T lymphocyte migration by activating GRK2 in pSS. We found that CD4 + GRK2, Th17 + CXCR3 was apparently increased and Treg + CXCR3 was significantly decreased in the spleen of 4W NOD mice without sicca symptom compared to ICR mice (control group). The protein levels of IFN-γ, CXCL9, 10, 11 increased in submandibular gland (SG) tissue accompanied by obvious lymphocytic infiltration and Th17 cells overwhelmingly infiltrated relative to Treg cells at the sicca symptom occurs, and we found that the proportion of Th17 cells was increased, whereas that of Treg cells was decreased in spleen. In vitro, we used IFN-γ to stimulate human salivary gland epithelial cells (HSGECs) co-cultured with Jurkat cells, and the results showed that CXCL9, 10, 11 was increased by IFN-γ activating JAK2/STAT1 signal pathway and Jurkat cell migration increased with the raised of cell membrane GRK2 expression. HSGECs with tofacitinib or Jurkat cells with GRK2 siRNA can reduce the migration of Jurkat cells. The results indicate that CXCL9, 10, 11 significantly increased in SG tissue through IFN-γ stimulating HSGECs, and the CXCL9, 10, 11/CXCR3 axis contributes to the progress of pSS by activating GRK2 to promote T lymphocyte migration.

Radioprotection of mice against X rays was achieved shortly after in three different laboratories, in Belgium, the US, and Britain, using cyanide, cysteine, and thiourea. The importance of sulfur-containing molecules for radioprotection was thus demonstrated from the very earliest experiments with living systems, although the reasons for selection of sulfur compounds were not apparent. Since 1952, other types of structures have been found with radioprotective abilities, including a number of commonly used pharmacological agents, but the most effective have been derivatives or analogs of the aminoalkyl mercaptans. By the time of the first International Symposium on Radiosensitizers and Radioprotective Drugs in 1964, it had become apparent that damage to DNA was a primary event in radiation damage. A number of dithiocarbamates have been radioprotective in mice, but the radioprotective activity is usually less than that of mercaptoethylamine (MEA) and its derivatives.

The aim of this prospective animal study is to investigate the influence of multiple administrations of macrocyclic ionic (gadoteric acid) and linear nonionic (gadodiamide) gadolinium-based contrast agents (GBCA) on submandibular gland tissue (SGT) of the rats. Twenty-four Sprague Dawley female rats were included the study. Group 1 was determined as a control group (n = 6). Group 2 was determined as saline group (n = 6). Group 3 was determined as Omniscan group (n = 6) and received only intraperitoneal (IP) 0.1mmol (0.2mL/kg)/kg gadodiamide for 8days. Group 4 was determined as Dotarem group (n = 6) and received only IP 0.1mmol (0.2mL/kg)/mg/kg gadoteric acid daily for 8days. On the 9th day of the administration, the rats were sedated with ketamine and xylazine through IP injection. The right SGT was removed after sedation. Histopathological and immunohistochemical changes in SGT were evaluated. The SGT of the Omniscan and Dotarem groups decreased SGT acini surface area, and serous acinar cells number were observed. On the other hand, no pathology was observed. Mucous acinar cells' caspase-3 positivity for the same markers in Omniscan and Dotarem sections was similar to the control group. However, Omniscan and Dotarem groups serous acinar cells were caspase-3 (+) staining. The intensity of serous acinar cells' caspase-3 (+) for the same markers in Dotarem sections was similar to the Omniscan group. The results also revealed in the analysis of the mean area of the acinus area of the SGT; there were significantly decreased Dotarem group rats when compared to control rats (p < 0.05). We consider that numerical increased apoptosis results arise from repeated doses of GBCAs. Being aware of this effect of the contrast agent may have significance for the chronic sialo-adenitis patients group when used for recurrent contrasted MRI for diagnosis of diseases like MS which requires in follow-up. We should be aware about the frequently contrasted MRI in routine investigations.

Neuropeptide Y in salivary glands of the rat: origin, release and secretory effects

We investigated whether sodium restriction or mineralocorticoid influence the release of submandibular kallikrein into the blood and/or the concentration of kallikrein in glandular tissue. For this we measured submandibular gland blood flow, arterial and submandibular gland venous kallikrein, and kallikrein in glandular homogenates of male Sprague-Dawley rats after one week of either low sodium or deoxycorticosterone acetate (DOCA) treatment. We also studied the effect of dexamethasone on the concentration of kallikrein in gland tissue and peripheral plasma. Kallikrein in plasma and in homogenates was measured by radioimmunoassay. Blood flow was determined by timed collections of venous outflow. Kallikrein release was calculated as the arteriovenous difference in kallikrein times the rate of submandibular gland plasma flow. The concentration of kallikrein in arterial plasma, the basal submandibular kallikrein release into blood, and the concentration of kallikrein in submandibular gland tissue were all higher during low sodium than during normal sodium intake (20.1 +/- 3.6 ng/ml vs 10.7 +/- 0.5, p less than 0.05; 0.40 +/- 0.09 ng/min/100 g bw vs 0.18 +/- 0.02, p less than 0.05, and 81.6 +/- 5.5 micrograms/mg protein vs 65.1 +/- 4.0, p less than 0.05, respectively). In contrast, DOCA treatment did not affect the concentration of kallikrein in arterial plasma, the basal release of kallikrein from the submandibular gland into blood, or the concentration of kallikrein in the gland. Dexamethasone in doses that did not affect the normal growth of the animals had no significant effect on the concentration of kallikrein either in submandibular gland tissue or in peripheral plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Occasionally, obtaining an adequate or acceptable postmortem blood specimen for drug analysis is not possible due to factors such as decomposition, exsanguination, or embalming. Submandibular salivary gland tissue, one of three major types of salivary gland tissue in the oral cavity of humans, has been reported to be a viable alternative postmortem specimen for toxicological testing. In this study, we evaluated the performance of the Randox Evidence Investigator instrument and Randox DOA (Drugs of Abuse) Ultra Whole Blood Array for the semi-quantitative determination of 21 immunoassays in an alternative matrix, submandibular salivary gland tissue. We analyzed 132 submandibular salivary gland tissue specimens and compared the generated results to concomitantly collected postmortem whole blood specimen results. Oxycodone 2, meprobamate, barbiturate, benzodiazepine assay 1, zolpidem, and buprenorphine all showed perfect agreement (Cohen's kappa score = 1.00) between the submandibular salivary gland tissue results and the postmortem whole blood results; dextromethorphan, fentanyl, benzoylecgonine, methamphetamine, tricyclic antidepressants, oxycodone 1, and opiate showed an almost perfect agreement (Cohen's kappa score = 0.81-0.99); methadone, generic opioids, and amphetamine exhibited substantial agreement (Cohen's kappa score = 0.61-0.80). Tramadol demonstrated fair agreement (Cohen's kappa score = 0.41-0.60). The lowest measure of agreement was observed with cannabinoids, meeting criteria for slight agreement (Cohen's kappa score = 0.01-0.20). An application of the techniques described in this study could be implemented in postmortem toxicology laboratories as well as medical examiners offices to provide preliminary drugs of abuse test results that can be used to direct additional testing. This study highlights the successful integration of a novel specimen matrix and an "off-label" use of an established analytical technique.

Secretory IgA in the saliva is essential for protection from mucosally transmitted pathogens and maintaining homeostasis at mucosal surfaces of the oral cavity. Expression of submandibular gland polymeric Ig receptor (pIgR) is essential for IgA secretion. In the present study, we investigated the influence of indigestible carbohydrates on IgA production in the salivary gland and saliva. Five-week-old rats were fed a fibre-free diet (control), or a diet with 5% (w/w) fructo-oligosaccharide (FOS) or a combination of 2·5% (w/w) polydextrose (PDX) and 2·5% (w/w) lactitol for 21-d. IgA concentrations in the caecal digesta, submandibular gland tissue, and saliva in the FOS and PDX+lactitol diet groups were significantly higher than those in the control group (P<0·05). The increase in IgA in the submandibular gland tissue was confirmed using immunohistochemical analysis. However, the IgA concentrations of serum did not differ between the FOS or PDX+lactitol groups and the control group (P=0·5). In the FOS and PDX+lactitol groups, the pIgR mRNA (pIgR/β-actin) expression level in the submandibular gland tissue was significantly higher than that in the control group (P<0·05). The present study suggests that indigestible carbohydrates play an important role in the increase in IgA concentrations in the submandibular gland tissue, saliva, and caecal digesta.