Xerostomia (dry mouth) is frequently experienced by patients treated with radiotherapy for head and neck cancers or with Sjögren's syndrome, with no permanent cure existing for this debilitating condition. To this end, in vitro platforms are needed to test therapies directed at salivary (fluid-secreting) cells. However, since these are highly differentiated secretory cells, the maintenance of their differentiated state while expanding in numbers is challenging. In this study, the efficiency of three reversible thermo-ionically crosslinked gels: (1) alginate-gelatin (AG), (2) collagen-containing AG (AGC), and (3) hyaluronic acid-containing AG (AGHA), to recapitulate a native-like environment for human salivary gland (SG) cell expansion and 3D spheroid formation was compared. Although all gels were of mechanical properties comparable to human SG tissue (~11 kPa) and promoted the formation of 3D spheroids, AGHA gels produced larger (>100 cells/spheroid), viable (>93%), proliferative, and well-organized 3D SG spheroids while spatially and temporally maintaining the high expression of key SG proteins (aquaporin-5, NKCC1, ZO-1, α-amylase) for 14 days in culture. Moreover, the spheroids responded to agonist-induced stimulation by increasing α-amylase secretory granules. Here, we propose alternative low-cost, reproducible, and reversible AG-based 3D hydrogels that allow the facile and rapid retrieval of intact, highly viable 3D-SG spheroids.
Humans ; Hydrogels/chemistry* ; Acinar Cells/cytology* ; Spheroids, Cellular/cytology* ; Salivary Glands/cytology* ; Gelatin/chemistry* ; Collagen/chemistry* ; Alginates/chemistry* ; Cell Culture Techniques/methods* ; Hyaluronic Acid/chemistry* ; Cell Proliferation ; Cell Survival ; Cells, Cultured

Background: A fine needle aspiration biopsy has been established as a safe, minimally invasive procedure in evaluation of salivary gland lesions. The complex overlapping cytomorphology of these lesions are challenging for pathologists, hence the introduction of an evidence-based system, the Milan System of Reporting Salivary Gland Cytopathology, to improve overall patient care. The study was taken up to reclassify salivary gland lesions from previous FNA biopsies in order to determine sensitivity, specificity, positive and negative predictive values of FNA, and evaluate the risk of malignancy of the various categories of the Milan system. Methodology: This was a 6-year retrospective descriptive study in a tertiary medical center. All salivary gland FNA cases were reviewed by two pathologists, and re-classified into the six categories of the Milan System. The number of false positive, false negative, true positive and true negative cases were obtained by comparing with the final histopathology diagnosis, and the risk of malignancy per category were calculated. Results: A total of 76 cases were reviewed and the overall average of the two readers diagnostic accuracy were 85.02% (95% CI: 84.50-85.60%), sensitivity and specificity were 80.77% (95% CI: 79.90-81.60%) and 86.19% (95% CI: 85.70-86.70%), respectively; positive and negative predictive values were 62.16% (95% CI: 60.70-63.60%) and 94.17% (95% CI: 94.00-94.40%), respectively. Conclusion The Milan System category with highest risk of malignancy was Malignant (Category VI – 100%). FNAB is still a reliable tool for clinicians, and use of the Milan System of Reporting Salivary Gland Cytopathology is beneficial in increasing efficacy of communication among clinicians to improve patient care.
Cytology ; Salivary Glands

Background: The Milan System for Reporting Salivary Gland Cytopathology (MSRGC) aims to increase the overall effectiveness of salivary gland FNAB by defining six general diagnostic categories with corresponding Rates of Malignancies (ROM). This study aims to use this system to categorize salivary gland FNAB in the Philippine General Hospital and stratify ROM per category. Methodology: In this study a total of 326 cases have been collected and reviewed, of which 154 (47.2%) had either surgical or clinical follow-up. The cases were assigned a Milan category by 3 cytopathologists blinded from the original diagnoses and from each other’s readings. Results: The overall sensitivity, specificity, PPV, and NPV in detecting neoplasm is at 71.6%, 90.9%, 88.3%, and 76.9%, respectively. On the other hand, the sensitivity, specificity, PPV, and NPV in detecting malignancy is at 52%, 92.9%, 59.1%, and 90.7%, respectively. The computed ROM is as follows: Category I 7.89%, Category II 9.43%, Category III 20%, Category IVa 10.53%, Category IVb 60%, Category V 75%, and Category VI 100%. Conclusion The overall diagnostic utility of salivary gland FNAB, as well as the computed ROM per diagnostic category are comparable to internationally published literature. This study also validates the MSRSGC as a valuable tool in stratifying ROM in salivary gland lesions.
Cytology ; Salivary Glands

We report the case of a patient who presented with cystic lymphoid hyperplasia of the right parotid gland as the index diagnosis of HIV infection. Histological examination of the excised parotid gland revealed a solid-cystic lymphoepithelial lesion with a non-keratinous squamous epithelium, which grew into the lymphoid component via anastomosing cords and islands. These anastomosing cords and islands contained variably abundant B cells, several subepithelial multinucleated histiocytes, salivary ducts infiltrated by small lymphocytes, and a dense lymphoid infiltrate containing lymphoid follicles with enlarged, irregular germinal centres.
Adult ; B-Lymphocytes ; cytology ; Biopsy ; Epithelial Cells ; cytology ; Epithelium ; metabolism ; HIV Infections ; diagnosis ; Humans ; Hyperplasia ; pathology ; virology ; Immunohistochemistry ; Lymphocytes ; cytology ; Male ; Parotid Gland ; pathology ; virology ; Salivary Glands ; pathology ; Tomography, X-Ray Computed

Lysophosphatidic acid (LPA) is a bioactive lysophospholipid involved in numerous physiological responses. However, the expression of LPA receptors and the role of the Hippo signaling pathway in epithelial cells have remained elusive. In this experiment, we studied the functional expression of LPA receptors and the associated signaling pathway using reverse transcriptase-PCR, microspectrofluorimetry, western blotting and immunocytochemistry in salivary gland epithelial cells. We found that LPA receptors are functionally expressed and involved in activating the Hippo pathway mediated by YAP/TAZ through Lats/Mob1 and RhoA/ROCK. Upregulation of YAP/TAZ-dependent target genes, including CTGF, ANKRD1 and CYR61, has also been observed in LPA-treated cells. In addition, based on data suggesting that tumor necrosis factor (TNF)-alpha induces cell apoptosis, LPA upregulates TNF-induced caspase-3 and cleaved Poly(ADP-ribose)polymerase (PARP). However, small interfering RNA treatment to Yes-associated protein (YAP) or transcriptional co-activator with a PDZ-binding motif (TAZ) significantly decreased TNF-alpha- and LPA-induced apoptosis, suggesting that YAP and TAZ modulate the apoptotic pathway in salivary epithelial cells.
Adaptor Proteins, Signal Transducing/genetics/metabolism ; *Apoptosis ; Cell Line ; Epithelial Cells/*cytology/metabolism ; Gene Expression Regulation ; Humans ; Intracellular Signaling Peptides and Proteins/genetics/metabolism ; Lysophospholipids/*metabolism ; Phosphoproteins/genetics/metabolism ; Protein-Serine-Threonine Kinases/*metabolism ; RNA Interference ; RNA, Small Interfering/genetics ; Receptors, Lysophosphatidic Acid/genetics/*metabolism ; Salivary Glands/*cytology/metabolism ; *Signal Transduction ; Tumor Necrosis Factor-alpha/metabolism ; rho-Associated Kinases/metabolism ; rhoA GTP-Binding Protein/metabolism

Salivary function in mammals may be defective for various reasons, such as aging, Sjogren's syndrome or radiation therapy in head and neck cancer patients. Recently, tissue-specific stem cell therapy has attracted public attention as a next-generation therapeutic reagent. In the present study, we isolated tissue-specific stem cells from the human submandibular salivary gland (hSGSCs). To efficiently isolate and amplify hSGSCs in large amounts, we developed a culture system (lasting 4-5 weeks) without any selection. After five passages, we obtained adherent cells that expressed mesenchymal stem cell surface antigen markers, such as CD44, CD49f, CD90 and CD105, but not the hematopoietic stem cell markers, CD34 and CD45, and that were able to undergo adipogenic, osteogenic and chondrogenic differentiation. In addition, hSGSCs were differentiated into amylase-expressing cells by using a two-step differentiation method. Transplantation of hSGSCs to radiation-damaged rat salivary glands rescued hyposalivation and body weight loss, restored acinar and duct cell structure, and decreased the amount of apoptotic cells. These data suggest that the isolated hSGSCs, which may have characteristics of mesenchymal-like stem cells, could be used as a cell therapy agent for the damaged salivary gland.
Amylases/genetics/metabolism ; Animals ; Antigens, CD/genetics/metabolism ; Apoptosis ; Cell Differentiation ; Humans ; Male ; Mesenchymal Stromal Cells/*cytology/metabolism ; Radiation Injuries, Experimental ; Rats ; Rats, Wistar ; *Regeneration ; Salivary Glands/cytology/injuries/physiology/*surgery ; *Salivation ; *Stem Cell Transplantation

Adenoviridae ; genetics ; Animals ; Dental Research ; Genetic Therapy ; Genetic Vectors ; History, 20th Century ; Humans ; National Institute of Dental and Craniofacial Research (U.S.) ; history ; organization & administration ; Salivary Gland Diseases ; therapy ; Salivary Glands ; cytology ; growth & development ; injuries ; physiology ; United States