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Background: Modernization has made individuals prefer processed and cooked foods (soft food), but this eating habit may have negative effects on the oral cavity. However, laboratory animals fed with soft diet are commonly used in an attempt to clarify this issue, and various oral tissues, including the salivary glands have been examined. In this review, we summarize the findings of previous studies concerning the responses of salivary glands to daily intake of soft diet. Highlight: The weight of the parotid glands decreased in rodents fed with soft diet (liquid or powder). In atrophic parotid glands, acinar cell shrinkage is histologically observed and the DNA content is reduced, showing that the atrophy is caused by a decrease in the size and number of acinar cells. Immunohistochemical examinations showed that the decrease in the acinar cell number was induced by suppression of acinar cell proliferation and acceleration of apoptosis. The atrophic parotid glands recovered following a change from soft to pellet diet. Other salivary glands, such as the submandibular, sublingual, and palatine glands, responded only slightly to the soft diet feeding. Conclusion: Accumulated research data showed that a soft diet negatively affects the parotid glands much more than other salivary glands and that atrophic parotid glands are able to recover by switching to a hard diet. Therefore, it should be emphasized that good eating habits are important for not only digestion but also the health of oral tissues, including the salivary glands.

The loss of salivary gland function caused by radiation therapy of the head and neck or autoimmune disease such as Sjögren’s syndrome is a serious condition that affects a patient’s quality of life. Due to the combined exocrine and endocrine functions of the salivary gland, gene transfer to the salivary glands holds the potential for developing therapies for disorders of the salivary gland and the expression of therapeutic proteins via the exocrine pathway to the mouth, upper gastrointestinal tract, or endocrine pathway, systemically, into the blood. Recent clinical success with viral vector–mediated gene transfer for the treatment of irradiation-induced damage to the salivary glands has highlighted the need for the development of novel vectors with acinar cell tropism able to result in stable long-term transduction. Previous studies with adeno-associated virus (AAV) focused on the submandibular gland and reported mostly ductal cell transduction. In this study, we have screened AAV vectors for acinar cell tropism in the parotid gland utilizing membrane-tomato floxed membrane-GFP transgenic mice to screen CRE recombinase encoding AAV vectors of different clades to rapidly identify capsid isolates able to transduce salivary gland acinar cells. We determined that AAVRh10 and a novel isolate found as a contaminant of a laboratory stock of simian adenovirus SV15, AAV44.9, are both able to transduce parotid and sublingual acinar cells. Persistence and localization of transduction of these AAVs were tested using vectors encoding firefly luciferase, which was detected 6 months after vector administration. Most luciferase expression was localized to the salivary gland compared to that of distal organs. Transduction resulted in robust secretion of recombinant protein in both blood and saliva. Transduction was species specific, with AAVRh10 having stronger transduction activity in rats compared with AAV44.9 or AAV2 but weaker in human primary salivary gland cells. This work demonstrates efficient transduction of parotid acinar cells by AAV that resulted in secretion of recombinant protein in both serum and saliva., Graphical Abstract, Salivary glands have both endocrine and exocrine secretory function and could serve as a depot organ for gene therapy. In this study we have identified AAV vectors that display both durable and localized expression in the salivary gland and are able to secrete recombinant protein into both serum and saliva.

The signaling events controlling proliferation, survival, and apoptosis during mammary epithelial acinar morphogenesis remain poorly characterized. By imaging single-cell ERK activity dynamics in MCF10A acini, we find that these fates depend on the average frequency of non-periodic ERK pulses. High pulse frequency is observed during initial acinus growth, correlating with rapid cell motility. Subsequent decrease in motility correlates with lower ERK pulse frequency and quiescence. Later, during lumen formation, coordinated ERK waves emerge across multiple cells of an acinus, correlating with high and low ERK pulse frequency in outer surviving and inner dying cells respectively. Optogenetic entrainment of ERK pulses causally connects high ERK pulse frequency with inner cell survival. Acini harboring the PIK3CA H1047R mutation, commonly observed in breast cancer, display increased ERK pulse frequency, inner cell survival and loss of lumen formation. Thus, fate decisions during acinar morphogenesis are fine-tuned by different spatio-temporal coordination modalities of ERK pulse frequency.

The prevalence of insomnia, depression and anxiety have recently been increasing (Kessler et al., 2005) with approximately 18.4% of the Brazilian population having experienced depression at least once during their lifetimes. These data characterize Brazil as the developing country with the largest number of people suffering from this disease (WHO, 2018). Depression is commonly accompanied by anxiety disorders and chronic psychiatric conditions with an estimated prevalence of approximately 29% (Kessler et al., 2005), which results in functional impairment that is associated with significant social costs. Treatment of these conditions includes psychological therapy and antidepressants. These drugs, such as benzodiazepines (BZDs), have been widely studied since the 1960s (Llorente et al., 2000) (WHO, 2018). BZDs are used by 9–25% of the elderly population aged 65 years of age and over, mainly among women (15–21.7% in Brazil, 9.9% in the United States, 24% in Canada and 25% in Sweden) (Jorm et al., 2000, Alvarenga et al., 2008, Johnell and Fastbom, 2009, Leggett et al., 2015). BZDs can be divided into two categories, anxiolytics and hypnotics, and can be used as anticonvulsants due to their muscle relaxant effects (De Almeida et al., 2008). As with all central-acting drugs, BZDs have side effects, such as decreased psychomotor activity, memory loss, physical dependence on long-term treatments, (Longo et al., 2000, Uzun et al., 2010) and hyposalivation and/or xerostomia, which is one of the main adverse effects described in the literature (Guggenheimer and Moore, 2003, Lambrecht et al., 2013, Raghavan et al., 2014). BZDs are among the drugs with the highest rates of complaints by patients in dental offices (Smith and Burtner, 1994) where these drugs are prescribed to reduce anxiety related to dental treatments of non-cooperative patients (Zaclikevis et al., 2009). Among the licensed BZDs, Lorazepam is one of the most widely used drugs for both insomnia and anxiety (Amato et al., 2010), particularly among elderly individuals with anxiety disorders and related conditions (Pomara et al., 2015). Previous studies, by our research group, demonstrated the damage Lorazepam and midazolam, another BDZ commonly prescribed for anxiety, caused to the acinar cells of parotid glands (Rinaldi et al., 2015, Mattioli et al., 2016, Rinaldi et al., 2018). We observed that the chronic use of BZDs significantly decreased the number and proliferation of acinar cells, reduced salivary flow and increased cell volume. In light of this, we hypothesize that the reduction in the number of acinar cells caused by Lorazepam in the salivary glands is possibly due to an apoptotic effect on these cells of the parotid glands of rats caused by the chronic use of this drug. Apoptosis plays a fundamental role in the elimination of unwanted, damaged or infected cells in multicellular organisms and serves as an important biological process in cellular development, differentiation and proliferation (So et al., 2016). The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end-labeling (TUNEL) (Tilly, 1996) technique is the gold standard test employed to detect apoptotic cells (Burattini et al., 2009, Heisler-Taylor et al., 2018). Because of the its wide-spread use of TUNEL, there is no need to use another technique to detect apoptotic cells. The literature lacks studies demonstrating Lorazepam-induced apoptosis in acinar cells. The aim of this study was to evaluate the apoptotic action of a BZD on the acinar cells of the parotid glands of rats by quantification of the number of apoptotic cell nuclei (NCA), the number of acinar cells (N) and the immunoexpression of nuclear proliferation cell proliferation (PCNA) protein.

Introduction: Submandibular glands are exposed to many effects due to diseases and therapeutic interventions. A study evaluating the effect of submandibular gland dysfunction on the parotid gland has not been presented in the literature. Objective: The aim of this study was to evaluate the histopathological changes in the parotid gland following submandibular gland failure. Methods: Three groups of seven randomly selected female New Zealand rabbits weighing 2500–3000 g were studied. Unilateral and bilateral submandibular glands were removed in Groups 1 and 2, respectively. No procedure was performed in Group III, the control group. The parotid glands were removed 30 days later. Histological parameters were evaluated and graded between 0 (none) and 3 (severe). Differences between groups were compared using the Mann–Whitney U test. Results: Mean mucus accumulation in acinar cells was 2.57 ± 0.53 and 1.71 ± 0.75 in Groups 1 and 2, respectively (p 0.05). This value was 0.28 ± 0.48 in Group 3, which was significantly lower than in Groups 1 and 2 (p 0,05). Esse valor foi de 0,28 ± 0,48 no Grupo 3, significativamente menor do que nos Grupos 1 e 2 (p

Background & Aims The Onecut 1 transcription factor (Oc1, a.k.a. HNF6) promotes differentiation of endocrine and duct cells of the pancreas; however, it has no known role in acinar cell differentiation. We sought to better understand the role of Oc1 in exocrine pancreas development and to identify its direct transcriptional targets. Methods Pancreata from Oc1Δpanc (Oc1fl/fl;Pdx1-Cre) mouse embryos and neonates were analyzed morphologically. High-throughput RNA-sequencing was performed on control and Oc1-deficient pancreas; chromatin immunoprecipitation sequencing was performed on wild-type embryonic mouse pancreata to identify direct Oc1 transcriptional targets. Immunofluorescence labeling was used to confirm the RNA-sequencing /chromatin immunoprecipitation sequencing results and to further investigate the effects of Oc1 loss on acinar cells. Results Loss of Oc1 from the developing pancreatic epithelium resulted in disrupted duct and acinar cell development. RNA-sequencing revealed decreased expression of acinar cell regulatory factors (Nr5a2, Ptf1a, Gata4, Mist1) and functional genes (Amylase, Cpa1, Prss1, Spink1) at embryonic day (e) 18.5 in Oc1Δpanc samples. Approximately 1000 of the altered genes were also identified as direct Oc1 targets by chromatin immunoprecipitation sequencing, including most of the previously noted genes. By immunolabeling, we confirmed that Amylase, Mist1, and GATA4 protein levels are significantly decreased by P2, and Spink1 protein levels were significantly reduced and mislocalized. The pancreatic duct regulatory factors Hnf1β and FoxA2 were also identified as direct Oc1 targets. Conclusions These findings confirm that Oc1 is an important regulator of both duct and acinar cell development in the embryonic pancreas. Novel transcriptional targets of Oc1 have now been identified and provide clarity into the mechanisms of Oc1 transcriptional regulation in the developing exocrine pancreas. Oc1 can now be included in the gene-regulatory network of acinar cell regulatory genes. Oc1 regulates other acinar cell regulatory factors and acinar cell functional genes directly, and it can also regulate some acinar cell regulatory factors (eg, Mist1) indirectly. Oc1 therefore plays an important role in acinar cell development., Graphical abstract

Summary The potential of reprogrammed β cells derived from pancreatic exocrine cells to treat diabetes has been demonstrated in animal models. However, the precise mechanisms and regulators involved in this process are not clear. Here, we describe a method that allows mechanistic studies of this process in primary exocrine cultures using adenoviral expression vectors. This rapid 5-day protocol, provides the researcher with a highly controlled experimental system in which the effects of different compounds or genetic manipulations can be studied. For complete details on the use and execution of this protocol, please refer to Elhanani et al. (2020)., Graphical Abstract, Highlights • In vitro isolation, infection, and culturing of mouse pancreatic exocrine cells • PDX1, NGN3, and MafA induce reprogramming of exocrine to β-like cells in vitro • Reprogrammed cells express endocrine genes and produce and secrete insulin • A rapid highly controlled system for mechanistic studies of β cell reprogramming, The potential of reprogrammed β cells derived from pancreatic exocrine cells to treat diabetes has been demonstrated in animal models. However, the precise mechanisms and regulators involved in this process are not clear. Here, we describe a method that allows mechanistic studies of this process in primary exocrine cultures using adenoviral expression vectors. This rapid 5-day protocol, provides the researcher with a highly controlled experimental system in which the effects of different compounds or genetic manipulations can be studied.

Salisphere-derived adult epithelial cells have been used to improve saliva production of irradiated mouse salivary glands. Importantly, optimization of the cellular composition of salispheres could improve their regenerative capabilities. The Rho Kinase (ROCK) inhibitor, Y27632, has been used to increase the proliferation and reduce apoptosis of progenitor cells grown in vitro. In this study, we investigated whether Y27632 could be used to improve expansion of adult submandibular salivary epithelial progenitor cells or to affect their differentiation potential in different media contexts. Application of Y27632 in medium used previously to grow salispheres promoted expansion of Kit+ and Mist1+ cells, while in simple serum-containing medium Y27632 increased the number of cells that expressed the K5 basal progenitor marker. Salispheres derived from Mist1CreERT2; R26TdTomato mice grown in salisphere media with Y27632 included Mist1-derived cells. When these salispheres were incorporated into 3D organoids, inclusion of Y27632 in the salisphere stage increased the contribution of Mist1-derived cells expressing the proacinar/acinar marker, Aquaporin 5 (AQP5), in response to FGF2-dependent mesenchymal signals. Optimization of the cellular composition of salispheres and organoids can be used to improve the application of adult salivary progenitor cells in regenerative medicine strategies. Keywords: Salivary gland, Salisphere, Organoid, Proacinar differentiation, Y27632

Continuously feeding a liquid diet to growing rodents strongly inhibits parotid gland growth, due to suppressed growth of acinar cells. This study investigated whether a liquid diet had a similar effect on submandibular and sublingual glands of growing rats. Rats were weaned on day 21 after birth and then fed a pellet diet in the control group and a liquid diet in the experimental group for 0, 1, 2, 4, and 8 weeks. Their submandibular and sublingual glands were excised, weighed, and examined histologically, immunohistochemically (using antibodies to 5'-bromo-2-deoxyuridine and cleaved caspase 3), and ultra structurally. The submandibular glands did not significantly differ between the control and experimental groups at all tested points. Only at Week 8, acinar cell area and 5'-bromo-2-deoxyuridine-labeling index of acinar cells in sublingual glands were significantly lower in the experimental group than in the control group. These results show that a liquid diet during rats' growth period had no effect on acinar cells in their submandibular glands, and only a slight effect on acinar cells in their sublingual glands of growing rats, in contrast to the marked effect of a liquid diet on parotid glands.

Background & Aims Alcohol abuse is the major cause of experimental and human pancreatitis but the molecular mechanisms remain largely unknown. We investigated the role of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, in the pathogenesis of alcoholic pancreatitis. Methods Using a chronic plus acute alcohol binge (referred to as Gao-binge) mouse model, we analyzed pancreas injury, autophagic flux, zymogen granule removal, TFEB nuclear translocation and lysosomal biogenesis in GFP-LC3 transgenic mice, acinar cell-specific Atg5 knockout (KO) and TFEB KO mice as well as their matched wild type mice. Results We found that Gao-binge alcohol induced typical features of pancreatitis in mice with increased serum amylase and lipase activities, pancreatic edema, infiltration of inflammatory cells, accumulation of zymogen granules (ZGs) and expression of inflammatory cytokines. While Gao-binge alcohol increased the number of autophagosomes, it also concurrently inhibited TFEB nuclear translocation and TFEB-mediated lysosomal biogenesis resulting in insufficient autophagy. Acinar cell-specific Atg5 KO and acinar cell-specific TFEB KO mice developed severe inflammatory and fibrotic pancreatitis in both Gao-binge alcohol and control diet-fed mice. In contrast, TFEB overexpression inhibited alcohol-induced pancreatic edema, accumulation of zymogen granules and serum amylase and lipase activities. In line with our findings in mice, decreased LAMP1 and TFEB nuclear staining were also observed in human alcoholic pancreatitis tissues. Conclusions our results indicate that TFEB plays a critical role in maintaining pancreatic acinar cell homeostasis. Impairment of TFEB-mediated lysosomal biogenesis by alcohol may lead to insufficient autophagy and promote alcohol-induced pancreatitis., Graphical abstract

Preclinical studies have suggested that the pancreatic tumor microenvironment both inhibits and promotes tumor development and growth. Here we establish the role of stromal fibroblasts during acinar-to-ductal metaplasia (ADM), an initiating event in pancreatic cancer formation. The transcription factor V-Ets avian erythroblastosis virus E26 oncogene homolog 2 (ETS2) was elevated in smooth muscle actin–positive fibroblasts in the stroma of pancreatic ductal adenocarcinoma (PDAC) patient tissue samples relative to normal pancreatic controls. LSL-Kras G12D/+ ; LSL-Trp53 R172H/+ ; Pdx-1-Cre (KPC) mice showed that ETS2 expression initially increased in fibroblasts during ADM and remained elevated through progression to PDAC. Conditional ablation of Ets- 2 in pancreatic fibroblasts in a Kras G12D -driven mouse ADM model decreased the amount of ADM events. ADMs from fibroblast Ets- 2–deleted animals had reduced epithelial cell proliferation and increased apoptosis. Surprisingly, fibroblast Ets-2 deletion significantly altered immune cell infiltration into the stroma, with an increased CD8+ T-cell population, and decreased presence of regulatory T cells (Tregs), myeloid-derived suppressor cells, and mature macrophages. The mechanism involved ETS2-dependent chemokine ligand production in fibroblasts. ETS2 directly bound to regulatory sequences for Ccl3 , Ccl4 , Cxcl4 , Cxcl5 , and Cxcl10 , a group of chemokines that act as potent mediators of immune cell recruitment. These results suggest an unappreciated role for ETS2 in fibroblasts in establishing an immune-suppressive microenvironment in response to oncogenic Kras G12D signaling during the initial stages of tumor development.

The current paradigm of pancreatic neoplastic transformation proposes an initial step whereby acinar cells convert into acinar-to-ductal metaplasias, followed by progression of these lesions into neoplasias under sustained oncogenic activity and inflammation. Understanding the molecular mechanisms driving these processes is crucial to the early diagnostic and prevention of pancreatic cancer. Emerging evidence indicates that transcription factors that control exocrine pancreatic development could have either, protective or facilitating roles in the formation of preneoplasias and neoplasias in the pancreas. We previously identified that the homeodomain transcription factor Prox1 is a novel regulator of mouse exocrine pancreas development. Here we investigated whether Prox1 function participates in early neoplastic transformation using in vivo, in vitro and in silico approaches. We found that Prox1 expression is transiently re-activated in acinar cells undergoing dedifferentiation and acinar-to-ductal metaplastic conversion. In contrast, Prox1 expression is largely absent in neoplasias and tumors in the pancreas of mice and humans. We also uncovered that Prox1-heterozygosis markedly increases the formation of acinar-to-ductal-metaplasias and early neoplasias, and enhances features associated with inflammation, in mouse pancreatic tissues expressing oncogenic Kras. Furthermore, we discovered that Prox1-heterozygosis increases tissue damage and delays recovery from inflammation in pancreata of mice injected with caerulein. These results are the first demonstration that Prox1 activity protects pancreatic cells from acute tissue damage and early neoplastic transformation. Additional data in our study indicate that this novel role of Prox1 involves suppression of pathways associated with inflammatory responses and cell invasiveness.

The goal of this study was to evaluate the potential activation of the nuclear factor erythroid 2-related factor and the antioxidant-responsive element (Nrf2–ARE) signaling pathway in response to melatonin in isolated mouse pancreatic acinar cells. Changes in intracellular free Ca2+ concentration were followed by fluorimetric analysis of fura-2-loaded cells. The activations of PKC and JNK were measured by Western blot analysis. Quantitative reverse transcription–polymerase chain reaction was employed to detect the expression of Nrf2-regulated antioxidant enzymes. Immunocytochemistry was employed to determine nuclear location of phosphorylated Nrf2, and the cellular redox state was monitored following MitoSOX Red-derived fluorescence. Our results show that stimulation of fura-2-loaded cells with melatonin (1 µM to 1 mM), in the presence of Ca2+ in the extracellular medium, induced a slow and progressive increase of [Ca2+]c toward a stable level. Melatonin did not inhibit the typical Ca2+ response induced by CCK-8 (1 nM). When the cells were challenged with indoleamine in the absence of Ca2+ in the extracellular solution (medium containing 0.5 mM EGTA) or in the presence of 1 mM LaCl3, to inhibit Ca2+ entry, we could not detect any change in [Ca2+]c. Nevertheless, CCK-8 (1 nM) was able to induce the typical mobilization of Ca2+. When the cells were incubated with the PKC activator PMA (1 µM) in the presence of Ca2+ in the extracellular medium, we observed a response similar to that noted when the cells were challenged with melatonin 100 µM. However, in the presence of Ro31-8220 (3 µM), a PKC inhibitor, stimulation of cells with melatonin failed to evoke changes in [Ca2+]c. Immunoblots, using an antibody specific for phospho-PKC, revealed that melatonin induces PKCα activation, either in the presence or in the absence of external Ca2+. Melatonin induced the phosphorylation and nuclear translocation of the transcription factor Nrf2, and evoked a concentration-dependent increase in the expression of the antioxidant enzymes NAD(P)H-quinone oxidoreductase 1, catalytic subunit of glutamate-cysteine ligase, and heme oxygenase-1. Incubation of MitoSOX Red-loaded pancreatic acinar cells in the presence of 1 nM CCK-8 induced a statistically significant increase in dye-derived fluorescence, reflecting an increase in oxidation, that was abolished by pretreatment of cells with melatonin (100 µM) or PMA (1 µM). On the contrary, pretreatment with Ro31-8220 (3 µM) blocked the effect of melatonin on CCK-8-induced increase in oxidation. Finally, phosphorylation of JNK in the presence of CCK-8 or melatonin was also observed. We conclude that melatonin, via modulation of PKC and Ca2+ signaling, could potentially stimulate the Nrf2-mediated antioxidant response in mouse pancreatic acinar cells., This work was supported by Junta de Extremadura-FEDER. Patricia Santofimia-Castaño was granted a fellowship from Fundacion Tatiana Perez de Guzman el Bueno. J.P.B. is funded by the Spanish Ministerio de Economia y Competitividad (SAF2013-41177-R), the Instituto de Salud Carlos III (RD12/0043/0021), the SP3-People-MC-ITN programme of the European Commission (608381), the National Institute on Drug Abuse (National Institutes of Health; 1R21DA037678-01), and the European Regional Development Fund.

Lipids play a role in acute pancreatitis (AP) progression. We investigate the ability of pancreatic acinar cells to trigger inflammatory response in the presence of lipid compounds generated in necrotic areas of peripancreatic adipose tissue (AT) during AP induced in rats by 5% sodium taurocholate. Lipid composition of AT was analyzed by HPLC-mass spectrometry. Acinar inflammatory response to total lipids as well as to either the free fatty acid (FFA) fraction or their chlorinated products (Cl-FFAs) was evaluated. For this, mRNA expression of chemokine (C-C motif) ligand 2 (CCL2) and P-selectin as well as the activation of MAPKs, NF-κB and STAT-3 were analyzed in pancreatic acini. Myeloperoxidase (MPO) activity, as an inducer of Cl-FFA generation, was also analyzed in AT. MPO activity significantly increased in necrotic (AT-N) induced changes in lipid composition of necrotic fat, such as increase in FFA and phospholipid (PL) content, generation of Cl-FFAs and increases in saturated FFAs and in the poly-:mono-unsaturated FFA ratio. Total lipids from AT-N induced overexpression of CCL2 and P-selectin in pancreatic acini as well as MAPKs phosphorylation and activation of NF-κB and STAT3. FFAs, but not Cl-FFAs, up-regulated CCL2 and P-selectin in acinar cells. We conclude that FFAs are capable of up-regulating inflammatory mediators in pancreatic acini and given that they are highly produced during AP, mainly may contribute to the inflammatory response triggered in acinar cells by fat necrosis. No role is played by Cl-FFAs generated as a result of neutrophil infiltration. © 2014 Elsevier B.V., This work was supported by a grant from the University of Salamanca, Spain (USAL 13/18KAL7)