Mathematical modeling of pulmonary acinus structure: Verification of acinar shape effects on pathway structure using rat lungs.

The pulmonary acinus is the gas exchange unit in the lung and has a very complex microstructure. The structure model is essential to understand the relationship between structural heterogeneity and mechanical phenomena at the acinus level with computational approaches. We propose an acinus structure model represented by a cluster of truncated octahedra in conical, double-conical, inverted conical, or chestnut-like conical confinement to accommodate recent experimental information of rodent acinar shapes. The basis of the model is the combined use of Voronoi and Delaunay tessellations and the optimization of the ductal tree assuming the number of alveoli and the mean path length as quantities related to gas exchange. Before applying the Voronoi tessellation, controlling the seed coordinates enables us to model acinus with arbitrary shapes. Depending on the acinar shape, the distribution of path length varies. The lengths are more widely spread for the cone acinus, with a bias toward higher values, while most of the lengths for the inverted cone acinus primarily take a similar value. Longer pathways have smaller tortuosity and more generations, and duct length per generation is almost constant irrespective of generation, which agrees well with available experimental data. The pathway structure of cone and chestnut-like cone acini is similar to the surface acini's features reported in experiments. According to space-filling requirements in the lung, other conical acini may also be acceptable. The mathematical acinus structure model with various conical shapes can be a platform for computational studies on regional differences in lung functions along the lung surface, underlying respiratory physiology and pathophysiology.

Pulmonary acini exhibit complex changes during postnatal rat lung development.

Pulmonary acini represent the functional gas-exchanging units of the lung. Due to technical limitations, individual acini cannot be identified on microscopic lung sections. To overcome these limitations, we imaged the right lower lobes of instillation-fixed rat lungs from postnatal days P4, P10, P21, and P60 at the TOMCAT beamline of the Swiss Light Source synchrotron facility at a voxel size of 1.48 µm. Individual acini were segmented from the three-dimensional data by closing the airways at the transition from conducting to gas exchanging airways. For a subset of acini (N = 268), we followed the acinar development by stereologically assessing their volume and their number of alveoli. We found that the mean volume of the acini increases 23 times during the observed time-frame. The coefficients of variation dropped from 1.26 to 0.49 and the difference between the mean volumes of the fraction of the 20% smallest to the 20% largest acini decreased from a factor of 27.26 (day 4) to a factor of 4.07 (day 60), i.e. shows a smaller dispersion at later time points. The acinar volumes show a large variation early in lung development and homogenize during maturation of the lung by reducing their size distribution by a factor of 7 until adulthood. The homogenization of the acinar sizes hints at an optimization of the gas-exchange region in the lungs of adult animals and that acini of different size are not evenly distributed in the lungs. This likely leads to more homogeneous ventilation at later stages in lung development.

TAZ inhibits acinar cell differentiation but promotes immature ductal cell proliferation in adult mouse salivary glands.

There are currently no treatments for salivary gland diseases, making it vital to understand signaling mechanisms operating in acinar and ductal cells so as to develop regenerative therapies. To date, little work has focused on elucidating the signaling cascades controlling the differentiation of these cell types in adult mammals. To analyze the function of the Hippo-TAZ/YAP1 pathway in adult mouse salivary glands, we generated adMOB1DKO mice in which both MOB1A and MOB1B were TAM-inducibly deleted when the animals were adults. Three weeks after TAM treatment, adMOB1DKO mice exhibited smaller submandibular glands (SMGs) than controls with a decreased number of acinar cells and an increased number of immature dysplastic ductal cells. The mutants suffered from reduced saliva production accompanied by mild inflammatory cell infiltration and fibrosis in SMGs, similar to the Sjogren's syndrome. MOB1-deficient acinar cells showed normal proliferation and apoptosis but decreased differentiation, leading to an increase in acinar/ductal bilineage progenitor cells. These changes were TAZ-dependent but YAP1-independent. Biochemically, MOB1-deficient salivary epithelial cells showed activation of the TAZ/YAP1 and ß-catenin in ductal cells, but reduced SOX2 and SOX10 expression in acinar cells. Thus, Hippo-TAZ signaling is critical for proper ductal and acinar cell differentiation and function in adult mice.

Pancreatitis initiated pancreatic ductal adenocarcinoma: Pathophysiology explaining clinical evidence.

Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant lethal disease due to its asymptomatic at its early lesion of the disease and drug resistance. Target therapy associated with molecular pathways so far seems not to produce reasonable outcomes. Understanding of the molecular mechanisms underlying inflammation-initiated tumorigenesis may be helpful for development of an effective therapy of the disease. A line of studies showed that pancreatic tumorigenesis was resulted from pancreatitis, which was caused synergistically by various pancreatic cells. This review focuses on those players and their possible clinic implications, such as exocrine acinar cells, ductal cells, and various stromal cells, including pancreatic stellate cells (PSCs), macrophages, lymphocytes, neutrophils, mast cells, adipocytes and endothelial cells, working together with each other in an inflammation-mediated microenvironment governed by a myriad of cellular signaling networks towards PDAC.

Protocol for Studying Reprogramming of Mouse Pancreatic Acinar Cells to ß-like Cells.

The potential of reprogrammed ß 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).

Cholinergic axons regulate type I acini in salivary glands of Ixodes ricinus and Ixodes scapularis ticks.

Regulatory factors controlling tick salivary glands (SGs) are direct upstream neural signaling pathways arising from the tick's central nervous system. Here we investigated the cholinergic signaling pathway in the SG of two hard tick species. We reconstructed the organization of the cholinergic gene locus, and then used in situ hybridization to localize mRNA encoding choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) in specific neural cells in the Ixodes synganglion. Immunohistochemical staining revealed that cholinergic axonal projections exclusively reached type I acini in the SG of both Ixodes species. In type I acini, the rich network of cholinergic axons terminate within the basolateral infoldings of the lamellate cells. We also characterized two types (A and B) of muscarinic acetylcholine receptors (mAChRs), which were expressed in Ixodes SG. We pharmacologically assessed mAChR-A to monitor intracellular calcium mobilization upon receptor activation. In vivo injection of vesamicol-a VAChT blocker-at the cholinergic synapse, suppressed forced water uptake by desiccated ticks, while injection of atropine, an mAChR-A antagonist, did not show any effect on water volume uptake. This study has uncovered a novel neurotransmitter signaling pathway in Ixodes SG, and suggests its role in water uptake by type I acini in desiccated ticks.

Osmotic Gradients in Epithelial Acini Increase Mechanical Tension across E-cadherin, Drive Morphogenesis, and Maintain Homeostasis.

Epithelial cells spontaneously form acini (also known as cysts or spheroids) with a single, fluid-filled central lumen when grown in 3D matrices. The size of the lumen is dependent on apical secretion of chloride ions, most notably by the CFTR channel, which has been suggested to establish pressure in the lumen due to water influx. To study the cellular biomechanics of acini morphogenesis and homeostasis, we used MDCK-2 cells. Using FRET-force biosensors for E-cadherin, we observed significant increases in the average tension per molecule for each protein in mature 3D acini as compared to 2D monolayers. Increases in CFTR activity resulted in increased E-cadherin forces, indicating that ionic gradients affect cellular tension. Direct measurements of pressure revealed that mature acini experience significant internal hydrostatic pressure (37 ± 10.9 Pa). Changes in CFTR activity resulted in pressure and/or volume changes, both of which affect E-cadherin tension. Increases in CFTR chloride secretion also induced YAP signaling and cellular proliferation. In order to recapitulate disruption of acinar homeostasis, we induced epithelial-to-mesenchymal transition (EMT). During the initial stages of EMT, there was a gradual decrease in E-cadherin force and lumen pressure that correlated with lumen infilling. Strikingly, increasing CFTR activity was sufficient to block EMT. Our results show that ion secretion is an important regulator of morphogenesis and homeostasis in epithelial acini. Furthermore, this work demonstrates that, for closed 3D cellular systems, ion gradients can generate osmotic pressure or volume changes, both of which result in increased cellular tension.

TRPV4 channel opening mediates pressure-induced pancreatitis initiated by Piezo1 activation.

Elevated pressure in the pancreatic gland is the central cause of pancreatitis following abdominal trauma, surgery, endoscopic retrograde cholangiopancreatography, and gallstones. In the pancreas, excessive intracellular calcium causes mitochondrial dysfunction, premature zymogen activation, and necrosis, ultimately leading to pancreatitis. Although stimulation of the mechanically activated, calcium-permeable ion channel Piezo1 in the pancreatic acinar cell is the initial step in pressure-induced pancreatitis, activation of Piezo1 produces only transient elevation in intracellular calcium that is insufficient to cause pancreatitis. Therefore, how pressure produces a prolonged calcium elevation necessary to induce pancreatitis is unknown. We demonstrate that Piezo1 activation in pancreatic acinar cells caused a prolonged elevation in intracellular calcium levels, mitochondrial depolarization, intracellular trypsin activation, and cell death. Notably, these effects were dependent on the degree and duration of force applied to the cell. Low or transient force was insufficient to activate these pathological changes, whereas higher and prolonged application of force triggered sustained elevation in intracellular calcium, leading to enzyme activation and cell death. All of these pathological events were rescued in acinar cells treated with a Piezo1 antagonist and in acinar cells from mice with genetic deletion of Piezo1. We discovered that Piezo1 stimulation triggered transient receptor potential vanilloid subfamily 4 (TRPV4) channel opening, which was responsible for the sustained elevation in intracellular calcium that caused intracellular organelle dysfunction. Moreover, TRPV4 gene-KO mice were protected from Piezo1 agonist- and pressure-induced pancreatitis. These studies unveil a calcium signaling pathway in which a Piezo1-induced TRPV4 channel opening causes pancreatitis.

Tamoxifen suppresses pancreatic ß-cell proliferation in mice.

Tamoxifen is a mixed agonist/antagonist estrogen analogue that is frequently used to induce conditional gene deletion in mice using Cre-loxP mediated gene recombination. Tamoxifen is routinely employed in extremely high-doses relative to typical human doses to induce efficient gene deletion in mice. Although tamoxifen has been widely assumed to have no influence upon ß-cells, the acute developmental and functional consequences of high-dose tamoxifen upon glucose homeostasis and adult ß-cells are largely unknown. We tested if tamoxifen influences glucose homeostasis in male mice of various genetic backgrounds. We then carried out detailed histomorphometry studies of mouse pancreata. We also performed gene expression studies with islets of tamoxifen-treated mice and controls. Tamoxifen had modest effects upon glucose homeostasis of mixed genetic background (F1 B6129SF1/J) mice, with fasting hyperglycemia and improved glucose tolerance but without overt effects on fed glucose levels or insulin sensitivity. Tamoxifen inhibited proliferation of ß-cells in a dose-dependent manner, with dramatic reductions in ß-cell turnover at the highest dose (decreased by 66%). In sharp contrast, tamoxifen did not reduce proliferation of pancreatic acinar cells. ß-cell proliferation was unchanged by tamoxifen in 129S2 mice but was reduced in C57Bl6 genetic background mice (decreased by 59%). Gene expression studies revealed suppression of RNA for cyclins D1 and D2 within islets of tamoxifen-treated mice. Tamoxifen has a cytostatic effect on ß-cells, independent of changes in glucose homeostasis, in mixed genetic background and also in C57Bl6 mice. Tamoxifen should be used judiciously to inducibly inactivate genes in studies of glucose homeostasis.

Mechanical Stabilization of the Glandular Acinus by Linker of Nucleoskeleton and Cytoskeleton Complex.

The nucleoskeleton and cytoskeleton are important protein networks that govern cellular behavior and are connected together by the linker of nucleoskeleton and cytoskeleton (LINC) complex. Mutations in LINC complex components may be relevant to cancer, but how cell-level changes might translate into tissue-level malignancy is unclear. We used glandular epithelial cells in a three-dimensional culture model to investigate the effect of perturbations of the LINC complex on higher order cellular architecture. We show that inducible LINC complex disruption in human mammary epithelial MCF-10A cells and canine kidney epithelial MDCK II cells mechanically destabilizes the acinus. Lumenal collapse occurs because the acinus is unstable to increased mechanical tension that is caused by upregulation of Rho-kinase-dependent non-muscle myosin II motor activity. These findings provide a potential mechanistic explanation for how disruption of LINC complex may contribute to a loss of tissue structure in glandular epithelia.

Cholecystokinin (CCK) Regulation of Pancreatic Acinar Cells: Physiological Actions and Signal Transduction Mechanisms.

Pancreatic acinar cells synthesize and secrete about 20 digestive enzymes and ancillary proteins with the processes that match the supply of these enzymes to their need in digestion being regulated by a number of hormones (CCK, secretin and insulin), neurotransmitters (acetylcholine and VIP) and growth factors (EGF and IGF). Of these regulators, one of the most important and best studied is the gastrointestinal hormone, cholecystokinin (CCK). Furthermore, the acinar cell has become a model for seven transmembrane, heterotrimeric G protein coupled receptors to regulate multiple processes by distinct signal transduction cascades. In this review, we briefly describe the chemistry and physiology of CCK and then consider the major physiological effects of CCK on pancreatic acinar cells. The majority of the review is devoted to the physiologic signaling pathways activated by CCK receptors and heterotrimeric G proteins and the functions they affect. The pathways covered include the traditional second messenger pathways PLC-IP3-Ca2+ , DAG-PKC, and AC-cAMP-PKA/EPAC that primarily relate to secretion. Then there are the protein-protein interaction pathways Akt-mTOR-S6K, the three major MAPK pathways (ERK, JNK, and p38 MAPK), and Ca2+ -calcineurin-NFAT pathways that primarily regulate non-secretory processes including biosynthesis and growth, and several miscellaneous pathways that include the Rho family small G proteins, PKD, FAK, and Src that may regulate both secretory and nonsecretory processes but are not as well understood. © 2019 American Physiological Society. Compr Physiol 9:535-564, 2019.

Optimal diameter reduction ratio of acinar airways in human lungs.

In the airway network of a human lung, the airway diameter gradually decreases through multiple branching. The diameter reduction ratio of the conducting airways that transport gases without gas exchange is 0.79, but this reduction ratio changes to 0.94 in acinar airways beyond transitional bronchioles. While the reduction in the conducting airways was previously rationalized on the basis of Murray's law, our understanding of the design principle behind the acinar airways has been far from clear. Here we elucidate that the change in gas transfer mode is responsible for the transition in the diameter reduction ratio. The oxygen transfer rate per unit surface area is maximized at the observed geometry of acinar airways, which suggests the minimum cost for the construction and maintenance of the acinar airways. The results revitalize and extend the framework of Murray's law over an entire human lung.

Modeling Airflow and Particle Deposition in a Human Acinar Region.

The alveolar region, encompassing millions of alveoli, is the most vital part of the lung. However, airflow behavior and particle deposition in that region are not fully understood because of the complex geometrical structure and intricate wall movement. Although recent investigations using 3D computer simulations have provided some valuable information, a realistic analysis of the air-particle dynamics in the acinar region is still lacking. So, to gain better physical insight, a physiologically inspired whole acinar model has been developed. Specifically, air sacs (i.e., alveoli) were attached as partial spheroids to the bifurcating airway ducts, while breathing-related wall deformation was included to simulate actual alveolar expansion and contraction. Current model predictions confirm previous notions that the location of the alveoli greatly influences the alveolar flow pattern, with recirculating flow dominant in the proximal lung region. In the midalveolar lung generations, the intensity of the recirculating flow inside alveoli decreases while radial flow increases. In the distal alveolar region, the flow pattern is completely radial. The micron/submicron particle simulation results, employing the Euler-Lagrange modeling approach, indicate that deposition depends on the inhalation conditions and particle size. Specifically, the particle deposition rate in the alveolar region increases with higher inhalation tidal volume and particle diameter. Compared to previous acinar models, the present system takes into account the entire acinar region, including both partially alveolated respiratory bronchioles as well the fully alveolated distal airways and alveolar sacs. In addition, the alveolar expansion and contraction have been calculated based on physiological breathing conditions which make it easy to compare and validate model results with in vivo lung deposition measurements. Thus, the current work can be readily incorporated into human whole-lung airway models to simulate/predict the flow dynamics of toxic or therapeutic aerosols.

Impaired Exocrine Pancreatic Function Associates With Changes in Intestinal Microbiota Composition and Diversity.

BACKGROUND & AIMS: Changes in intestinal microbiome composition are associated with inflammatory, metabolic, and malignant disorders. We studied how exocrine pancreatic function affects intestinal microbiota. METHODS: We performed 16S ribosomal RNA gene sequencing analysis of stool samples from 1795 volunteers from the population-based Study of Health in Pomerania who had no history of pancreatic disease. We also measured fecal pancreatic elastase by enzyme-linked immunosorbent assay and performed quantitative imaging of secretin-stimulated pancreatic fluid secretion. Associations of exocrine pancreatic function with microbial diversity or individual genera were calculated by permutational analysis of variance or linear regression, respectively. RESULTS: Differences in pancreatic elastase levels associated with significantly (P < .0001) greater changes in microbiota diversity than with participant age, body mass index, sex, smoking, alcohol consumption, or dietary factors. Significant changes in the abundance of 30 taxa, such as an increase in Prevotella (q < .0001) and a decrease of Bacteroides (q < .0001), indicated a shift from a type-1 to a type-2 enterotype. Changes in pancreatic fluid secretion alone were also associated with changes in microbial diversity (P = .0002), although to a lesser degree. CONCLUSIONS: In an analysis of fecal samples from 1795 volunteers, pancreatic acinar cell, rather than duct cell, function is presently the single most significant host factor to be associated with changes in intestinal microbiota composition.

ARID1A, a SWI/SNF subunit, is critical to acinar cell homeostasis and regeneration and is a barrier to transformation and epithelial-mesenchymal transition in the pancreas.

OBJECTIVE: Here, we evaluate the contribution of AT-rich interaction domain-containing protein 1A (ARID1A), the most frequently mutated member of the SWItch/sucrose non-fermentable (SWI/SNF) complex, in pancreatic homeostasis and pancreatic ductal adenocarcinoma (PDAC) pathogenesis using mouse models. DESIGN: Mice with a targeted deletion of Arid1a in the pancreas by itself and in the context of two common genetic alterations in PDAC, Kras and p53, were followed longitudinally. Pancreases were examined and analysed for proliferation, response to injury and tumourigenesis. Cancer cell lines derived from these models were analysed for clonogenic, migratory, invasive and transcriptomic changes. RESULTS: Arid1a deletion in the pancreas results in progressive acinar-to-ductal metaplasia (ADM), loss of acinar mass, diminished acinar regeneration in response to injury and ductal cell expansion. Mutant Kras cooperates with homozygous deletion of Arid1a, leading to intraductal papillary mucinous neoplasm (IPMN). Arid1a loss in the context of mutant Kras and p53 leads to shorter tumour latency, with the resulting tumours being poorly differentiated. Cancer cell lines derived from Arid1a-mutant tumours are more mesenchymal, migratory, invasive and capable of anchorage-independent growth; gene expression analysis showed activation of epithelial-mesenchymal transition (EMT) and stem cell identity pathways that are partially dependent on Arid1a loss for dysregulation. CONCLUSIONS: ARID1A plays a key role in pancreatic acinar homeostasis and response to injury. Furthermore, ARID1A restrains oncogenic KRAS-driven formation of premalignant proliferative IPMN. Arid1a-deficient PDACs are poorly differentiated and have mesenchymal features conferring migratory/invasive and stem-like properties.

Glucocorticoid receptor regulates expression of microRNA-22 and downstream signaling pathway in apoptosis of pancreatic acinar cells.

AIM: To elucidate the underlying mechanism that microRNA-22 (miR-22) promotes the apoptosis of rat pancreatic acinar cells (AR42J) and the elements that regulate the expression of miR-22. METHODS: One hundred nanomoles per liter of caerulein (Cae) was administrated to induce the apoptosis of AR42J cells and the apoptosis rate was detected by flow cytometry analysis. An amylase assay kit was used to measure the amylase expression level in the supernatant. Quantitative real-time PCR (qRT-PCR) was adopted to measure miR-22 expression. We used online tools to predict the potential transcription promoter of miR-22 and the binding sites, which was further identified by using luciferase reporter analysis, chromatin immunoprecipitation (ChIP) and ChIP-qPCR assays. Then, a mimic of miR-22, Nr3c1 plasmid encoding the glucocorticoid receptor (GR), and si-Nr3c1 were used to transfect AR42J cells, respectively. The mRNA expression of miR-22, Nr3c1, and Erb-b2 receptor tyrosine kinase 3 (ErbB3) was confirmed by qRT-PCR and the apoptosis rate of AR42J cells was detected by flow cytometry analysis. Western blot was used to detect the expression of ErbB3, GR, PI3k, PI3k-p85α, Akt, p-Akt, Bad, Bax, Bcl-xl, Bcl-2, and cleaved caspase3. RESULTS: After inducing apoptosis of AR42J cells in vitro, the expression of miR-22 was significantly increased by 2.20 ± 0.26 and 4.19 ± 0.54 times, respectively, at 3 h and 6 h in comparison with the control group. As revealed by qRT-PCR assay, the expression of miR-22 was 78.25 ± 6.61 times higher in the miR-22 mimic group relative to the miRNA control group, accompanied with an obviously increased acinar cell apoptosis rate (32.53 ± 1.15 vs 18.07 ± 0.89, P = 0.0006). The upregulation of miR-22 could suppress its target gene, ErbB3, and the phosphorylation of PI3k and Akt. Furthermore, we predicted the potential transcription promoter of miR-22 and the binding sites using online tools. Luciferase reporter analysis and site-directed mutagenesis indicated that the binding site (GACAGCCATGTACA) of the GR, which is encoded by the Nr3c1 gene. Downregulation of the expression of GR could upregulate the expression of miR-22, which further promoted the apoptosis of AR42J cells. CONCLUSION: GR transcriptionally represses the expression of miR-22, which further promotes the apoptosis of pancreatic acinar cells by downregulating the downstream signaling pathway.

A simulation study of diffusion-convection interaction and its effect on multiple breath washout indices.

Experimental studies of acinar ventilation heterogeneity (Sacin) derived from the multiple breath washout have shown the potential of Sacin to pick up structural change in lung disease. Recent Sacin data suggest that even when intra-acinar structure is unaltered, the combination of convection, diffusion and number of acini fed by patent terminal bronchioles can modify Sacin. We show here how Sacin is affected by structural features such as the secondary alveolar septa, intra-acinar ramification and number of ventilated acini. The simulations also predict relationships between respective alterations in Sacin and washout indices such as lung clearance index (LCI) and alveolar mixing efficiency (AME). This was verified experimentally, with highly significant correlations between Sacin and LCI (r = +0.85;p < 0.001) and between Sacin and AME (r = - 0.92; p < 0.001). We have shown how acinar ventilation heterogeneity can be affected by a reduction of number of ventilated acini, a change in overall alveolization or in intra-acinar alveolization pattern, via their impact on the balance between convection and diffusion at acinar level.

Arid1a restrains Kras-dependent changes in acinar cell identity.

Mutations in members of the SWI/SNF chromatin remodeling family are common events in cancer, but the mechanisms whereby disruption of SWI/SNF components alters tumorigenesis remain poorly understood. To model the effect of loss of function mutations in the SWI/SNF subunit Arid1a in pancreatic ductal adenocarcinoma (PDAC) initiation, we directed shRNA triggered, inducible and reversible suppression of Arid1a to the mouse pancreas in the setting of oncogenic KrasG12D. Arid1a cooperates with Kras in the adult pancreas as postnatal silencing of Arid1a following sustained KrasG12D expression induces rapid and irreversible reprogramming of acinar cells into mucinous PDAC precursor lesions. In contrast, Arid1a silencing during embryogenesis, concurrent with KrasG12D activation, leads to retention of acinar cell fate. Together, our results demonstrate Arid1a as a critical modulator of Kras-dependent changes in acinar cell identity, and underscore an unanticipated influence of timing and genetic context on the effects of SWI/SNF complex alterations in epithelial tumorigenesis.

Myoepithelial cell-driven acini contraction in response to oxytocin receptor stimulation is impaired in lacrimal glands of Sjögren's syndrome animal models.

The purpose of the present studies was to investigate the impact of chronic inflammation of the lacrimal gland, as occurs in Sjögren's syndrome, on the morphology and function of myoepithelial cells (MECs). In spite of the importance of MECs for lacrimal gland function, the effect of inflammation on MECs has not been well defined. We studied changes in MEC structure and function in two animal models of aqueous deficient dry eye, NOD and MRL/lpr mice. We found a statistically significant reduction in the size of MECs in diseased compared to control lacrimal glands. We also found that oxytocin receptor was highly expressed in MECs of mouse and human lacrimal glands and that its expression was strongly reduced in diseased glands. Furthermore, we found a significant decrease in the amount of two MEC contractile proteins, α-smooth muscle actin (SMA) and calponin. Finally, oxytocin-mediated contraction was impaired in lacrimal gland acini from diseased glands. We conclude that chronic inflammation of the lacrimal gland leads to a substantial thinning of MECs, down-regulation of contractile proteins and oxytocin receptor expression, and therefore impaired acini contraction. This is the first study highlighting the role of oxytocin mediated MEC contraction on lacrimal gland function.

The newly established bovine endometrial gland cell line (BEGC) forms gland acini in vitro and is only IFNτ-responsive (MAPK42/44 activation) after E2 and P4-pre-incubation.

INTRODUCTION: Uterine glands (UG) are crucial for the establishment of ruminant pregnancy and influenced (orchestrated manner) by estrogen (E2), progesterone (P4) and interferon tau (IFNτ). In the study we established a bovine endometrial glandular cell line (BGEC) and tested its functional reactivity (signaling) to IFNτ. METHODS: BGEC was characterized by light microscopy (LM), epithelial markers (ezrin, CK18) [immunofluorescence (IF)/immunohistochemistry (IHC)] and ultrastructure (TEM/SEM) (apical microvilli). In vitro formation of gland acini and transepithelial-electric-resistance (TEER) measurements (EVOM) were done. The expression of mRNA-transcripts (RT-PCR) of steroid receptors (PR, PGRMC1/2, ESR1/2) and the IFNτ-system (IFNAR1/2, IRF1, 2, 9) was checked. BEGC was stimulated with IFNτ (10 ng/ml;1000 ng/ml) (15 min) after steroid pre-treatment [10 pg/ml E2 (two days)/20 ng/ml P4 (two days)]. Activation of MAPK42/44;STAT1 was evaluated (densitometrical Western Blot). RESULTS: BGEC cells expressed epithelial markers and possessed apical microvilli. High TEER-values could be measured (2320-2620 ohm/cm2). The assembled BEGC acini (25 days) were similar to UG in vivo (markers/ultrastructure). All transcripts (steroid receptors/IFNτ-system) could be detected in BEGC (mRNA). MAPK42/44 were significantly activated after E2/P4 pre-treatment and IFNτ stimulation (10 ng/ml) (p < 0.05), whilst 1000 ng/ml IFNτ did not activate MAPK42/44. Neither a STAT1 (by IFNτ) nor an activation (MAPK42/44;STAT1) by IFNτ-only was observed. DISCUSSION: BGEC retains its epithelial phenotype in culture and forms gland acini in vitro thereby confirming its glandular character. Cells were only reactive to (low) IFNτ concentrations when pre-treated with steroids thereby closely resembling implantation physiology in vivo. BEGC can be used as a bovine implantation model to study embryo-maternal communication during early pregnancy in cattle.