Effects of pro-inflamatory cytokines on polarized rat parotid Par-C10 monolayers [abstract]

Abstract only availableSjögren's syndrome (SS), an autoimmune disorder, is distinguished by inflammation and salivary gland cell death, leading to xerostomia (dry mouth). The G protein-coupled P2Y2 receptor (P2Y2R) is up-regulated in response to damage or stress in salivary epithelium. Pro-inflammatory cytokines associated with SS can be produced by infiltrating lymphocytes or salivary epithelium. Correlations have been found between lymphocytic infiltration and increased production of pro-inflammatory cytokines such as interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-Éø (TNFα) and interferon-γ (IFNγ) and decreased function of exocrine glands in SS. Recent data has shown that P2Y2R activation enhances the activity of metalloproteases that release TNFα. OBJECTIVES: To study the effects of cytokines on polarized salivary epithelium. METHODS: Polarized rat parotid (Par-C10) monolayers were used to perform these studies. Cytokines released by UTP-induced P2Y2R activation were identified by ELISA. To evaluate the role of cytokines associated with SS on epithelial integrity, epithelial resistance was determined and correlated with the expression and distribution of tight junction (TJ) proteins by immunofluorescence and Western analysis, respectively. RESULTS: Activation of P2Y2Rs in Par-C10 monolayers induced the release of TNFα. The cytokines TNFα and IFNγ, but not IL-6 or IL1β, decreased the resistance of Par-C10 cells. However, the expression/distribution of the TJ protein ZO-1 was unaffected. CONCLUSIONS: The data support a hypothesis that P2Y2R expression and activation in salivary gland cells contribute to epithelial dysfunction in SS by generating pro-inflammatory cytokines that regulate ion transport and epithelial integrity in salivary glands. Future studies will determine the role of cytokines on the expression and distribution of other TJ molecules including occludin, claudins and junctional adhesion molecules. These studies may lead to better therapeutic strategies for minimizing autoimmune-associated dysfunction of salivary gland that contributes to xerostomia in SS patient.Life Sciences Undergraduate Research Opportunity Progra

Up-regulation of the P2Y2 receptor by cytokines in neuronal cells

Abstract only availableAlzheimer's Disease (AD) is characterized by inflammation and neurodegeneration in the brain due to the presence of extracellular amyloid beta (A β) plaques and neurofibrillary tangles. Microglial and astrocyte cells associated with these plaques and tangles have been shown to release cytokines in AD patients, which have a proinflammatory effect on the brain. The P2Y2 receptor (P2Y2R) is a receptor protein that is up-regulated in response to damage or stress in a variety of tissues, including blood vessels and salivary gland epithelium. Recently our laboratory has shown that activation of the P2Y2R enhances α -secretase-dependent amyloid precursor protein (APP) processing. APP is proteolytically processed by β - and γ -secretases to release neurodegenerative A β. Alternatively, APP can be cleaved within the A β domain by α -secretase releasing the non-amyloidogenic product, sAPP α, which has been shown to have neuroprotective properties. Primary neurons have low P2Y2R expression, however, it has been demonstrated that cytokines up-regulate P2Y2R in smooth muscle cells. Therefore, this study will explore if cytokines up-regulate P2Y2R expression in primary rat neurons and in SH-SY5Y human neuroblastoma cells. Primary rat neurons and SH-SY5Y human neuroblastoma cells were plated on glass cover slips 24 or 48 hours with individual treatment, or a combination of, human interleukin-1 β (IL1- β), tumor necrosis factor α (TNF α), and interferon γ (IF γ). P2Y2R activity was measured by increases in intracellular calcium concentration ([Ca2+]i ) in response to the P2Y2R agonist UTP. Results support the hypothesis that P2Y2R is up-regulated by cytokines in neuronal cells. Furthermore, real-time PCR results indicate a two-fold increase in P2Y2R mRNA after cytokine treatment. Therefore, activation of the up-regulated P2Y2R in stressed neurons generates a neuroprotective (sAPP α) rather than neurodegenerative (A β) peptide. These results could have a substantial impact on the understanding and treatment of neurological disorders such as AD.Life Sciences Undergraduate Research Opportunity Progra

Phosphorylation of EGFR, ERK 1/2 and downstream transcription factors after P2Y2 receptor activation in a human submandibular gland cell line

Abstract only availableP2 nucleotide receptors mediate a variety of biological responses and are activated by the extracellular nucleotides adenosine triphosphate (ATP), adenosine diphosphate (ADP), uridine triphosphate (UTP), uridine diphosphate (UDP). The P2Y2 nucleotide receptor is a seven transmembrane spanning domain receptor activated by the nucleotides ATP and UTP, and is up-regulated in a variety of tissues in response to injury or stress. For example, the P2Y2 receptors are not normally expressed in salivary glands, but upon disruption of tissue homeostasis, the P2Y2 receptors are up-regulated. Sjogren's disease is an autoimmune disorder that affects salivary and lacrimal glands resulting in a decreased ability to produce saliva and tears. Previous work by our lab has shown that the P2Y2 receptor is up-regulated in submandibular glands of a Sjogren's syndrome mouse model, suggesting that it may be up-regulated in human Sjogren's syndrome. The goal of this project is to analyze the function of P2Y2 receptors in salivary gland tissues. HSG cells, which endogenously express P2Y2 receptors and are derived from a human submandibular gland tumor, were utilized as a cell model to analyze downstream signaling pathways in response to UTP. Our results show that UTP, the P2Y2 receptor selective agonist, causes phosphorylation of the epidermal growth factor receptor (EGFR), extracellular regulated kinases (ERK 1/2) and the downstream transcription factors p90RSK, and ELK, suggesting that P2Y2 receptors may play a role in gene transcription in salivary gland tissues.NSF-REU Biology & Biochemistr

P2Y2 nucleotide receptors mediate inflammatory responses in mouse salivary gland cells

Abstract only availableSjögren's syndrome (SS) is a chronic inflammatory autoimmune disease characterized by destruction of salivary and lacrimal glands leading to xerostomia (dry mouth) and xerophthalmia (dry eyes). Although the mechanisms involved have not been adequately elucidated, the diminished function of exocrine glands in SS is often associated with lymphocytic infiltration of the tissue. Aberrant expression of specific adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1) and intracellular cell adhesion molecule-1 (ICAM-1) is also observed in salivary gland with SS, which enable salivary epithelium to interact directly with infiltrating lymphocytes. P2Y2 nucleotide receptor (P2Y2R) is G protein-couple receptor that is activated by extracellular ATP and UTP. P2Y2R expression and activity is up-regulated in response to damage or stress in a variety of tissues, including submandibular glands (SMGs), where it mediates a complex set of cellular responses to injury of disease. Additionally, P2Y2R activation up-regulates VCAM-1 expression in dispersed rat SMG cell culture and human submandibular gland (HSG) cells. Our objective is to investigate weather P2Y2R up-regulation correlates with increased expression of adhesion molecules in SMGs from a mouse model for SS (C57BL/6.NOD-Aec1Aec2) as compared with normal mouse strain (C57BL/6). P2Y2R expression was measured by RT-PCR and adhesion molecules expression was determined by Western blot analysis. Salivary flow was preformed by cannulation of individual glands. We could see that P2Y2R expression and ICAM-1 expression were both up-regulated in the SMGs from a mouse model for SS as compared with normal mouse strain. And salivary flow was decreased in salivary glands from a mouse model for SS. These results suggest that P2Y2R mediate inflammatory responses related to secretory dysfunction in the mouse model for SS. Our ultimate goal would be to translate all this information to the human salivary gland in order to understand SS and to develop new therapies for salivary dysfunction in SS.Gyeongsang National Universit

Interventions novatrices pour faciliter une transition harmonieuse à la vie adulte pour les personnes présentant des incapacités et leur famille

Le présent projet visait le développement, en se basant sur les meilleures pratiques, les initiatives les plus probantes et les modèles de services les plus efficaces, d’un processus de transition vers une intégration socioprofessionnelle et communautaire plus harmonieux pour les jeunes DITSA ajusté aux besoins individuels des différents profils (OPHQ, 2003 ; OPHQ, 2015; MSSS, 2016). Ce processus de transition implique une préparation précoce qui cimente plus durablement le cheminement du jeune vers son projet de vie. La démarche a été réalisée en concertation avec l’ensemble des acteurs impliqués auprès du jeune adulte et de sa famille afin d’obtenir une vision commune et réaliste des besoins, des solutions et des priorités menant vers une intégration socioprofessionnelle et communautaire durable et satisfaisante

P2Y2 receptors Transactivate the EGFR/ERB1 and ERB3 Growth Factor Receptors in Human Salivary Gland Cells [abstract]

Abstract only availableThe epidermal growth factor receptor (EGFR/ERB1) plays a key role in the regulation of epithelial cell development, differentiation and in the pathophysiology of hyperproliferative diseases such as cancer. Transactivation of the EGFR/ERB1 by G-protein coupled receptors has been shown to be dependent on proteolytic cleavage of membrane ligands such as heparin binding epidermal growth factor (HBEGF), EGF, transforming growth factor (TGF-), epiregulin, amphiregulin and betacellulin. Utilizing the human submandibular gland (HSG) cell line, we found that activation of the P2Y2 nucleotide receptor (P2Y2R) by its agonist UTP caused a time-dependent activation of EGFR/ERB1; however, neutralizing antibodies to the known ligands to EGFR/ERB1 failed to inhibit the UTP-induced phosphorylation of EGFR/ERB1. EGFR/ERB1 phosphorylation can also be induced by heterodimerization with one of the other ERB family members, ERB2, ERB3, and ERB4. HSG cells express ERB2 and ERB3 but not ERB4. Since ERB2 is a ligandless receptor, ERB3 is the likely dimerizing partner. Our results indicate that P2Y2R activation by UTP phosphorylates ERB3. Heregulin, the only known ligand for ERB3 is expressed in HSGs. Therefore, our results suggest that P2Y2R activation stimulates the formation of ERB3-EGFR/ERB1 heterodimers by cleavage of heregulin and its binding to ERB3

Strategies to reduce waiting times in outpatient rehabilitation services for adults with physical disabilities : a systematic literature review

Objective: Identifying effective strategies to reduce waiting times is a crucial issue in many areas of health services. Long waiting times for rehabilitation services have been associated with numerous adverse effects in people with disabilities. The main objective of this study was to conduct a systematic literature review to assess the effectiveness of service redesign strategies to reduce waiting times in outpatient rehabilitation services for adults with physical disabilities. Methods: We conducted a systematic review, searching three databases (MEDLINE, CINAHL and EMBASE) from their inception until May 2021. We identified studies with comparative data evaluating the effect of rehabilitation services redesign strategies on reducing waiting times. The Mixed Methods Appraisal Tool was used to assess the methodological quality of the studies. A narrative synthesis was conducted. Results: Nineteen articles including various settings and populations met the selection criteria. They covered physiotherapy (n = 11), occupational therapy (n = 2), prosthetics (n = 1), exercise physiology (n = 1) and multidisciplinary (n = 4) services. The methodological quality varied (n = 10 high quality, n = 6 medium, n = 3 low); common flaws being missing information on the pre-redesign setting and characteristics of the populations. Seven articles assessed access processes or referral management strategies (e.g. self-referral), four focused on extending/modifying the roles of service providers (e.g. to triage) and eight changed the model of care delivery (e.g. mode of intervention). The different redesign strategies had positive effects on waiting times in outpatient rehabilitation services. Conclusions: This review highlights the positive effects of many service redesign strategies. These findings suggest that there are several effective strategies to choose from to reduce waiting times and help better respond to the needs of persons experiencing physical disabilities

P2X7 nucleotide receptors mediate caspase-8/9/3-dependent apoptosis in rat primary cortical neurons

Apoptosis is a major cause of cell death in the nervous system. It plays a role in embryonic and early postnatal brain development and contributes to the pathology of neurodegenerative diseases. Here, we report that activation of the P2X7 nucleotide receptor (P2X7R) in rat primary cortical neurons (rPCNs) causes biochemical (i.e., caspase activation) and morphological (i.e., nuclear condensation and DNA fragmentation) changes characteristic of apoptotic cell death. Caspase-3 activation and DNA fragmentation in rPCNs induced by the P2X7R agonist BzATP were inhibited by the P2X7R antagonist oxidized ATP (oATP) or by pre-treatment of cells with P2X7R antisense oligonucleotide indicating a direct involvement of the P2X7R in nucleotide-induced neuronal cell death. Moreover, Z-DEVD-FMK, a specific and irreversible cell permeable inhibitor of caspase-3, prevented BzATP-induced apoptosis in rPCNs. In addition, a specific caspase-8 inhibitor, Ac-IETD-CHO, significantly attenuated BzATP-induced caspase-9 and caspase-3 activation, suggesting that P2X7R-mediated apoptosis in rPCNs occurs primarily through an intrinsic caspase-8/9/3 activation pathway. BzATP also induced the activation of C-jun N-terminal kinase 1 (JNK1) and extracellular signal-regulated kinases (ERK1/2) in rPCNs, and pharmacological inhibition of either JNK1 or ERK1/2 significantly reduced caspase activation by BzATP. Taken together, these data indicate that extracellular nucleotides mediate neuronal apoptosis through activation of P2X7Rs and their downstream signaling pathways involving JNK1, ERK and caspases 8/9/3

Characterization of a P2Y2 nucleotide receptor antibody by Western blot analysis [abstract]

Abstract only availableFaculty Mentor: Dr. Gary Weisman, BiochemistryP2 nucleotide receptors modulate a wide range of physiological responses following their activation by extracellular nucleotides (Ralevic V et al., Pharmacol. Rev. 1998; 50: 413-492). The G protein-coupled P2Y2 nucleotide receptor (P2Y2R) subtype is fully activated by equivalent concentrations of ATP or UTP and is up-regulated in salivary gland models of stress and disease (Turner JT et al., Am. J. Physiol. 1997; 273: C1100-C1107; Ahn JS et al., Am. J. Physiol. 2000; 279: C286-C294; Schrader AM et al., Arch. Oral. Biol. 2005; 50: 533-540), in blood vessels after balloon angioplasty, and in collared carotid arteries where they promote intimal hyperplasia and inflammation by increasing smooth muscle cell proliferation and leukocyte infiltration (Seye CI et al., Arterioscler. Thromb. Vasc. Biol. 1997; 17: 3602-3610; Seye CI et al., 2002; Circulation 106: 2720-2726). Since a reliable anti-P2Y2R antibody is not currently available, determination of the presence of the P2Y2R in cells and tissues has been limited to P2Y2R mRNA quantification by reverse transcription-polymerase chain reaction (RT-PCR) or in situ hybridization of cells or tissues using P2Y2R-specific riboprobes. Alternatively, the functional activity of the P2Y2R in freshly isolated cells or established cell cultures can be determined by measuring changes in the intracellular free calcium concentration in response to ATP or UTP. Recently, a commercially-available anti-rat P2Y2R antibody has been produced by Alamone Laboratories (Jerusalem, Israel). The purpose of this study is to characterize the specificity of the Alamone antibody for the P2Y2R in human, rat and mouse tissues. Preliminary results from Western blot analysis of cell lysates from the rat ParC10 salivary gland cell line that expresses endogenous P2Y2Rs indicate a single band with an approximate size of 45 kD. Furthermore, a primary preparation of rat submandibular gland acinar cells cultured for 48 h also yielded a 45 kD band in Western analysis, whereas freshly prepared (0 time) acini did not show any bands, consistent with the observation that the P2Y2R is upregulated in submandibular gland acini as a function of time of culture. Additional experiments are underway to evaluate the specificity of the antibody with cells from P2Y2R knock-out mice and human 1321N1 astrocytoma cells expressing the recombinant human P2Y2R