epidermal-growth-factor and Sjogren-s-Syndrome

The pathogenesis of Sjögren's syndrome (SS) involves multiple factors including genetic background, cell death, and exocrine dysfunction. We here discuss apoptotic control in exocrine glands in SS by showing various pro- and anti-apoptotic pathways. Although the membrane-bound and soluble form of the Fas/Fas ligand system is a leading player with activation of the death domain and caspase 8/3 cleavage, the role of soluble Fas/FasL (including its polymorphism) in apoptosis is controversial. The tumor necrosis factor related apoptosis-inducing ligand (TRAIL)-mediated apoptosis of salivary gland epithelial cells (SGECs) involves a mitochondrial pathway that includes caspase 9 cleavage. The involvement of innate immunity cells such as toll-like receptors (TLRs) has been investigated; TLR2-4 and TLR7-9 are associated with the induction of inflammation in exocrine glands of SS patients. TLR3 has the potential to induce the apoptosis of SS patients' SGECs. Linkage of epidermal growth factor (EGF) was shown in exocrine glands in SS, and it inhibited the Fas/FasL system with the help of cell-survival factors. TLR3 has dual actions to cause inflammation as well as apoptosis, which are inhibited by EGF. In conclusion, apoptosis in exocrine glands of SS patients is tightly controlled by balance of pro-apoptotic signals and growth factor.

Topics: Animals; Apoptosis; Caspase 9; Cell Survival; Epidermal Growth Factor; Exocrine Glands; Fas Ligand Protein; fas Receptor; Humans; Sjogren's Syndrome; Toll-Like Receptors

Amphiregulin (AREG) is a well-characterized member of the epidermal growth factor (EGF) family and is one of the ligands of the EGF receptor (EGFR). AREG plays a key role in mammalian development and in the control of branching morphogenesis in various organs. Furthermore, AREG participates in a wide range of physiological and pathological processes activating the major intracellular signalling cascades governing cell survival, proliferation and motility. In this article, we review current advances in exocrine glands morphogenesis, focusing on the salivary gland, and discuss the essential aspects of AREG structure, function and regulation, and its differential role within the EGFR family of ligands. Finally, we identify emerging aspects in AREG research applied to mammary gland development and the salivary gland autoimmune disease, Sjögren's syndrome.

Topics: Amphiregulin; Animals; Epidermal Growth Factor; Epithelial Cells; Exocrine Glands; Fibroblasts; Gene Expression Regulation, Developmental; Humans; Morphogenesis; Salivary Glands; Signal Transduction; Sjogren's Syndrome

Sjögren's syndrome (SS) is a chronic inflammatory autoimmune disease characterized by lymphocytic infiltration of the exocrine glands, especially the salivary and lacrimal glands. As a result of salivary gland dysfunction, most patients with SS have xerostomia, related to a reduced salivary flow rate. In addition to the discomfort due to xerostomia, dry mouth can cause various intraoral manifestations such as refractory stomatitis, ulcer and atrophic changes in the oral mucosa and tongue, and patients' quality of life (QOL) is impaired severely. These manifestations are believed to be caused mainly by a decrease in the clearance in the oral cavity owing to hyposalivation. However, since saliva has several beneficial physiological effects on the intraoral environment, qualitative changes in sialochemistry should also be considered a cause of the refractory intraoral manifestations in SS. Salivary epidermal growth factor (EGF) is considered an important cytoprotective factor against injuries, and it contributes to wound healing in the oral cavity. We evaluated changes in salivary EGF levels and assessed the association between salivary EGF levels and the severity of intraoral manifestations in SS patients. The results showed that the salivary EGF levels decreased with the progression of SS, and this deterioration in saliva quality as well as hyposalivation could play a role in the pathogenesis of refractory intraoral manifestations in SS patients. Our findings provide new target for therapeutic intervention in SS.

Topics: Adult; Aged; Biomarkers; Disease Progression; Epidermal Growth Factor; Female; Humans; Male; Middle Aged; Saliva; Severity of Illness Index; Sjogren's Syndrome

To assess the efficacy of sodium hyaluronate as vehicle for diluting autologous serum.. The concentration and temporal stability of EGF, TGF-β, PDGF-AB and albumin in fresh and frozen samples of autologous serum diluted with sodium hyaluronate and saline solution, as well as the pH, osmolarity and density was studied. In parallel, the clinic effects of autologous serum diluted to 20% with sodium hyaluronate were compared with another solution of autologous serum diluted to 20% with saline in a prospective, comparative, randomized and double-blind study in 26 patients (52 eyes) with Sjögren syndrome. Patients underwent a complete ophthalmic assessment including tear film evaluation and corneal and conjunctival impression cytology at the beginning of the study and 2 months later.. The growth factor (GF) concentration remained stable during 1 month at 4°C both in fresh and defrosted samples without any differences being found between both preparations. No differences were found related to osmolarity, pH and density between these preparations before and after frosting. Autologous serum diluted with sodium hyaluronate caused a significant improvement of the tear film stability, fluorescein and rose Bengal stain, break-up time, corneal and conjunctival squamous metaplasia as well as in the patient subjective perception.. Sodium hyaluronate is an excellent vehicle for diluting autologous serum due to the gradual release of GF and increasing their duration and effect on the ocular surface. Preparations diluted with sodium hyaluronate are better tolerated by patients and require a lower number of drops administrations.

Topics: Adult; Aged; Double-Blind Method; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Female; Humans; Hyaluronic Acid; Hydrogen-Ion Concentration; Male; Middle Aged; Ophthalmic Solutions; Osmolar Concentration; Pharmaceutical Vehicles; Platelet-Derived Growth Factor; Prospective Studies; Serum; Serum Albumin; Sjogren's Syndrome; Transforming Growth Factor beta

The diagnosis and monitoring of Sjögren syndrome (SS) is often difficult, requiring a multidisciplinary approach with invasive procedures. Our aim is to elucidate the tear protein alterations of dry eye disease (DED) with primary SS (pSS) and secondary SS (sSS) with the long-term instillation of eyedrops. We collected clinical demographics and tear fluid (TF) samples from DED patients with no autoimmune diseases (non-SS-DED), pSS-DED, and sSS-DED patients, followed by TF screening with tandem mass tagging-labeling gel-free proteomics assay. Bioinformatic analysis via Ingenuity Pathway Analysis was used to identify functional pathways and interacting networks. Validation of candidate proteins with enzyme-linked immunosorbent assay on the tear samples was done. The top functional pathways of the two comparisons (sSS-DED vs. pSS-DED and sSS-DED vs. non-SS-DED) were both associated with inflammation and stress-related signaling. After constructing an interaction network model with the selected candidate proteins, five proteins were identified. A Disintegrin and Metalloproteinase domain-containing protein 10 (ADAM10) was found to be an important candidate biomarker in all groups, followed by epidermal growth factor (EGF) in TF. This study revealed novel DED markers, ADAM10 and EGF, in differentiating between primary and secondary SS patients from tears by in-depth proteomic analysis.

Topics: Dry Eye Syndromes; Epidermal Growth Factor; Humans; Ophthalmic Solutions; Proteomics; Sjogren's Syndrome; Tears

Aberrant angiogenesis plays a role in the pathogenesis of Sjögren's syndrome (SS).. The aim of this study was to compare the levels of vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) in stimulatory parotid saliva and in serum in healthy subjects (HS), patients with primary SS (pSS) and secondary SS (sSS) and to evaluate the expression of EGF, proangiogenic VEGF165 and antiangiogenic VEGF165 b mRNA isoforms. Additionally, we determined the salivary levels of serine/arginine splicing factor (SRSF1), which regulates VEGF165 and VEGF165 b expression.. The study comprised 34 women (16 with pSS and 18 with sSS) and healthy subjects for blood and saliva sampling. EGF and VEGF levels in saliva and serum and salivary SRSF1 levels were determined by enzyme-linked immunosorbent assay (ELISA). The expression of VEGF165 , VEGF165 b and EGF in peripheral blood mononuclear cells (PBMC) was evaluated by quantitative polymerase chain reaction (qPCR).. There were no differences in the levels of EGF, VEGF, SRSF1 and in the expression of the EGF, VEGF165 and VEGF165 b between HS and SS patients, or between pSS and sSS patients. The salivary levels of VEGF165 and EGF were significantly higher in pSS, sSS and HS than serum levels. Levels of SRSF1 correlated positively with VEGF and EGF levels. Levels of EGF, VEGF and SRSF1 correlated with each other.. The balance of VEGF isoforms is not disturbed in SS. Saliva is more sensitive for the detection of EGF and VEGF than serum, but salivary levels of those proteins are not representative for SS.

Topics: Biomarkers; Epidermal Growth Factor; Female; Healthy Volunteers; Humans; Leukocytes, Mononuclear; Polymerase Chain Reaction; Saliva; Salivary Glands, Minor; Sjogren's Syndrome; Vascular Endothelial Growth Factor A

Sjögren syndrome was chosen as a clinical model to study acinar salivary deficiencies in the development of laryngopharyngeal reflux (LPR). The objective of this prospective cohort study was to compare salivary epidermal growth factor (EGF) concentrations of patients with Sjögren syndrome with and without LPR and gastroesophageal reflux disease (GERD) with normal controls. LPR was diagnosed with positive scores on the Reflux Symptom Index and Reflux and Reflux Finding Score, corroborated by esophagogastroduodenoscopy and/or 24-hour pH-metry. Salivary EGF concentrations of both unstimulated and mechanically stimulated saliva were established using enzyme-linked immunosorbent assay, and the significance level was set at 95%. Twenty-one patients and 19 controls were studied. All patients had LPR and 60% also had GERD. The mean salivary EGF concentration of unstimulated and stimulated saliva in the control group was 1,751.37 pg/ml and 544.76 pg/ml, respectively. Unstimulated and stimulated salivary EGF concentrations in the study group were 2,534.65 pg/ml and 920.69 pg/ml, respectively. These differences were not statistically significant. Body mass index, presence of erosive esophagitis, or severity of hyposalivation did not significantly influence salivary EGF concentrations. LPR and GERD are highly prevalent in patients with Sjögren syndrome. Unlike previous studies in which significant EGF deficiencies were found in patients with reflux laryngitis and GERD, patients with Sjögren syndrome seem to have reflux caused by a decrease in clearance capacity and not in specific salivary components.

Topics: Acinar Cells; Adult; Brazil; Cohort Studies; Endoscopy, Digestive System; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Esophageal pH Monitoring; Female; Gastroesophageal Reflux; Humans; Laryngopharyngeal Reflux; Male; Middle Aged; Prospective Studies; Saliva; Salivary Glands; Sjogren's Syndrome; Statistics as Topic; Symptom Assessment

Healthy human labial salivary glands produce epidermal growth factor (EGF). In Sjögren's syndrome (SS), EGF staining is diminished. SS is also associated with chronic autoimmune corpus gastritis. We therefore hypothesized that EGF secretion would be diminished in SS and that this could affect gastric target cells.. Salivary EGF secretion in SS was compared to that in healthy controls using an enzyme-linked immunosorbent assay (ELISA). EGF receptor (EGFR) immunoreactive cells in the gastric corpus of healthy human subjects were analysed using immunostaining.. Salivary secretion of EGF was diminished in SS patients (232.4, range 52.6-618.4, vs. 756.6, range 105.3-1631.6 pg/min, p = 0.002). Proton-pump positive parietal cells were mostly EGFR immunoreactive whereas very few pepsinogen I (PGI)-positive cells were EGFR positive.. As EGF is relatively acid resistant, salivary gland-derived EGF might participate in an exo/endocrine mode of parietal cell maintenance in the gastric corpus. Deficiency of salivary gland-derived EGF in SS patients may cause impairment of gastric parietal cells resulting in exposure of immunogenic cryptic antigens and loss of immunological self-tolerance.

Topics: Adult; Aged; Autoimmune Diseases; Case-Control Studies; Chief Cells, Gastric; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; ErbB Receptors; Female; Gastric Mucosa; Gastritis; Humans; Immunohistochemistry; Middle Aged; Parietal Cells, Gastric; Saliva; Sjogren's Syndrome; Young Adult

To assess changes in salivary epidermal growth factor (EGF) levels within three years and investigate the correlation between these changes and the severity of intraoral manifestations in patients with Sjögren's syndrome (SS).. Twenty-three SS patients (14 primary SS and 9 secondary SS) and 14 controls were followed up for three years. Salivary EGF concentration was measured using an enzyme-linked immunosorbent assay, and intraoral manifestations were evaluated using a short version of the Oral Health Impact Profile (OHIP-14). Changes in salivary flow rate, EGF level, and severity of intraoral manifestations were analyzed, along with associations among them.. The OHIP-14 score significantly increased and the total salivary EGF output significantly decreased after three years in the SS group (10.2 ± 8.8 vs. 12.6 ± 9.2, p = 0.040; 10158.4 ± 9820.9 vs. 8352.8 ± 7813.3 pg/10 min, p = 0.032), though the salivary flow rate did not change. The decrease in total EGF output was especially high in patients with long disease duration and poor oral health-related quality of life (OHRQoL). In patients with poor OHRQoL, the change in total EGF output significantly correlated with the OHIP-14 score (r = - 0.847, p = 0.008). However, there was no correlation between the change in salivary flow rate and the OHIP-14 score.. The rapid decrease in salivary EGF level contributes to the progression of intraoral manifestations of SS.

Topics: Adult; Aged; Aged, 80 and over; Epidermal Growth Factor; Female; Follow-Up Studies; Humans; Male; Middle Aged; Quality of Life; Saliva; Severity of Illness Index; Sjogren's Syndrome; Surveys and Questionnaires

To assess changes in salivary epidermal growth factor (EGF) levels and the correlation between these levels and the severity of intraoral manifestations in Sjögren's syndrome (SS).. Forty SS patients and 23 controls were enrolled. Salivary EGF concentration was measured using an enzyme-linked immunosorbent assay, and intraoral manifestations were evaluated using a short version of the Oral Health Impact Profile (OHIP-14). The associations among salivary flow rate, EGF levels and the severity of intraoral manifestations were analyzed.. The total salivary EGF output was significantly decreased in the SS patients compared with the controls (9237.6 ± 8447.0 vs. 13296.9 ± 7907.1 pg/10 min, respectively, p = 0.033). In the SS patients, total EGF output and salivary flow rate showed a strong positive correlation (rs = 0.824, p = 0.0005), while total EGF output and disease duration showed a negative correlation (rs = -0.484, p = 0.008). Further, total EGF output was significantly correlated with the OHIP-14 score (rs = -0.721, p = 0.012).. The salivary flow rate and EGF levels are decreased in SS, and this deterioration in saliva quality causes refractory intraoral manifestations. Our findings have provided new therapeutic targets for SS.

Topics: Adult; Aged; Aged, 80 and over; Epidermal Growth Factor; Female; Humans; Male; Middle Aged; Saliva; Salivation; Severity of Illness Index; Sjogren's Syndrome

To investigate the correlation between epidermal growth factor (EFG) and atrophic glossitis (AG) in patients with Sjoigren's syndromes (SS) and explore its pathogenesis.. Ninety-three patients with SS (60 with AG and 33 without AG) and 20 normal were selected. The concentrations of EGF in saliva were analyzed by ELISA. The expressions of EGF receptor (EGFR) in the epithelial cells of the tongue were assayed by immunohistochemistry. The differences among each group were analyzed with SPSS19.0 software package.. The saliva EGF concentrations in SS was lower than that in normal control group(P<0.0001),and EGF concentrations in SS with AG was significantly lower than that in SS without AG (P=0.024). EGF levels in saliva gradually decreased in the mild, moderate and severe atrophic glossitis groups, and there were significant differences among each group(P<0.05). EGFR in the epithelial cells of tongue was lower in SS with moderate and severe AG than in the control group(P=0.009, P=0.037), and there was a significant correlation between EGF and the degree of AG (r=-0.673, P<0.01).. Saliva EGF concentrations decrease significantly in patients with SS and it is closely related to the morbidity of atrophic glossitis.

Topics: EGF Family of Proteins; Epidermal Growth Factor; ErbB Receptors; Glossitis; Humans; Immunohistochemistry; Saliva; Sjogren's Syndrome

Muscarinic type 3 receptor (M3R) plays a pivotal role in the induction of glandular fluid secretions. Although M3R is often the target of autoantibodies in Sjögren's syndrome (SjS), chemical agonists for M3R are clinically used to stimulate saliva secretion in patients with SjS. Aside from its activity in promoting glandular fluid secretion, however, it is unclear whether activation of M3R is related to other biological events in SjS. This study aimed to investigate the cytoprotective effect of chemical agonist-mediated M3R activation on apoptosis induced in human salivary gland (HSG) cells. Carbachol (CCh), a muscarinic receptor-specific agonist, abrogated tumor necrosis factor α/interferon γ-induced apoptosis through pathways involving caspase 3/7, but its cytoprotective effect was decreased by a M3R antagonist, a mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (ERK) inhibitor, a phosphatidylinositol 3-kinase/Akt inhibitor, or an epidermal growth factor receptor (EGFR) inhibitor. Ligation of M3R with CCh transactivated EGFR and phosphorylated ERK and Akt, the downstream targets of EGFR. Inhibition of intracellular calcium release or protein kinase C δ, both of which are involved in the cell signaling of M3R-mediated fluid secretion, did not affect CCh-induced ERK or Akt phosphorylation. CCh stimulated Src phosphorylation and binding to EGFR. A Src inhibitor attenuated the CCh/M3R-induced cytoprotective effect and EGFR transactivation cascades. Overall, these results indicated that CCh/M3R induced transactivation of EGFR through Src activation leading to ERK and Akt phosphorylation, which in turn suppressed caspase 3/7-mediated apoptotic signals in HSG cells. This study, for the first time, proposes that CCh-mediated M3R activation can promote not only fluid secretion but also survival of salivary gland cells in the inflammatory context of SjS.

Topics: Apoptosis; Calcium; Carbachol; Caspase 3; Caspase 7; Cell Survival; Cells, Cultured; Epidermal Growth Factor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Humans; Interferon-gamma; Phosphorylation; Protein Kinase C-delta; Proto-Oncogene Proteins c-akt; Receptor, Muscarinic M3; Salivary Glands; Signal Transduction; Sjogren's Syndrome; src-Family Kinases; Transcriptional Activation; Tumor Necrosis Factor-alpha

Non-obese diabetic (NOD) mice develop Sjögren's-like disease (SS-like) with loss of saliva flow and increased lymphocytic infiltrates in salivary glands (SGs). There are recent reports using multipotent mesenchymal stromal cells (MSCs) as a therapeutic strategy for autoimmune diseases due to their anti-inflammatory and immunomodulatory capabilities. This paper proposed a combined immuno- and cell-based therapy consisting of: A) an injection of complete Freund's adjuvant (CFA) to eradicate autoreactive T lymphocytes, and B) transplantations of MSCs to reselect lymphocytes. The objective of this was to test the effectiveness of CD45(-)/TER119(-) cells (MSCs) in re-establishing salivary function and in reducing the number of lymphocytic infiltrates (foci) in SGs. The second objective was to study if the mechanisms underlying a decrease in inflammation (focus score) was due to CFA, MSCs, or CFA+MSCs combined.. Donor MSCs were isolated from bones of male transgenic eGFP mice. Eight week-old female NOD mice received one of the following treatments: insulin, CFA, MSC, or CFA+MSC (combined therapy). Mice were followed for 14 weeks post-therapy. CD45(-)/TER119(-) cells demonstrated characteristics of MSCs as they were positive for Sca-1, CD106, CD105, CD73, CD29, CD44, negative for CD45, TER119, CD11b, had high number of CFU-F, and differentiated into osteocytes, chondrocytes and adipocytes. Both MSC and MSC+CFA groups prevented loss of saliva flow and reduced lymphocytic infiltrations in SGs. Moreover, the influx of T and B cells decreased in all foci in MSC and MSC+CFA groups, while the frequency of Foxp3(+) (T(reg)) cell was increased. MSC-therapy alone reduced inflammation (TNF-α, TGF-β), but the combination of MSC+CFA reduced inflammation and increased the regenerative potential of SGs (FGF-2, EGF).. The combined use of MSC+CFA was effective in both preventing saliva secretion loss and reducing lymphocytic influx in salivary glands.

Topics: Adjuvants, Immunologic; Animals; Antigens, CD; Antigens, Ly; Combined Modality Therapy; Epidermal Growth Factor; Female; Fibroblast Growth Factor 2; Freund's Adjuvant; Immunohistochemistry; Lymphocytes; Male; Membrane Proteins; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, Transgenic; Reverse Transcriptase Polymerase Chain Reaction; Saliva; Salivary Glands; Sjogren's Syndrome; Transforming Growth Factor alpha; Treatment Outcome; Tumor Necrosis Factor-alpha

Non-obese diabetic (NOD) mice develop Sjögren's-like syndrome (Ss) and a gradual loss of saliva secretory function. Our previous study showed that injections of matched normal spleen cells with Complete Freund's Adjuvant (CFA) reversed salivary gland dysfunction in 14-week-old NOD mice, which had established Ss. The spleen and bone marrow are closely related organs, and both are among the first sites of hematopoiesis during gestation. Noticing a rapidly increasing number of clinical trials using bone marrow (BM) cells treatments for autoimmune diseases, we tested if BM cells can prevent Ss and restore salivary glands' function. We injected CFA and MHC class I-matched normal BM cells in 7-week-old NOD mice, which had not yet developed Ss. We found at week 52 post-treatment that all NOD mice receiving BM cells and CFA had a recovery of salivary flow and were protected from Ss and diabetes. BM cells-treated mice had their salivary function restored quantitatively and qualitatively. Saliva flow was higher (p<0.05) in BM cells-transplanted mice when compared to control mice, which continued to deteriorate over time. Total proteins, epidermal growth factor, amylase, and electrolytes concentrations in saliva of BM cells-treated mice were not significantly changed at week 44 and 52 post-therapy when compared to pre-therapy (when the mice did not have Ss). Restoration of salivary flow could have resulted from a combination of rescue and paracrine effects from BM cells. This study suggests that a combined immuno- and cell-based therapy can permanently prevent Ss and restored salivary function in NOD mice.

Topics: Amylases; Animals; Bone Marrow Cells; Cell- and Tissue-Based Therapy; Epidermal Growth Factor; Immunohistochemistry; In Situ Hybridization, Fluorescence; Mice; Mice, Inbred NOD; Salivary Glands; Sjogren's Syndrome

Epidermal growth factor (EGF) exerts tropic effects on salivary epithelial cells. We examined EGF-mediated signaling pathways in the salivary epithelial cells of patients with Sjögren's syndrome (SS). We compared the immunohistochemical expression of EGF receptor (EGF-R), phosphatidylinositol 3-kinase (PI3K), Akt and nuclear factor kappa B (NF-kappaB) in the labial salivary glands of SS patients (n = 6) with those of control subjects (n = 2). EGF-mediated signaling pathways were further studied in vitro (n = 3) using primary salivary epithelial cells; NF-kappaB p65 nuclear translocation and Akt phosphorylation were examined by immunofluorescence and western blotting, respectively. The phosphorylation of EGF-R and Akt, and the nuclear expression of NF-kappaB p65, were increased in situ in the salivary epithelial cells of SS patients compared with those of control subjects. Epidermal growth factor induced rapid EGF-R phosphorylation and NF-kappaB p65 nuclear translocation in primary salivary epithelial cells in vitro. However, EGF also induced late Akt phosphorylation (after 12 h). Chemical inhibition of PI3K-Akt by LY294002/wortmannin did not affect EGF-mediated NF-kappaB p65 nuclear translocation; and NF-kappaB inhibition by Bay 11-7082 did not suppress Akt phosphorylation. Our data suggest that EGF stimulates both the PI3K-Akt pathway and NF-kappaB via distinct mechanisms, promoting tropic effects in SS salivary epithelial cells.

Topics: Biopsy; Case-Control Studies; Cells, Cultured; Enzyme Activation; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Female; Fluorescein-5-isothiocyanate; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Humans; Immunohistochemistry; NF-kappa B; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Recombinant Proteins; Rhodamines; Salivary Glands, Minor; Sjogren's Syndrome

To verify the hypothesis that protein concentrations, such as lactoferrin, epidermal growth factor (EGF), and aquaporin 5 (AQP5), in tears are abnormal in patients with dry eye.. Prospective case-control study.. One hundred three dry eye patients were divided into three groups: dry eye not associated with the Sjögren syndrome (non-SS; n = 71), Sjögren syndrome (SS; n = 23), and Stevens-Johnson syndrome (SJS; n = 9). Sixteen normal control subjects were also checked. The concentrations of lactoerrin, EGF, and AQP5 were measured by enzyme-linked immunosorbent assay.. The concentration of lactoferrin was significantly decreased in tears of non-SS (P =.0001), SS (P =.00005), and SJS (P =.0006) patients compared with control subjects. The concentration of EGF was significantly decreased in non-SS (P =.0005), SS (P =.00002), and SJS (P =.0001) patients compared with control subjects. The concentration of AQP5 was significantly increased in tears of only SS patients (P =.01) compared with control subjects and increased in tears of only SS patients compared with non-SS patients (P =.007).. The decrease in both lactoferrin and EGF was found not only in SS patients but also in non-SS patients, indicating that tear components in dry eyes differ in their quantity and quality. Quantification of AQP5 increased only in SS patients, suggesting that AQP5 protein leaks into the tears when acinar cells of the lacrimal gland are damaged by lymphocytic infiltration.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Aquaporin 5; Aquaporins; Case-Control Studies; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Eye Proteins; Female; Humans; Lactoferrin; Male; Membrane Proteins; Middle Aged; Prospective Studies; Sjogren's Syndrome; Stevens-Johnson Syndrome; Tears

Epithelial cells appear to play an important role in the initiation and maintenance of autoimmune lesions in the salivary glands of patients with Sjogren's syndrome. Therefore, the detailed study of immunological function of salivary gland epithelial cells (SGEC) may provide useful information for the understanding of Sjögren's syndrome pathogenesis. In this report we aimed to formulate a protocol for the establishment of human non-neoplastic SGEC lines as a tool for the study of the physiology and pathophysiology of these cells. Pointing towards a practical approach, we sought to establish SGEC lines from quite a limited amount of biopsy tissue obtained during the diagnostic evaluation of patients. Herein, the favorable conditions for the long-term maintenance of human non-neoplastic SGEC lines are presented and involve the successive application of a serum-containing and a serum-free culture medium, supplemented with essential epithelial growth factors. This protocol has been found reliable and convenient, as attested by the reproducible establishment of non-neoplastic SGEC lines. The analysis of SGEC phenotypic features, as well as a coculture system for the study of interactions between epithelial cells and lymphocytes, are also described. Such techniques may provide valuable means for the functional and molecular investigation of human SGEC and particularly for the study of Sjögren's syndrome and other disorders of glandular epithelia.

Topics: Apoptosis; Autoimmune Diseases; Biopsy; CD4-Positive T-Lymphocytes; Cell Communication; Cell Culture Techniques; Cell Division; Coculture Techniques; Culture Media; Culture Media, Serum-Free; Epidermal Growth Factor; Epithelial Cells; Humans; Leukemia, T-Cell; Phenotype; Reproducibility of Results; Salivary Glands; Sjogren's Syndrome; T-Lymphocytes; Time Factors

Salivary epithelial cells from patients with primary Sjögren's syndrome (SS) undergo Fas-mediated apoptosis. Bcl-2 and Bcl-xL are apoptosis suppressing oncogenes. Very little is known about the role of these oncogene molecules in salivary epithelial cells. To investigate the possible prevention of salivary glandular destruction in SS by Bcl-2 and Bcl-xL, stable transfectants expressing these molecules were made from HSY cells, a human salivary epithelial cell line. HSY cells were transfected with an expression vector for human Bcl-2 or Bcl-xL. Stable transfectants were selected and apoptosis was induced by anti-Fas antibody. Apoptosis was quantified by propidium iodide staining followed by flow cytometry. Caspase activity was detected by immunohistochemical analysis and enzyme cleavage of DEVD-AMC, a fluorescent substrate. Response to carbachol, a muscarinic receptor agonist, and EGF was measured by Ca2+ mobilization and influx. Fas-mediated apoptosis was significantly inhibited in Bcl-2 and Bcl-xL transfectants compared to wild-type and control transfectants (empty vector). Surprisingly, caspase activity was not inhibited in Bcl-2 and Bcl-xL transfectants. Activation of the Fas pathway in the Bcl-2 and Bcl-xL transfectants by antibody also inhibited carbachol and EGF responsiveness (i.e., Ca2+ mobilization and/or influx) by 50-60%. This Fas-mediated inhibition of cell activation was partially or completely restored by specific peptide interference of caspase enzyme activity. The prevention of Fas-mediated apoptosis by the overexpression of Bcl-2 and Bcl-xL in salivary gland epithelial cells results in injured cells expressing caspase activity and unable to respond normally to receptor agonists. Such damaged cells may exist in SS patients and could explain the severe dryness out of proportion to the actual number of apoptotic cells seen on salivary gland biopsy.

Topics: Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Calcium; Carbachol; Cardiotonic Agents; Caspases; Ceramides; Epidermal Growth Factor; Epithelial Cells; fas Receptor; Flow Cytometry; GTP-Binding Proteins; Humans; Immunohistochemistry; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Salivary Glands; Signal Transduction; Sjogren's Syndrome; Transfection; Tumor Cells, Cultured

To evaluate the efficacy of autologous serum application for the treatment of dry eye in Sjögren's syndrome.. The stability of essential components (EGF, vitamin A, and TGF-beta) in preserved serum were examined following preservation at 4 degrees C and -20 degrees C. In a primary clinical trial, 12 patients with Sjögren's syndrome were treated with autologous serum (diluted to 20% with sterile saline) for 4 weeks, and vital staining of the ocular surface was compared before and after treatment. The effects of serum on mucin (MUC-1) expression were observed in cultured conjunctival epithelial cells in vitro.. EGF, vitamin A, and TGF-beta were well preserved for up to 1 month in the refrigerator at 4 degrees C and up to 3 months in the freezer at -20 degrees C. Rose bengal and fluorescein scores improved significantly from the initial scores of 5.3 and 5.6 to 1.7 and 2.5 after 4 weeks, respectively. The additive effect of human serum for cultured conjunctival epithelial cells showed significant MUC-1 upregulation on the cell surface.. Autologous serum application is a safe and efficient way to provide essential components to the ocular surface in the treatment of dry eye associated with Sjögren's syndrome.

Topics: Blood; Dry Eye Syndromes; Epidermal Growth Factor; Female; Humans; Male; Middle Aged; Mucin-1; Ophthalmic Solutions; Sjogren's Syndrome; Tears; Transforming Growth Factor beta; Vitamin A

To compare epidermal growth factor (EGF) concentration in tear fluid and levels of inflammatory cytokines in the conjunctival epithelium of patients with Sjögren's syndrome keratoconjunctivitis sicca with those of normal controls.. Schirmer 1 tear testing, corneal fluorescein staining and conjunctival impression cytology for quantitation of goblet cell density were performed in ten patients with Sjögren's syndrome-associated keratoconjunctivitis sicca and ten asymptomatic normal controls. ELISA was used to detect the concentration of EGF in tear fluid and interleukin 6 in lysates of conjunctival cytology specimens obtained from all subjects. The levels of RNA transcripts encoding inflammatory cytokines [interleukin 1alpha_(IL-1alpha), interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor alpha_(TNF-alpha), and transforming growth factor beta1 (TGF-beta1)] as well as a housekeeping gene (G3PDH) were evaluated in conjunctival cytology specimens taken from all subjects by semiquantitative competitive reverse transcriptase polymerase chain reaction (RT-PCR).. Decreased tear fluid EGF concentration was noted in Sjögren's syndrome patients (mean 0.68 +/- 0.59 ng/ml) compared to controls (mean 1.66 +/- 0.45 ng/ml, P = 0.004). Significantly increased levels of IL-1alpha, IL-6, IL-8, TNF-alpha and TGF-beta1 RNA transcripts were found in the conjunctival epithelium of Sjögren's syndrome patients compared to controls (P < 0.05), while the level of G3PDH was similar in both groups. The concentration of IL-6 protein was significantly higher in Sjögren's syndrome conjunctiva samples (P = 0.012). Tear EGF concentration correlated with Schirmer 1 scores (rho 0.767, P < 0.001), corneal fluorescein staining scores (rho -0.562, P = 0.01), conjunctival goblet cell density (rho 0.661, P = 0.001) and the levels of IL-1alpha_and IL-8 RNA in the conjunctival epithelium (rho -0.677 and -0.747, respectively, P = 0.001). Both IL-1alpha_and IL-8 RNA in the conjunctival epithelium increased as Schirmer 1 scores decreased (P

Topics: Adult; Aged; Cell Count; Conjunctiva; Cytokines; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Epithelial Cells; Female; Humans; Interleukins; Keratoconjunctivitis Sicca; Male; Middle Aged; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sjogren's Syndrome; Tears; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) affect cells through binding to a shared EGF receptor (EGF-R), which is a transmembrane protein with tyrosine kinase activity. They exert trophic effects on vascular endothelial, salivary acinar, and ductal and mucosal epithelial cells. In Sjögren's syndrome (SS) focal sialadenitis leads to salivary gland tissue damage, diminished salivary flow, and changes in the oral epithelium, a complex referred to as xerostomia. We compared the localization of EGF, TGF-alpha, and EGF-R in labial salivary glands in SS and in healthy controls.. Labial salivary gland tissues of 12 patients with SS and 7 healthy controls were stained with the immunohistochemical peroxidase-antiperoxidase method for EGF, TGF-alpha, and EGF-R.. Immunoreactivity for both EGF and TGF-alpha was found in endothelial cells of blood vessels and in some ductal epithelial cells. TGF-alpha, but not EGF, was also found in some acinar cells. EGF-R was found in endothelial, acinar, and salivary duct epithelial cells. There was no difference in the expression of EGF-R between diseased and healthy specimens, but both EGF and TGF-alpha were diminished in SS.. The interrelated localization of EGF-R and its ligands, EGF and TGF-alpha, suggests an autocrine, juxtacrine, and paracrine mitogenic/trophic role for them and thus a role in the maintenance of the secretory and excretory cells of the normal salivary glands. The trophic effects on acinar cells seem not to be mediated by EGF, but more likely by TGF-alpha. The diminished expression of EGF and TGF-alpha indicates a failure of this trophic system in SS, which may contribute to the acinar atrophy and secondary changes thereof, including atrophy of the oral mucosa.

Topics: Adolescent; Adult; Aged; Blood Vessels; Epidermal Growth Factor; Epithelium; ErbB Receptors; Female; Humans; Immunohistochemistry; Male; Middle Aged; Salivary Ducts; Salivary Glands, Minor; Sjogren's Syndrome; Transforming Growth Factor alpha

Topics: Arthritis, Rheumatoid; Epidermal Growth Factor; Humans; Radioimmunoassay; Saliva; Sjogren's Syndrome; Stomach Ulcer