Salivary Gland Samples Research Articles

Effect of Pilocarpine on Minor Salivary Glands of Rabbits: A Histological Study

Pilocarpine is a parasympathomimetic agent with mild β-adrenergic increase the rate of salivation. It is commonly used for treatment of patients with Sjögren'ssyndrome and post radiation salivary gland hypofunction. The intention of the present work was to describe the effects of Pilocarpine drug which is used as a drug with parasympathomimetic effect and the histological changes during treatment with it. Methods: Eighteen male rabbits were used in this experiment to show the effect of Pilocarpine. Different doses of drug 3mg/kg and 5mg/kg were given to treated groups (A and B) within the therapeutic limits. The experimental animals were injected intraperitoneally twice daily, for two and six weeks. Samples of the minor salivary glands were processed for light microscopy. Sections of the minor salivary glands were stained with H&E and PAS stains. Statistical analysis was followed to measure the diameter of the secretory acini. Results: The most noticeable changes were significant increase in the diameter of the secretory acini, and vacuolation with foamy appearance of the cells of the acini in treated groups. The PAS positive reaction in the acini was increased as the dose of the drug increased. Conclusion: Pilocarpine as sialogogues drug simulates the effect of parasympathomimetic drugs. The structural histological alterations noticed in this study substantiate the use of Pilocarpine as prophylactic and therapeutic agents in patients suffering from xerostomia, patients receiving radiotherapy at head and neck malignancies, and some diseases like Sjögren's syndrome

MicroRNA-mediated Regulation of Mucin-type O-glycosylation Pathway: A Putative Mechanism of Salivary Gland Dysfunction in Sjögren Syndrome.

To investigate microRNA (miRNA) that is potentially implicated in primary Sjögren syndrome (pSS)-related salivary hypofunction in labial salivary glands and to study miRNA-mediated mechanisms underlying oral dryness and altered rheology, focusing on the mucin O-glycosylation pathway. We performed miRNA expression profiling in minor salivary gland samples of patients with pSS presenting a different impairment in their unstimulated salivary flow rate. A computational in silico analysis was performed to identify genes and pathways that might be modulated by the deregulated miRNA that we had identified. To confirm in silico analysis, expression levels of genes encoding for glycosyltransferases and glycan-processing enzymes were investigated using Human Glycosylation-RT2 Profiler PCR Array. Among 754 miRNA analyzed, we identified 126 miRNA that were significantly deregulated in pSS compared to controls, with a trend that was inversely proportional with the impairment of salivary flow rates. An in silico approach pinpointed that several upregulated miRNA in patients with pSS target important genes in the mucin O-glycosylation. We confirmed this prediction by quantitative real-time PCR, highlighting the downregulation of some glycosyltransferase and glycosidase genes in pSS samples compared to controls, such as GALNT1, responsible for mucin-7 glycosylation. Collectively, our data suggest that the expression of different predicted miRNA-target genes in the mucin type O-glycan biosynthesis pathway is altered in pSS patients with low salivary flow and that the miRNA expression profile could influence the glycosidase expression levels and consequently the rheology in pSS.

Comparison of Oxidative Effects of Two Different Administration Form of Oxybutynin in the Potential Target Tissues.

Oxybutynin is an important anticholinergic agent that prevents uncontrolled contractions in the treatment of overactive bladder (OAB). However, drugs containing oxybutynin have significant side effects such as dry eyes, dry mouth, increased heart rate, constipation, blurred vision, and confusion. In recent years, new delivery methods for this agent are being searched. One of them is vaginal delivery. In this study, we aimed to compare the effects of oxybutynin on oxidative parameters in the potential target tissues of the oral and vaginal delivery. Female New Zealand white rabbits (n=12) were divided into two groups: oral delivery and vaginal delivery. The animals were sacrificed 48 h after administration and nitric oxide (NOx), thiobarbituric acid-reactive substances (TBARs), and glutathione (GSH) levels were determined spectrophotometrically in the aorta, salivary gland, and small intestine tissue samples. Vaginal delivery significantly decreased NOx levels in all tissue samples as compared to oral delivery (p < 0.05). Moreover, it reduced TBARs levels in salivary gland and aorta tissue samples (p < 0.05). In the light on these findings, it can be said that vaginal delivery may decrease the oxidant-induced side effects of oxybutynin as compared to oral delivery.

Overexpression Cathepsin D Contributes to Perineural Invasion of Salivary Adenoid Cystic Carcinoma.

Objective: Cathepsin D (CTSD) is a pivotal orchestrator in the occurrence and development of tumors. Recently, CTSD was detected in salivary adenoid cystic carcinoma (SACC). However, its functional role in perineural invasion (PNI) of SACC remained elusive. We conducted the present study to detect the expression of CTSD in SACC, analyze the correlation between CTSD expression and prognosis of SACC patients and elucidate the role of CTSD in occurrence of PNI in SACC to lay the foundation for further studies.Methods: Immunohistochemical analysis was conducted to assess CTSD and Ki67 expression in 158 SACC samples and 20 normal salivary gland samples adjacent to carcinoma. Meanwhile, the correlation between CTSD and PNI of SACC specimens was analyzed using Wilcoxon test. QRT-PCR, immunofluorescence and western blot analysis were used to examine the levels of CTSD mRNA and protein in SACC-LM cell line. SiRNA-mediated CTSD silence was performed. Scratch wound healing assay, transwell invasion assay and DRG co-culture assay of PNI was used to detect the ability of migration, invasion and PNI. FITC-phalloidin was used to detect cytoskeletal organization.Results: Our data demonstrated that the positive expression of CTSD was observed in 74.1% (117/158) of SACC cases, and the expression of CTSD was significantly correlated with the PNI (p < 0.05). The ability of migration, invasion, and PNI could be inhibited significantly by siRNA-mediated CTSD silence (p < 0.01). Furthermore, siRNA-mediated CTSD silence inhibited cytoskeletal organization and pseudo foot formation in SACC-LM cells.Conclusion: Our results suggested that an association between PNI and expression of CTSD existed. CTSD may promote PNI of SACC accompanied by cytoskeletal organization and pseudo foot formation.

HIF-1α, NOTCH1, ADAM12, and HB-EGF are overexpressed in mucoepidermoid carcinoma

Intratumoral hypoxia (IH) occurs during cellular proliferation of malignant tumors. This phenomenon is characterized by a decrease in oxygen levels in the neoplastic microenvironment. Throughout this condition, the proteins HIF-1α, NOTCH1, ADAM12, and HB-EGF can be activated, triggering signaling pathways associated with tumor invasiveness through invadopodia formation. This study aimed to evaluate the immunostaining of HIF-1α, NOTCH1, ADAM12, and HBEGF in 19 cases of mucoepidermoid carcinoma (MEC) and 10 samples of salivary glands (control group). The immunoperoxidase technique was employed to detect the proteins of interest. The Student t test was used to compare immunoexpression between MEC samples and the control group. Protein immunostaining was statistically significantly higher in MEC samples than in the control group (P < .01), and the proteins were especially overexpressed in epidermoid cells of MEC. We suggest that there is an association between the NOTCH1 signaling pathway activated by IH and the biologic behavior of MEC.

Interleukin-25 Axis Is Involved in the Pathogenesis of Human Primary and Experimental Murine Sjögren's Syndrome.

To investigate the role of the interleukin-25 (IL-25)/IL-17 receptor B (IL-17RB) axis in experimental Sjögren's syndrome (SS) and in patients with primary SS and primary SS-associated lymphoma. Expression of IL-25, IL-17RB, IL-17B, and tumor necrosis factor receptor-associated factor 6 (TRAF6) was analyzed on minor salivary gland (SG) samples from patients with primary SS and on parotid gland samples from patients with primary SS-associated B cell non-Hodgkin's lymphoma (NHL). IL-17RB expression and the frequencies of natural group 2 innate lymphoid cells (ILC2s), inflammatory ILC2s, and M2-polarized macrophages were assessed by flow cytometry in SG mononuclear cells and peripheral blood mononuclear cells (PBMCs). Tissue distribution of ILC2s was studied by confocal microscopy. The role of recombinant IL-25 and of rituximab in modulating IL-25 expression was investigated in in vitro studies. IL-25/IL-17RB and TRAF6 expression and the role of IL-25 inhibition were also studied in the experimental murine model of SS. Activation of the IL-25/IL-17RB/TRAF6 axis correlated with the focus score and was observed in patients with primary SS and in patients with primary SS-associated NHL. A significant increase in the frequency of inflammatory ILC2s was observed both in SG mononuclear cells and in PBMCs. IL-25 stimulation of isolated SG mononuclear cells and PBMCs from patients and controls resulted both in inflammatory ILC2 expansion and in increased autoantibody production. Rituximab modulated expression of inflammatory ILC2s and IL-25 in primary SS. SG protein-immunized mice developed overt SS symptoms with increased IL-25 expression and increased frequency of CD4+IL-17RB+TRAF6+ cells. IL-25 neutralization attenuated disease progression and tissue pathology in mice with experimental SS. IL-25 may promote the inflammatory state in primary SS and may be a potential target for novel disease-modifying therapeutic strategies in patients with primary SS.

Image analysis of fibrosis in labial salivary glands of patients with systemic autoimmune diseases. Close correlation of lobular fibrosis to seropositive rheumatoid arthritis and increased anti-CCP and RF titres in the serum

Lobular fibrosis in labial salivary glands of patients with systemic autoimmune disease is a rarely examined and rather neglected histological change. Its significance and disease association is poorly understood. Our aim was to explore the clinical correlations of fibrosis in labial salivary gland samples using objective methods and laboratory parameters. Labial salivary gland samples from more than 300 patients over a 3-year period were selected from the archives of the pathology department, histologically examined, digitised, image analysed and statistically evaluated to identify the presence and intensity of lobular fibrosis, its relation to age, clinical diagnoses of systemic autoimmune disease and the presence of rheumatoid factor (RF), anti-cyclic citrullinated peptide (CCP), antinuclear antibodies (ANAs), and anti-dsDNA serum markers. Significant correlationwas found between lobular fibrosis and the presence of autoimmune disease (p= 0.023), mainly seropositive rheumatoid arthritis (p < 0.001). Also significant association was found between the fibrosis and the presence of serum anti-CCP (p < 0.001) and IgA/IgG/IgM-RF (p < 0.001, p < 0.001 and p= 0.008, respectively). Significant association was explored between the anti-dsDNA positivity and the negative histology groups (p= 0.033) and between the ANA positivity and the inflammation only group (p= 0.021). The results suggest that lobular fibrosis tends to associate to certain systemic autoimmune diseases, mainly seropositive rheumatoid arthritis, and seems to be rare in labial salivary gland biopsiesof autoimmune diseases characterised by presence of anti-dsDNA. The close correlation of ANA positivity and the inflammation only histology was not surprising, since the majority of patients (62%) have Sjögren's syndrome, known for its inflammatory infiltrate. These findings emphasise that evaluation of lobular fibrosis and inflammation in histological samples of labial salivary gland biopsiesare equally important.

Dynamics of Boophilus Ticks and its Role in Transmission of Piroplasms at Behaira District

Dynamics of Boophilus tick was investigated at Behaira district. A total number of (86) out of (839) cattle (10.25%) were infected with Boophilus spp. The prevalence of Boophilus spp infection predominated in summer season (21.33%) followed by autumn (10.78%) and last spring (6.34%) and the lowest burden was in winter. Concerning the locality, El- Dalangat was representing the highest locality infected with tick vector (17.39%) and according to age, the animals less than one year was the lowest one infected with tick (5.17%) then animals which were aged 1-3 years (9.8%) and animals more than 3 years (10.75%).The neck and dewlap representing the highest predilection seats of Boophilus spp. (81.39%) in cattle and the lowest one was udder (2.94%). Hemolymph and salivary gland samples of 56 Boophilus spp were extracted, stained by Giemsa and microscopically inspected under oil immersion lens. the total infection of Hemolymph with ookinete was (28.57%) that increased in summer season (32.14%) and according to salivary gland smears of Boophilus spp. showed that the total infection rate was (21.43%) and increased in summer (25%) also winter had no infection for both. Electron microscopic examination of salivary gland of Boophilus spp. showed a prescence of electron-dense and more irregular-shaped sporozoites were detected in some infected cells.

Oncogenic DNA viruses found in salivary gland tumors

BackgroundPrevious investigations studying the association of DNA viruses with salivary gland tumors (SGTs) have led to conflicting results. The aim of this study was to determine the prevalence of different DNA viruses by using a highly sensitive assay in a multi-center series of over 100 fresh frozen salivary gland samples. MethodsDNA was isolated from 84 SGTs (80 parotid tumors and 4 submandibular gland tumors) and 28 normal salivary tissue samples from 85 patients in Northeast Italy. Using a highly sensitive type-specific multiplex genotyping assay, we analyzed the samples for the presence of DNA from 62 different viruses including 47 papillomaviruses, 10 polyomaviruses, and 5 herpesviruses. ResultsWe observed a high prevalence of beta human papillomavirus DNA in malignant tumors. In contrast, polyomavirus DNA was present in benign, malignant, and non-tumor control samples. Most striking was the significant distribution of herpesvirus DNA in the SGT samples, in particular the high prevalence of Epstein-Barr type 1 and type 2 DNA in Warthin’s tumor samples. ConclusionOur data provides evidence for the presence of DNA viruses in SGTs. Mechanistic studies are needed to further attribute tumor formation to these viruses.

The impact of temperature and Wolbachia infection on vector competence of potential dengue vectors Aedes aegypti and Aedes albopictus in the transmission of dengue virus serotype 1 in southern Taiwan

BackgroundWe evaluated the impact of temperature and Wolbachia infection on vector competence of the local Aedes aegypti and Ae. albopictus populations of southern Taiwan in the laboratory.ResultsAfter oral infection with dengue serotype 1 virus (DENV-1), female mosquitoes were incubated at temperatures of 10, 16, 22, 28 and 34 °C. Subsequently, salivary gland, head, and thorax-abdomen samples were analyzed for their virus titer at 0, 5, 10, 15, 20, 25 and 30 days post-infection (dpi) by real-time RT-PCR. The results showed that Ae. aegypti survived significantly longer and that dengue viral genome levels in the thorax-abdomen (103.25 ± 0.53–104.09 ± 0.71 PFU equivalents/ml) and salivary gland samples (102.67 ± 0.33–103.89 ± 0.58 PFU equivalents/ml) were significantly higher at high temperature (28–34 °C). The survival of Ae. albopictus was significantly better at 16 or 28 °C, but the virus titers from thorax-abdomen (100.70–102.39 ± 1.31 PFU equivalents/ml) and salivary gland samples (100.12 ± 0.05–101.51 ± 0.31 PFU equivalents/ml) were significantly higher at 22–28 °C. Within viable temperature ranges, the viruses were detectable after 10 dpi in salivary glands and head tissues in Ae. aegypti and after 5–10 dpi in Ae. albopictus. Vector competence was measured in Ae. albopictus with and without Wolbachia at 28 °C. Wolbachia-infected mosquitoes survived significantly better and carried lower virus titers than Wolbachia-free mosquitoes. Wolbachia coinfections (92.8–97.2%) with wAlbA and wAlbB strains were commonly found in a wild population of Ae. albopictus.ConclusionsIn southern Taiwan, Ae. aegypti is the main vector of dengue and Ae. albopictus has a non-significant role in the transmission of dengue virus due to the high prevalence of Wolbachia infection in the local mosquito population of southern Taiwan.

Mucoepidermoid carcinoma-associated expression of MUC5AC, MUC5B and mucin-type carbohydrate antigen sialyl-Tn in the parotid gland

ObjectivesThe aberrant expression of mucins and mucin-type carbohydrates has been described in many types of cancer, including mucoepidermoid carcinoma (MEC), a malignant salivary gland tumor. In this study, we examined the aberrant expression patterns of mucins (MUC1, MUC4, MUC5AC and MUC5B), simple mucin-type carbohydrate antigens (Tn, sialyl-Tn and T) and mature carbohydrate antigens (Lewisa and sulfo-Lewisa antigens) in MEC originating from the parotid gland, which normally does not secrete mucins. DesignWe conducted an immunohistochemical study to investigate the presence of mucins and carbohydrates in 24 MEC samples originating from the parotid gland and in surrounding normal tissue of the same gland in comparison 6 samples of normal salivary glands. The expression levels were compared with respect to the histological grading. Furthermore, 24 MEC samples from non-parotid salivary glands were included. ResultsWe observed loss of topology of membrane-bound MUC1 and MUC4, and de novo expression of MUC5AC, MUC5B and sialyl-Tn in MEC that originated in the parotid gland. Furthermore, mucins MUC1, MUC4 and carbohydrate antigens Tn, sialyl-Tn, T, Lewisa and sulfo-Lewisa were overexpressed in MEC samples compared to surrounding normal salivary gland tissues. MUC1 was expressed in both low- and high grade MECs, whereas MUC4 was not expressed in high grade MECs of the parotid gland. ConclusionDuring the development of MEC in the parotid gland, the genes for gel-forming secretory mucins are switched on. Besides these MEC tissues overexpress short oligosaccharides, suggesting that the glycosylation machinery is altered.

Altered expression of apoptosis-regulating miRNAs in salivary gland tumors suggests their involvement in salivary gland tumorigenesis.

Salivary gland tumors comprise a heterogeneous group of lesions with different histological features and diverse clinical pathophysiology. They account for about 3% of all head and neck tumors. Apoptosis plays an important role during morphogenesis of glandular structures, including that of the salivary gland. Recent studies have demonstrated that several microRNAs (miRNAs) are involved in the control of apoptosis. The aim of the present study was to determine the expression of apoptosis-related miRNAs (miR-15a, miR-16, miR-17-5p, miR-20a, miR-21, miR-29, and miR-34) and their target mRNAs in 25 pleomorphic adenomas, 23 mucoepidermoid carcinomas, and 10 non-neoplastic salivary gland samples by real-time RT-PCR. We observed upregulation of miR-15a, miR-16, miR-17-5p, miR-21, miR-29, and miR-34a in pleomorphic adenomas. The expression of miR-21 and miR-34a was upregulated in 91 and 74% of mucoepidermoid carcinomas, respectively. Downregulation of miR-20a was observed in 75% of pleomorphic adenomas and in 57% of mucoepidermoid carcinomas. APAF1, BAX, BCL2, BID, CASP2, CASP8, DIABLO , and TP53 transcripts were upregulated in both tumor types. BAD transcripts were upregulated in pleomorphic adenomas. CASP3 and CASP6 transcripts were upregulated in mucoepidermoid carcinomas. BCL2, CASP2, CASP6, and CASP8 proteins were mostly absent in mucoepidermoid carcinomas but expressed in few cells in pleomorphic adenomas. Our study provides evidence of alterations in the expression of apoptosis-regulating miRNAs in salivary gland tumors, suggesting possible involvement of these microRNAs in salivary gland tumorigenesis.

Exploring the transcriptomic data of the Australian paralysis tick, Ixodes holocyclus

AbstractIxodes holocyclus is the paralysis tick commonly found in Australia. I. holocyclus does not cause paralysis in the primary host – bandicoots, but markedly affects secondary hosts such as companion animals, livestock and humans. Holocyclotoxins are the neurotoxin molecules in I. holocyclus responsible for paralysis symptoms. There is a limited understanding of holocyclotoxins due to the difficulties in purifying and expressing these toxins in vitro. Next-generation sequencing technologies were utilised for the first time to generate transcriptome data from two cDNA samples –salivary glands samples collected from female adult ticks engorged on paralysed companion animals and on bandicoots. Contig-encoded proteins in each library were annotated according to their best BLAST match against several databases and functionally assigned into six protein categories: housekeeping, transposable elements, pathogen-related, hypothetical, secreted and novel. The “secreted protein” category is comprised of ten protein families: enzymes, protease inhibitors, antigens, mucins, immunity-related, lipocalins, glycine-rich, putative secreted, salivary and toxin-like. Comparisons of contig representation between the two libraries reveal the differential expression of tick proteins collected from different hosts. This study provides a preliminary description of the I. holocyclus tick salivary gland transcriptome.

Potential role for inhibition of protein phosphatase 2A tumor suppressor in salivary gland malignancies.

The aetiology and pathogenesis of salivary gland malignancies remain unknown. To reveal novel molecular factors behind the development of salivary gland cancer, we performed gene expression analyses from Smgb-Tag mouse salivary gland samples. The overall purpose was to apply these results for clinical use to find new approaches for both possible therapeutic targets and more accurate diagnostic tools. Smgb-Tag mouse strain, in which salivary neoplasms arise through a dysplastic phase in submandibular glands, was investigated using genome-wide microarray expression analysis, ingenuity pathway analysis, RT-PCR, and immunohistochemistry. Thirty-eight human salivary gland adenoid cystic carcinoma samples were investigated using immunohistochemistry for validation purposes. Our genome-wide study showed that Ppp2r1b, a PP2A subunit encoding tumor suppressor gene, is underexpressed in submandibular gland tumors of Smgb-Tag mice. mTOR signaling pathway was significantly enriched and mTOR linked PP2A subunit gene B55 gamma was significantly underexpressed in the analyses. Furthermore, parallel immunohistochemical analysis of three PP2A inhibitors demonstrated that two PP2A inhibitors, CIP2A and SET, are highly expressed in both dysplastic and adenocarcinomatous tumors of the Smgb-Tag mice. In addition, all 38 investigated human salivary adenoid cystic carcinoma samples stained positively for CIP2A and most for SET. Finally, p-S6 staining showed activation of mTOR pathway in human adenoid cystic carcinoma samples. Our results suggest that PP2A inhibition either via PP2A subunit underexpression or PP2A inhibitor overexpression play an important role in the formation of salivary gland malignancy, potentially due to mTOR signaling activation.

Abstract 4015: miR-221 ans miR-222 expression in salivary gland tumors

Abstract Background: MicroRNAs (miRNAs) are a class of short noncoding RNAs, that regulate the gene expression by binding to the 3′ - untranslated region of target mRNAS and by blocking the translation or degradation of the mRNAs that mediate various pathophysiological process including the pathogenesis of diverse human cancers and important cellular functions such as proliferation, apoptosis, differentiation, and cell signaling. Salivary gland tumors are complex, due to their broad histological spectrum resulting from multiple tumor cell differentiation. We previously detected that the protein c-kit is involved in the process of salivary gland morphogenesis and tumorigenesis; other recent studies reported alterations in KIT gene in adenoid cystic carcinoma. The aim of the present study is to determine the expression of microRNAs targeting KIT in pleomorphic adenoma (PA) and adenoid cystic carcinoma (ACC), the most common benign and malignant salivary gland neoplasms, respectively, and that originate from the intercalated duct region. Material and Methods: Twenty-five pleomorphic adenoma, 14 adenoid cystic carcinoma and 10 non-neoplastic salivary gland samples were used and miRNA expression was evaluated by Real Time RT-PCR using TaqMan miRNA Assays (miR- 221 and 222). Results: Expression of miR-221 was detected in 13 CAC samples and 19 PA samples. In CAC, expression of miR-221 was downregulated in 4 out of 13 (30,77%) samples, upregulated in 4 out of 13 (30,77%) samples, and normal in 5 out of 13 (38,46%) samples. In PA, miR-221 expression was downregulated in 7 out of 19 (36,84%) samples, upregulated in 2 out of 19 (10,53%) samples, and normal in 10 out of 19 (52,63%) samples. Expression of miR-222 was detected in 14 CAC samples and 22 PA samples. In the majority of CAC samples, the expression of miR-222 was similar to that observed in non-neoplastic samples. In PA samples, expression of miR-222 was downregulated in 7 out of 22 (31,8%) samples, upregulated in 8 out of 22 (36,4%) samples, and normal in 7 out of 22 (31,8%) samples. Conclusion: The expression of miRNAs 221 and 222 do not present a definite pattern to discriminate PA from ACC. This might be due to different mechanisms involved in their tumor biology or due to their common site of origin (the intercalated duct) which could add some similar biological features to both neoplasms. Citation Format: Renata Carolina Fraga Ianez, Cláudia Coutinho-Camillo, Clóvis Pinto, Fernando Soares, Silvia Lourenço. miR-221 ans miR-222 expression in salivary gland tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4015. doi:10.1158/1538-7445.AM2015-4015

Abstract 242: Evaluation of apoptosis-related microRNAs in salivary gland tumors

Abstract MicroRNAs (miRNAs) are a class of short non-coding RNAs that regulates gene expression post-transcriptionally, being important modulators of several cellular functions such as proliferation, apoptosis, differentiation, and cell signaling. Salivary gland tumors comprise a heterogeneous group of lesions with different histologic features and diverse clinical behavior accounting for about 3% of all head and neck tumors. The low incidence of salivary gland tumors, coupled with the large amount of different lesions present in this group, makes the diagnosis a challenging activity. Therefore, the identification of molecular markers for diagnosis and prognosis is so important, especially for this group of neoplasms. Apoptosis plays an important role during during morphogenesis of glandular structures, including salivary gland development and recent studies demonstrated that several miRNAs are involved in the control of apoptosis. The aim of the present study was to determine the expression of apoptosis-related miRNAs in salivary gland tumors. With this goal, 60 samples have been selected: 25 pleomorphic adenomas (most common benign tumor), 25 mucoepidermoid carcinomas (most common malignant tumor) and 10 normal salivary gland samples. miRNA expression was examined by Real Time RT-PCR (qPCR) using TaqMan miRNA Assays (miR-15a, miR-16, miR-17-5p, miR-20a, miR-21, miR-29 and miR-34). The relative microRNA expression level was normalized based on the expression of RNU48. Experiments were performed in duplicate for each data point. Considering up- or down-regulation as a 2-fold difference in expression relative to the normal tissue, we observed up-regulation of miR-15a, miR-16, miR-17-5p, miR-21, miR-29 and miR-34a in 76.0%, 60.0%, 47.8%, 80.0%, 84.0% and 96.0% of the pleomorphic adenoma samples, respectively. Down-regulation of miR-20a was observed in 75.0% of the pleomorphic adenoma samples. Normal expression of miR-15a, miR-16, miR-17-5p and miR-29 was observed in the mucoepidermoid carcinoma samples. Down-regulation of miR-20a was observed in 56.5% of the mucoepidermoid samples. An increased expression if miR-21 was observed in 90.9% of these samples. miR-34a is up-regulated in 73.9% of the mucoepidermoid carcinoma samples, suggesting an important role in the pathogenesis of these salivary gland tumor. Our study provides evidence of alterations in the expression of apoptosis-regulating miRNAs in salivary gland tumors, suggesting a possible involvement of these microRNAs in salivary gland tumorigenesis. Citation Format: Bianca T. Flores, Silvia V. Lourenço, Maria A. Nagai, Luiz Paulo Kowalski, Fernando A. Soares, Cláudia Coutinho-Camillo. Evaluation of apoptosis-related microRNAs in salivary gland tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 242. doi:10.1158/1538-7445.AM2015-242

Classical swine fever virus marker vaccine strain CP7_E2alf: Shedding and dissemination studies in boars

Over the last decade, pestivirus chimaera CP7_E2alf has proven to be a most promising marker vaccine candidate against classical swine fever (CSF). To provide further background data for the risk assessment towards licensing and release, especially on presence of the vaccine chimaera in faeces, urine, and organs of the male reproductive tract, supplementary studies were carried out under controlled laboratory conditions. In detail, the shedding and dissemination pattern of Suvaxyn® CSF Marker (“CP7_E2alf”) was assessed in 12 adult boars after single intramuscular vaccination with a tenfold vaccine dose. Four and seven days post vaccination, six animals were subjected to necropsy and triplicate samples were obtained from reproductive and lymphatic organs as well as urine, faeces, blood, and several additional organs and matrices. The sampling days were chosen based on pre-existing data that indicated the highest probability of virus detection. Upon vaccination, neither local nor systemic adverse effects were observed in the experimental animals. It was confirmed that primary replication is restricted to the lymphatic tissues and especially the tonsil. While viral genome was detectable in several samples from lymphatic tissues at four and seven days post vaccination, infectious virus was only demonstrated at four days post vaccination in one tonsil sample and one parotid lymphnode. Sporadic detection at a very low level occurred in some replicates of liver, lung, bone marrow, and salivary gland samples. In contrast, viral genome was not detected in any sample from reproductive organs and accessory sex glands, in faeces, urine, or bile.The presented data on the dissemination of the vaccine virus CP7_E2alf in adult boars are supplementing existing safety and efficacy studies and indicate that the use of the vaccine is also safe in reproductive boars.

Identification of potential genomic biomarkers for Sjögren's syndrome using data pooling of gene expression microarrays.

Sjögren's syndrome (SS) is an autoimmune disease characterized by lymphocytic infiltration and destruction of salivary and lacrimal glands. The diagnosis of SS can be challenging due to lack of a specific test for the disease. The purpose of this study is to examine the accuracy of using gene expression profile for diagnosis of SS. We identified 9 publically available datasets that included gene expression data from saliva and salivary gland biopsy samples of 52 patients with SS and 51 controls. Out of these datasets, we compiled and pooled data from three datasets that included 37 and 29 samples from SS patients and healthy controls, respectively, which were designated as "training set." Then, we performed cross-listing in a group of independent gene expression datasets from patients with SS to identify consensus gene list of differentially expressed genes. We performed Linear Discriminant Analysis (LDA) to quantify the accuracy of discriminating genes to predict SS in both the "training set" and an independent group of datasets that was designated as "test set." We identified 55 genes as potential classifier genes to differentiate SS from healthy controls. An LDA by leave-one-out cross-validation method identified 19 genes (EPSTI1, IFI44, IFI44L, IFIT1, IFIT2, IFIT3, MX1, OAS1, SAMD9L, PSMB9, STAT1, HERC5, EV12B, CD53, SELL, HLA-DQA1, PTPRC, B2M, and TAP2) with highest classification accuracy rate (95.7 %). Moreover, we validated our results by reproducing the same gene expression profile as a discriminatory test in the "test set," which included data from salivary gland samples of 15 patients with SS and 22 controls with 94.6 % accuracy. We propose that gene expression profile in the saliva or salivary glands could represent a promising simple and reproducible diagnostic biomarker for SS.

Hsp27 (HSPB1) differential expression in normal salivary glands and pleomorphic adenomas and association with an increased Bcl2/Bax ratio.

Pleomorphic adenoma (PA) is the most common salivary gland neoplasm. The Hsp27 (HSPB1) is an antiapoptotic protein whose synthesis follows cytotoxic stresses and result in a transient increase in tolerance to subsequent cell injury. Although Hsp27 is expressed in a range of normal tissues and neoplasms, a wide variation in its expression exists among different cells and tissues types. In certain tumours of glandular origin (such as oesophageal adenocarcinomas), the level of Hsp27 is decreased. In the present study, Hsp27 protein levels were evaluated by enzyme-linked immunosorbent assay (ELISA) in a set of 18 fresh PA and 12 normal salivary gland samples. In addition, we tested if Hsp27 protein levels correlated with p53 expression and cell proliferation index, as well as with the transcriptional levels of Bcl-2-associated X protein (BAX), B cell lymphoma 2 (BCL2) and Caspase 3 in PA. We further tested the association between Hsp27 expression and PA tumour size. While all normal salivary gland samples expressed Hsp27 protein, only half of the PA samples expressed it, resulting in a reduced expression of Hsp27 in PA when compared with normal salivary glands (P = 0.003). The expression levels of this protein correlated positively with a higher messenger ribonucleic acid (mRNA) ratio of Bcl2/Bax (R = 0.631; P = 0.01). In conclusion, a decreased Hsp27 protein expression level in PA was found. In addition, Hsp27 levels correlated positively with the Bcl2/Bax mRNA ratio, suggesting an antiapoptotic effect.

The effect of temperature on the extrinsic incubation period and infection rate of dengue virus serotype 2 infection in Aedes albopictus.

Dengue fever is an acute mosquito-borne viral disease caused by dengue virus (DENV). Temperature may affect the efficiency of the mosquito vectors in spreading DENV. Aedes albopictus mosquitoes were infected orally with a DENV2 suspension and incubated at different temperatures. Subsequently, DENV2 antigen was collected from salivary gland and thorax-abdomen samples on different days postinfection and tested using an immunofluorescence assay to determine the extrinsic incubation period and infection rate. As the temperature increased, the extrinsic DENV2 incubation period in Ae. albopictus gradually shortened, and infection rates showed a tendency to initially increase, followed by a subsequent decrease.