In 2003 we reported the development of a microarray based high throughput screening technique for identifying gene expression patterns that correlated with a specific phenotype (Di Pasquale et al. 2003). This approach was termed comparative gene analysis (CGA). Since our initial publication we have continued to refine the bioinformatics aspect of this approach and identified several receptors for different viruses including AAVs and filoviruses such as ebola (Rhein et al 2016). This work has lead to a better understanding of the overall virus lifecyle of AAVs and lead to the reclassification of the family Parvoviridae (Cotmore et al. 2014). This work has also highlighted the role of protein glycosylation in the lifecycle of some viruses and suggested that alterations in host glycosylation could prevent virus attachment and maturation. As a genus, the dependoviruses use a diverse group of cell surface carbohydrates for attachment and entry. Despite the fact that a majority of adeno-associated viruses (AAVs) utilize sialic acid (SIA) for binding and transduction, this virus-carbohydrate interaction is poorly understood. Utilizing X-ray crystallography, two SIA binding regions were mapped for AAV5 (Afione et al 2015). The first site mapped to the depression in the center of the 3-fold axis of symmetry, while the second site was located under the _HI loop close to the 5-fold axis. Mutagenesis of amino acids 569 and 585 or 587 within the 3-fold depression resulted in elimination or alteration in SIA-dependent transduction, respectively. This change in SIA binding was confirmed using glycan microarrays. Mutagenesis of the second site identified a role in transduction that was SIA independent. Further studies of the mutants at the 3-fold site demonstrated a change in transduction activity and cell tropism in vivo as well as resistance to neutralization by a polyclonal antibody raised against the wild-type virus. Primary Sjgrens syndrome (pSS) is an autoimmune disease, characterized by lymphoid cell infiltration into the salivary and lacrimal glands, and affects 0.5% of the population in the United States of which 90% are women. The consequence of chronic immune cell activation in these exocrine glands is diminished secretory function, which leads to symptoms of dry mouth and dry eyes. In order to understand the environment of the salivary gland that might contribute to the gender bias associated with pSS we compared the transcriptome of male and female salivary glands from healthy individuals (Michael et al. 2012). Comparison of the transcriptome of minor salivary glands from normal male and female volunteers with that of salivary glands and other secretory epithelia identified a number of gender and tissue-specific gene expression patterns. These differences include, but are not limited to, a diverse set of genes involved in immune modulation, chemotactic control, inhibition of complement, metabolism, and neurogenesis. Analysis of these changes provides insight into the protective and predisposing molecular factors that may be involved in the development of pSS. Some of the gene changes observed in this study correlate with previously observed sexual dimorphisms in salivary gland function and also illustrate several new targets for further investigation. To identify epithelial changes in gene expression within female patients associated with the loss of gland function, custom microarrays were probed with complementary RNA (cRNA) isolated from minor salivary glands (MSGs) of female patients with pSS who had low focus scores and low salivary flow rates, and the results were compared with those obtained using cRNA from the MSGs of sex-matched healthy volunteers. A significant increase in expression of BMP-6 was observed in RNA isolated from pSS patients compared with healthy volunteers. Overexpression of BMP-6 locally in the salivary or lacrimal glands of mice resulted in the loss of fluid secretion as well as changes in the connective tissue of the salivary gland. Assessment of the fluid movement in either isolated acinar cells from mice overexpressing BMP-6 or a human salivary gland cell line cultured with BMP-6 revealed a loss in volume regulation in these cells. Lymphocytic infiltration in the submandibular gland of BMP-6 vector-treated mice was increased. No significant changes in the production of proinflammatory cytokines or autoantibodies associated with pSS (anti-Ro/SSA and anti-La/SSB) were found after BMP-6 overexpression. In addition to identifying BMP-6 expression in association with xerostomia and xerophthalmia in pSS, the present results suggest that BMP-6-induced salivary and lacrimal gland dysfunction in pSS may not be the direct effect of autoantibodies or immune activation associated with the disease but a down stream change in epithelial gland function triggered by BMP-6 expression. Our findings show that changes in the epithelia of pSS patients has an effect on salivary gland function. Recently, we identified a connection between expression of aquaporin 5, a water channel critical for salivary gland fluid secretion, and BMP-6. Increased expression of this cytokine is strongly associated with the most common symptom of primary Sjgren's syndrome, the loss of salivary gland function. This finding led us to develop a therapy for the treatment of Sjgren's syndrome by increasing the water permeability of the gland. Our study demonstrates that the targeted increase of gland permeability not only resulted in the restoration of secretory gland function but also resolved the hallmark salivary gland inflammation and systemic inflammation associated with disease. Secretory function also increased in the lacrimal gland, suggesting this local therapy could treat the systemic symptoms associated with primary Sjgren's syndrome (Lai et al 2016). Low-level, chronic viral infections have been suspect in the development of select autoimmune diseases, including primary Sjgren's syndrome (pSS). Multiple studies have shown stimulation of antiviral response pathways in pSS tissues suggestive of a viral infection. Yet, with this data in hand, a causal link between a viral infection and development of pSS had not been identified. Therefore, a study was designed to further define the viral landscape within pSS-affected salivary gland tissue to identify potential viral-mediated triggers in the pathogenesis of this autoimmune disease via viral specific microarrays and confirmation in animal models. Through this analysis, two distinct viral profiles were identified, including the increased presence of hepatitis delta virus (HDV) in 50% of pSS patients evaluated. Presence of HDV antigen and sequence were confirmed in minor salivary gland tissue. Patients with elevated HDV levels in salivary gland tissue were negative for detectible hepatitis B virus (HBV) surface antigen and antibodies to HBV or HDV. Expression of HDV antigens in vivo resulted in reduced stimulated saliva flow, increase in focal lymphocytic infiltrates, and development of autoantibodies. Identification of HDV in pSS patients and induction of a complete pSS-like phenotype in vivo provides further support of a viral-mediated etiopathology in the development of pSS (Weller et al 2016). We have gone on to further utilize this approach in the study of other types of systemic autoimmune diseases (SAID) using RNA obtained from 33 twins discordant for SAID and 33 matched controls (GAN et al 2016). This study suggests viral micro array analysis can be generalizable to evaluate shared viral agents and antiviral immune responses that may be involved in the development of disease.

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Dental & Craniofacial Research
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Corden, A; Handelman, B; Yin, H et al. (2017) Neutralizing antibodies against adeno-associated viruses in Sjögren's patients: implications for gene therapy. Gene Ther 24:241-244
Alevizos, I; Zheng, C; Cotrim, A P et al. (2017) Late responses to adenoviral-mediated transfer of the aquaporin-1 gene for radiation-induced salivary hypofunction. Gene Ther 24:176-186
Zeng, Mei; Szymczak, Mitchell; Ahuja, Malini et al. (2017) Restoration of CFTR Activity in Ducts Rescues Acinar Cell Function and Reduces Inflammation in Pancreatic and Salivary Glands of Mice. Gastroenterology 153:1148-1159
Alevizos, I; Zheng, C; Cotrim, A P et al. (2017) Immune reactivity after adenoviral-mediated aquaporin-1 cDNA transfer to human parotid glands. Oral Dis 23:337-346
Crispino, Giulia; Galindo Ramirez, Fabian; Campioni, Matteo et al. (2017) In vivo genetic manipulation of inner ear connexin expression by bovine adeno-associated viral vectors. Sci Rep 7:6567
Baum, Bruce J; Afione, Sandra; Chiorini, John A et al. (2017) Gene Therapy of Salivary Diseases. Methods Mol Biol 1537:107-123
Merling, Randall K; Kuhns, Douglas B; Sweeney, Colin L et al. (2017) Gene-edited pseudogene resurrection corrects p47phox-deficient chronic granulomatous disease. Blood Adv 1:270-278
Fong, Shelley; Chiorini, John A; Sneyd, James et al. (2017) Computational modeling of epithelial fluid and ion transport in the parotid duct after transfection of human aquaporin-1. Am J Physiol Gastrointest Liver Physiol 312:G153-G163
Rhein, Bethany A; Brouillette, Rachel B; Schaack, Grace A et al. (2016) Characterization of Human and Murine T-Cell Immunoglobulin Mucin Domain 4 (TIM-4) IgV Domain Residues Critical for Ebola Virus Entry. J Virol 90:6097-6111
Weller, Melodie L; Gardener, Matthew R; Bogus, Zoe C et al. (2016) Hepatitis Delta Virus Detected in Salivary Glands of Sjögren's Syndrome Patients and Recapitulates a Sjögren's Syndrome-Like Phenotype in Vivo. Pathog Immun 1:12-40

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