Fumi Mineshiba

Effect of human vasoactive intestinal peptide gene transfer in a murine model of Sjogren's syndrome.

Background: Sjögren’s syndrome (SS), an autoimmune exocrinopathy mainly affecting lachrymal and salivary glands, results in ocular and oral dryness (keratoconjunctivitis sicca and xerostomia). The aetiology and pathogenesis are largely unknown; currently, only palliative treatment is available. Objective: To determine whether gene transfer of vasoactive intestinal peptide (VIP), based on its immunomodulatory properties, might be useful in the management of SS. Methods: A recombinant serotype 2 adeno-associated virus encoding the human VIP transgene (rAAV2hVIP) was constructed and its efficacy tested in the female non-obese diabetic (NOD) mouse model for SS after retrograde instillation in submandibular glands (SMGs). 1010 particles/gland of rAAV2hVIP or rAAV2LacZ (encoding β-galactosidase; control vector) were administered at 8 weeks of age (before sialadenitis onset). Salivary flow rates were determined before vector delivery and at time of death (16 weeks). After death, saliva, serum, and SMGs were harvested. Salivary output, inflammatory infiltrates (focus scores), VIP protein expression, cytokine profile, and serum anti-VIP antibodies were analysed. Results: rAAV2hVIP significantly improved the salivary flow, increased SMG and serum expression of VIP, and reduced SMG cytokines interleukin (IL) 2, IL10, IL12 (p70), and tumour necrosis factor α, and serum RANTES, compared with the control vector. No difference in focus scores or apoptotic rates was found; neutralising antibodies were not detected. Conclusions: Local delivery of rAAV2hVIP can have disease modifying and immunosuppressive effects in SMGs of the NOD mouse model of SS. The new strategy of employing VIP prophylactically may be useful for both understanding and managing the salivary component of SS.

Transient TWEAK overexpression leads to a general salivary epithelial cell proliferation.

OBJECTIVES Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a multifunctional cytokine that has pro-apoptotic, pro-angiogenic and pro-inflammatory effects. In liver, TWEAK leads to proliferation of progenitor oval cells, but not of mature hepatocytes. This study evaluated the hypothesis that TWEAK overexpression in salivary glands would lead to the proliferation of a salivary progenitor cell. METHODS A recombinant, serotype 5 adenoviral vector encoding human TWEAK, AdhTWEAK, was constructed, initially tested in vitro, and then administered to male Balb/c mice via cannulation of Whartons duct. TWEAK expression in vivo was monitored as protein secreted into saliva and serum by enzyme-linked immunosorbent assays. Salivary cell proliferation was monitored by proliferating cell nuclear antigen staining and apoptosis was monitored using TUNEL staining. RESULTS AdhTWEAK administration led to a dose-dependent, transient TWEAK protein expression, detected primarily in saliva. Salivary epithelial cell proliferation was generalized, peaking on approximately days 2 and 3. TWEAK expression had no detectable effect on apoptosis of salivary epithelial cells. CONCLUSION Transient overexpression of TWEAK in murine salivary glands leads to a general proliferation of epithelial cells vs a selective stimulation of a salivary progenitor cell.

A Novel Hybrid Adenoretroviral Vector with More Extensive E3 Deletion Extends Transgene Expression in Submandibular Glands

Salivary glands are an attractive target for gene transfer. Salivary epithelial cells are considered to be highly differentiated and have low rates of cell division (~6 months), affording the opportunity to obtain relatively long-term transgene expression in the absence of genomic integration. Here, we report a novel modified hybrid adenoretroviral vector, which provides stable transgene expression in salivary epithelial cells in vivo for up to 6 months in the absence of genomic integration. This modified hybrid vector, Ad(ΔE1/3)LTR(2)EF1α-hEPO, encodes human erythropoietin (hEPO) and differs from a previously developed hybrid vector, AdLTR(2)EF1α-hEPO, by having more extensive E3 gene deletion. Following direct salivary gland gene transfer by retroductal cannulation, rats transduced with Ad(ΔE1/3)LTR(2)EF1α-hEPO had sustained, elevated serum hEPO levels and hematocrits for 6 months (length of experiment), as compared with ~2 months for animals administered the AdLTR(2)EF1α-hEPO vector. Immunohistochemistry demonstrated that this novel vector could transduce both acinar and ductal cells. Interestingly, the Ad(ΔE1/3)LTR(2)EF1α-hEPO vector evoked much weaker local (salivary gland) immune responses than seen after AdLTR(2)EF1α-hEPO vector delivery, which likely permits its significantly lengthened transgene expression in this tissue.

672. The Effect of Serine Protease Inhibitor 6 (SPI-6) Gene Transfer on a Mouse Model of Sj|[ouml]|gren's Syndrome

Top of pageAbstract Sj|[ouml]|grens syndrome (SS) is an autoimmune disease characterized by a focal and diffuse lymphoid cell infiltration into the salivary and lacrimal glands. This chronic immune cell activation leads to reduced secretory function with symptoms of xerostomia (dry mouth) and keratoconjunctivitis sicca (dry eyes). It has been hypothesized that these sequelae are due to apoptosis of the exocrine epithelial cells. Apoptosis can be induced by cytotoxic T-lymphocytes (CTLs), which contain the enzyme Granzyme B (GrB) in cytoplasmic granules. GrB initiates a caspase-independent apoptosis pathway and is inhibited by serine proteinase inhibitor 6 (SPI-6) in the mouse. In this study, we evaluated the effect of transgenic SPI-6 produced in salivary glands after gene transfer to non-obese diabetic (NOD) mice, which develop exocrine gland infiltrates and are a commonly used disease model for SS. We constructed two serotype 2 rAAV vectors, rAAVSPI-6 and rAAVLacZ (encoding |[beta]|-galactosidase; vector control) using the CMV promoter. rAAVs (1010 particles/gland) were administered directly into both submandibular glands of 8-week old female NOD mice (n=8) via retroductal delivery. Salivary flow rates were determined before vector delivery and at time of sacrifice (16-weeks old). Serum glucose levels and weight were measured weekly and diabetic mice (serum glucose|[ge]|400 mg/dL) were treated with insulin. After sacrifice, submandibular glands were harvested and analyzed for inflammatory infiltrates (focus scores), as well as SPI-6 expression and cytokine profile [mouse interleukins (IL)-2, 4, 6, 12, 18, interferon (IFN)-|[gamma]|, tumor necrosis factor-|[alpha]| and RANTES] in aqueous gland extracts. At 8 weeks, salivary flow rates were not different between mice assigned to the rAAVSPI-6 and rAAVLacZ groups. In contrast, at 16 weeks flow rates were significantly higher in the rAAVSPI-6-treated mice (p=0.018). Submandibular gland IL-10 levels were significantly increased (p=0.009), while IFN-|[gamma]| levels were significantly decreased (p=0.029), in the rAAVSPI-6 mice compared to the rAAVLacZ group. There were no differences in focus scores, serum glucose levels or animal weights between the two treatment groups at 16 weeks. Overall, these results suggest that SPI-6 gene transfer may be beneficial in limiting the autoimmune exocrinopathy of NOD mice.