Late responses to adenoviral-mediated transfer of the aquaporin-1 gene for radiation-induced salivary hypofunction.

We evaluated late effects of AdhAQP1 administration in five subjects in a clinical trial for radiation-induced salivary hypofunction (http://www.clinicaltrials.gov/ct/show/NCT00372320?order=). All were identified as initially responding to human aquaporin-1 (hAQP1) gene transfer. They were followed for 3-4 years after AdhAQP1 delivery to one parotid gland. At intervals we examined salivary flow, xerostomic symptoms, saliva composition, vector presence and efficacy in the targeted gland, clinical laboratory data and adverse events. All displayed marked increases (71-500% above baseline) in parotid flow 3-4.7 years after treatment, with improved symptoms for ~2-3 years. There were some changes in [Na+] and [Cl-] consistent with elevated salivary flow, but no uniform changes in secretion of key parotid proteins. There were no clinically significant adverse events, nor consistent negative changes in laboratory parameters. One subject underwent a core needle biopsy of the targeted parotid gland 3.1 years post treatment and displayed evidence of hAQP1 protein in acinar, but not duct, cell membranes. All subjects responding to hAQP1 gene transfer initially had benefits for much longer times. First-generation adenoviral vectors typically yield transit effects, but these data show beneficial effects can continue years after parotid gland delivery.

Effect of Tempol on the prevention of irradiation-induced mucositis in miniature pigs.

OBJECTIVE: The goals of this study were to (i) establish a useful miniature pig (minipig) model for irradiation-induced oral mucositis and (ii) evaluate the effect of Tempol to prevent its development. METHODS AND MATERIALS: Minipigs were irradiated with 6 Gy for five consecutive days targeting the entire oral cavity. To prevent radiation damage, minipigs were treated with 30 mg kg-1 Tempol 10 min before irradiation (n = 4), while the radiation-alone group was similarly injected with saline (n = 4). Lesions were graded using an oral mucositis score and visual inspection every 3 days, and biopsy of multiple sites was performed at day 18. Weight and chest and abdominal circumferences were measured every 3 days. RESULTS: Lesions began about 12 days after the first irradiation fraction and healed about 30 days after irradiation. Epithelial thickness was calculated on the lingual and buccal mucosa on the 18th day after the first irradiation fraction. Tempol provided modest protection from ulceration after irradiation using this treatment strategy. CONCLUSIONS: This study established a useful large animal model for irradiation-induced oral mucositis and showed modest beneficial effects of Tempol in limiting tissue damage. The latter finding may be potentially valuable in preventing oral mucositis in patients receiving irradiation for head and neck cancers.

Immune reactivity after adenoviral-mediated aquaporin-1 cDNA transfer to human parotid glands.

OBJECTIVES: The purpose of this study was to examine the humoral and cellular immune reactivity to adenoviral vector (AdhAQP1) administration in the human parotid gland over the first 42 days of a clinical gene therapy trial. METHODS: Of eleven treated subjects, five were considered as positive responders (Baum et al, 2012). Herein, we measured serum neutralizing antibody titers, circulating cytotoxic lymphocytes, and lymphocyte proliferation in peripheral blood mononuclear cells. Additionally, after adenoviral vector stimulation of lymphocyte proliferation, we quantified secreted cytokine levels. RESULTS: Responders showed little to modest immune reactivity during the first 42 days following gene transfer. Additionally, baseline serum neutralizing antibody titers to serotype 5-adenovirus generally were not predictive of a subject's response to parotid gland administration of AdhAQP1. Cytokine profiling from activated peripheral blood mononuclear cells could not distinguish responders and non-responders. CONCLUSIONS: The data are the first to describe immune responses after adenoviral vector administration in a human parotid gland. Importantly, we found that modest (2-3 fold) changes in systemic cell-mediated immune reactivity did not preclude positive subject responses to gene transfer. However, changes beyond that level likely impeded the efficacy of gene transfer.

Adenovirus-mediated hAQP1 expression in irradiated mouse salivary glands causes recovery of saliva secretion by enhancing acinar cell volume decrease.

Head and neck irradiation (IR) during cancer treatment causes by-stander effects on the salivary glands leading to irreversible loss of saliva secretion. The mechanism underlying loss of fluid secretion is not understood and no adequate therapy is currently available. Delivery of an adenoviral vector encoding human aquaporin-1 (hAQP1) into the salivary glands of human subjects and animal models with radiation-induced salivary hypofunction leads to significant recovery of saliva secretion and symptomatic relief in subjects. To elucidate the mechanism underlying loss of salivary secretion and the basis for AdhAQP1-dependent recovery of salivary gland function we assessed submandibular gland function in control mice and mice 2 and 8 months after treatment with a single 15-Gy dose of IR (delivered to the salivary gland region). Salivary secretion and neurotransmitter-stimulated changes in acinar cell volume, an in vitro read-out for fluid secretion, were monitored. Consistent with the sustained 60% loss of fluid secretion following IR, a carbachol (CCh)-induced decrease in acinar cell volume from the glands of mice post IR was transient and attenuated as compared with that in cells from non-IR age-matched mice. The hAQP1 expression in non-IR mice induced no significant effect on salivary fluid secretion or CCh-stimulated cell volume changes, except in acinar cells from 8-month group where the initial rate of cell shrinkage was increased. Importantly, the expression of hAQP1 in the glands of mice post IR induced recovery of salivary fluid secretion and a volume decrease in acinar cells to levels similar to those in cells from non-IR mice. The initial rates of CCh-stimulated cell volume reduction in acinar cells from hAQP1-expressing glands post IR were similar to those from control cells. Altogether, the data suggest that expression of hAQP1 increases the water permeability of acinar cells, which underlies the recovery of fluid secretion in the salivary glands functionally compromised post IR.

Persistence of hAQP1 expression in human salivary gland cells following AdhAQP1 transduction is associated with a lack of methylation of hCMV promoter.

In 2012, we reported that 5 out of 11 subjects in a clinical trial (NCT00372320) administering AdhAQP1 to radiation-damaged parotid glands showed increased saliva flow rates and decreased symptoms over the initial 42 days. AdhAQP1 is a first-generation, E1-deleted, replication-defective, serotype 5 adenoviral vector encoding human aquaporin-1 (hAQP1). This vector uses the human cytomegalovirus enhancer/promoter (hCMVp). As subject peak responses were at times much longer (7-42 days) than expected, we hypothesized that the hCMVp may not be methylated in human salivary gland cells to the extent previously observed in rodent salivary gland cells. This hypothesis was supported in human salivary gland primary cultures and human salivary gland cell lines after transduction with AdhAQP1. Importantly, hAQP1 maintained its function in those cells. Conversely, when we transduced mouse and rat cell lines in vitro and submandibular glands in vivo with AdhAQP1, the hCMVp was gradually methylated over time and associated with decreased hAQP1 expression and function in vitro and decreased hAQP1 expression in vivo. These data suggest that the hCMVp in AdhAQP1was probably not methylated in transduced human salivary gland cells of responding subjects, resulting in an unexpectedly longer functional expression of hAQP1.

Diversity within the genus Elymus (Poaceae: Triticeae) as investigated by the analysis of the nr5S rDNA variation in species with St and H haplomes.

The genus Elymus ("Ryegrass") is a repository for a range of species with a variety of haplome contents; hence the pejorative name "dustbin" genus. We have analyzed 1,059 sequences from 128 accessions representing 24 species to investigate the relationships among the StH haplomes-containing species described by Yen and Yang (Genus Elymus Beijing 5:58-362, 2013). Sequences were assigned to "unit classes" of orthologous sequences and subjected to a suite of analyses including BLAST (Basic Local Alignment Search Tool) searches, phylogenetic analysis and population genetic analysis to estimate species diversity. Our results support the genome analyses in Yen and Yang (Genus Elymus Beijing 5:58-362, 2013), i.e., genomic constitution StStHH including variants restricted to Elymus. Population genetic analysis of the 5S nrDNA sequence data revealed that the within-species variance component is roughly ±89 %; thus, we were unable to identify molecular markers capable to separate the 24 species analyzed. Separate phylogenetic analyses of the two unit classes and of all the data exhibit a trend only of the species to cluster on the phylograms. Finally, the analysis provides evidence for the multiple origins of American and Eurasian species.

Toward gene therapy for growth hormone deficiency via salivary gland expression of growth hormone.

OBJECTIVES: Salivary glands are useful targets for gene therapeutics. After gene transfer into salivary glands, regulated secretory pathway proteins, such as human growth hormone, are secreted into saliva, whereas constitutive secretory pathway proteins, such as erythropoietin, are secreted into the bloodstream. Secretion of human growth hormone (hGH) into the saliva is not therapeutically useful. In this study, we attempted to redirect the secretion of transgenic hGH from the saliva to the serum by site-directed mutagenesis. MATERIALS AND METHODS: We tested hGH mutants first in vitro with AtT20 cells, a model endocrine cell line that exhibits polarized secretion of regulated secretory pathway proteins. Selected mutants were further studied in vivo using adenoviral-mediated gene transfer to rat submandibular glands. RESULTS: We identified two mutants with differences in secretion behavior compared to wild-type hGH. One mutant, ΔN1-6 , was detected in the serum of transduced rats, demonstrating that expression of this mutant in the salivary gland resulted in its secretion through the constitutive secretory pathway. CONCLUSION: This study demonstrates that mutagenesis of therapeutic proteins normally destined for the regulated secretory pathway may result in their secretion via the constitutive secretory pathway into the circulation for potential therapeutic benefit.

AdLTR2EF1α-FGF2-mediated prevention of fractionated irradiation-induced salivary hypofunction in swine.

Patients frequently experience a loss of salivary function following irradiation (IR) for the treatment of an oral cavity and oropharyngeal cancer. Herein, we tested if transfer of fibroblast growth factor-2 (FGF2) cDNA could limit salivary dysfunction after fractionated IR (7.5 or 9 Gy for 5 consecutive days to one parotid gland) in the miniature pig (minipig). Parotid salivary flow rates steadily decreased by 16 weeks post-IR, whereas blood flow in the targeted parotid gland began to decrease ~3 days after beginning IR. By 2 weeks, post-IR salivary blood flow was reduced by 50%, at which point it remained stable for the remainder of the study. The single preadministration of a hybrid serotype 5 adenoviral vector encoding FGF2 (AdLTR2EF1a-FGF2) resulted in the protection of parotid microvascular endothelial cells from IR damage and significantly limited the decline of parotid salivary flow. Our results suggest that a local treatment directed at protecting salivary gland endothelial cells may be beneficial for patients undergoing IR for oral cavity and oropharyngeal cancer.

What does the nr5S DNA multigene family tell us about the genomic relationship between Dasypyrum breviaristatum and D. villosum (Triticeae: Poaceae)?

The genus Dasypyrum contains two species: the annual and widespread D. villosum (2x = 2n = 14) and the perennial and generally rare D. breviaristatum (2x = 2n = 14 and 4x = 2n = 28). The origin of the latter and its genome constitution have been subject of several studies. There is agreement that the genome of the diploid D. villosum (VV) is different from the diploid cytotype of D. breviaristatum (VbVb), but there is no agreement of the constitution of the tetraploid cytotype, specifically whether is it an autotetraploid or an allotetraploid. This is a long-standing disagreement that this study aims to resolve using the 5S nrDNA as a genomic marker. Our studies suggest that the 4x D. breviaristatum is an allotetraploid (VVVbVb).

Gene therapy.

Applications of gene therapy have been evaluated in virtually every oral tissue, and many of these have proved successful at least in animal models. While gene therapy will not be used routinely in the next decade, practitioners of oral medicine should be aware of the potential of this novel type of treatment that doubtless will benefit many patients with oral diseases.

What does the 5S rRNA multigene family tell us about the origin of the annual Triticeae (Poaceae)?

We have investigated the complex relationships among the annual genera within the tribe Triticeae through phylogenetic analyses of the 5S rRNA multigene family. Cloned sequences were assigned to groups of orthologous sequences, called unit classes, that were subjected to several analyses including BLAST (Basic Local Alignment Search Tool) searches to assess possible ancestral relationships with perennial genera; phylogenetic analyses using parsimony (Pars), maximum likelihood (ML), and Bayesian methods; and minimum reticulation networks from the Pars, ML, and Bayesian trees. In this study, we included genera with both annual and perennial species, such as Dasypyrum, Hordeum, and Secale. BLAST pointed to Pseudoroegneria (carrier of the St genome) and possibly Thinopyrum (carrier of the J genome) as the potential next of kin. However, Thinopyrum and Pseudoroegneria have never fallen together on the individual trees with the former generally associated with Crithopsis, Aegilops, Triticum, and Dasypyrum, while the latter is usually associated with the rest of the genera within Triticeae. The "long" unit classes placed Dasypyrum breviaristatum together with Dasypyrum villosum, whereas the "short" unit classes put them far apart on the trees. None of the gene trees alone was able to summarize the complex relationships among the genera, in line with previous results in the Triticeae. However, the application of tools designed to display phylogenetic networks was able to depict the complex links among the genera based on the short and the long gene trees, including the close link between Thinopyrum and Pseudoroegneria suggested by the phylogenetic analyses. In addition, our analyses provide support for the hypothesis that at least some annual Triticeae taxa are derived from their perennial relatives.

Phylogenetic relationships among the polyploid and diploid Aegilops species inferred from the nuclear 5S rDNA sequences (Poaceae: Triticeae).

Phylogenetic inferences of the polyploid Aegilops taxa were drawn based upon the analysis of 909 nuclear 5S rDNA sequences obtained from 15 Aegilops polyploid taxa (531 sequences new to this paper) and 378 sequences from our previous study on the diploid taxa. The 531 sequences can be split into two orthologous groups (unit classes), the long AE1 and short AE1 previously identified in the diploid set. An examination of the relationships between unit classes and their associated haplomes suggests that U haplome sequences found in Ae. umbellulata are the closest to the T sequences found in Amblyopyrum muticum and that sequences of the polyploid species expected to be the M type found in Ae. comos are more similar to the T haplome sequences, except in the three hexaploids Ae. glumiaristata, Ae. juvenalis, and Ae. vavilovii and the tetraploid Ae. crassa where they are found to be similar to the M haplome sequences. These three hexaploid taxa likely originated from the tetraploid Ae. crassa (DM), while the closest taxon to the fourth hexaploid, Ae. recta, is the tetraploid Ae. neglecta (UM). Based upon the distribution of the unit classes, several reticulate phylogenies depicting evolutionary relationships among diploid, tetraploid, and hexaploid taxa were constructed; however, none of these widely used methods could depict the expected reticulate relationship as previously drawn from cytogenetic analyses in this group of allopolyploid species. These results suggest that evolutionary relationships derived from models based upon the assumption of bifurcating species require careful interpretation when these same models are applied to species with reticulate evolution.

Including the p53 ELAV-like protein-binding site in vector cassettes enhances transgene expression in rat submandibular gland.

OBJECTIVE: ELAV-like proteins regulate mRNA stability and/or translation. We evaluated whether inclusion of binding sites for ELAV-like HuR proteins in vector cassettes could improve transgene expression in the salivary gland. METHODS: Western blots and immunofluorescence staining were used to determine whether HuR protein was expressed in salivary cells and tissue. HuR binding sites were inserted into the pACEF1α-luc-BGH expression plasmid. Cell lines were transfected with plasmids in vitro and luciferase expression measured. Rat submandibular glands were transfected in vivo with plasmids containing ELAV-like HuR protein-binding sites. An adenoviral vector with p53 ELAV-like HuR protein-binding site was generated and also tested in vivo. Four unique 29mer HuR shRNA constructs were used in A5 cells to evaluate whether there was a specific interaction between HuR protein and the p53 HuR protein-binding site. RESULTS: Salivary cells express HuR protein. Inclusion of the p53 ELAV-like HuR protein-binding site resulted in high luciferase activity in salivary cells in vitro, with similar results in vivo. In vitro shRNA data demonstrated that the high luciferase activity was mediated by the interaction between HuR protein and the p53 HuR protein-binding site. The AdEF1α-luc-p53BGH, including this binding site, mediated very high luciferase activity, ~4-fold that seen with the CMV promoter, in rat submandibular glands. CONCLUSIONS: Including the p53 ELAV-like protein-binding site in transgene cassettes may enhance therapeutic vectors intended for use with salivary glands.

AAV2-mediated transfer of the human aquaporin-1 cDNA restores fluid secretion from irradiated miniature pig parotid glands.

Previously (Shan et al, 2005), we reported that adenoviral vector-mediated transfer of the human aquaporin-1 (hAQP1) cDNA to minipig parotid glands following irradiation (IR) transiently restored salivary flow to near normal levels. This study evaluated a serotype 2, adeno-associated viral (AAV2) vector for extended correction of IR (single dose; 20 Gy)-induced, parotid salivary hypofunction in minipigs. At 16 weeks following the IR parotid salivary flow decreased by 85-90%. AAV2hAQP1 administration at week 17 transduced only duct cells and resulted in a dose-dependent increase in salivary flow to approximately 35% of pre-IR levels (to approximately 1 ml per 10 min) after 8 weeks (peak response). Administration of a control AAV2 vector or saline was without effect. Little change was observed in clinical chemistry and hematology values after AAV2hAQP1 delivery. Vector-treated animals generated high anti-AAV2 neutralizing antibody titers by week 4 (approximately 1:1600) and significant elevations in salivary (approximately 15%), but not serum, granulocyte macrophage colony-stimulating factor levels. Following vector administration, salivary [Na(+)] was dramatically increased, from approximately 10 to approximately 55 mM (at 4 weeks) and finally to 39 mM (8 weeks). The findings demonstrate that localized delivery of AAV2hAQP1 to IR-damaged parotid glands leads to increased fluid secretion from surviving duct cells, and may be useful in providing extended relief of salivary hypofunction in previously irradiated patients.

Spatial control of the actin cytoskeleton in Drosophila epithelial cells.

The actin cytoskeleton orders cellular space and transduces many of the forces required for morphogenesis. Here we combine genetics and cell biology to identify genes that control the polarized distribution of actin filaments within the Drosophila follicular epithelium. We find that profilin and cofilin regulate actin-filament formation throughout the cell cortex. In contrast, CAP-a Drosophila homologue of Adenylyl Cyclase Associated Proteins-functions specifically to limit actin-filament formation catalysed by Ena at apical cell junctions. The Abl tyrosine kinase also collaborates in this process. We therefore propose that CAP, Ena and Abl act in concert to modulate the subcellular distribution of actin filaments in Drosophila.

Convenient and reproducible in vivo gene transfer to mouse parotid glands.

OBJECTIVES: Published studies of gene transfer to mouse salivary glands have not employed the parotid glands. Parotid glands are the likely target tissue for most clinical applications of salivary gene transfer. The purpose of the present study was to develop a convenient and reproducible method of retroductal gene transfer to mouse parotid glands. METHODS: The volume for vector delivery was assessed by infusion of Toluidine Blue into Stensen's ducts of Balb/c mice after direct intraoral cannulation. Recombinant, serotype 5 adenoviral vectors, encoding either firefly luciferase or human erythropoietin (hEpo), were constructed and then administered to parotid glands (10(7) vector particles/gland). Transgene expression in vivo was measured by enzyme activity (luciferase) or an enzyme-linked immunosorbent assay (hEpo). Vector biodistribution was measured by real-time quantitative (Q) PCR. RESULTS: The chosen volume for mouse parotid vector delivery was 20µL. Little vector was detected outside of the targeted glands, with both QPCR and luciferase assays. Transgene expression was readily detected in glands (luciferase, hEpo), and serum and saliva (hEpo). Most secreted hEpo was detected in saliva. CONCLUSION: These studies show that mouse parotid glands can be conveniently and reproducibly targeted for gene transfer, and should be useful for pre-clinical studies with many murine disease models.

Transgenic α-1-antitrypsin secreted into the bloodstream from salivary glands is biologically active.

OBJECTIVES: Salivary glands are potentially a valuable target for gene therapeutics. Herein, we examined the expression and biochemical activity of human alpha-1-antitrypsin (hA1AT) produced in rodent submandibular glands after gene transfer. METHODS: A serotype 5 adenoviral vector (Ad.hA1AT) was constructed and first characterized by dose response and time course studies using SMIE cells in vitro. hA1AT expression was analysed by ELISA and the biologic activity determined by the inhibition of human neutrophil elastase (hNE) and formation of hA1AT-hNE complexes. Ad.hA1AT was administered to submandibular glands of rats and mice. The levels and activity of hA1AT were analysed in saliva, serum and gland extracts. Treatment with endoglycosidase H and Peptide N-Glycosidase F was used to assess N-linked glycosylation. RESULTS: Transgenic hA1AT, expressed in submandibular glands following Ad.hA1AT administration, was secreted into the bloodstream, N-glycosylated and biochemically active. CONCLUSION: After in vivo gene transfer, rodent salivary glands can produce a non-hormonal, transgenic, secretory glycoprotein exhibiting complex and conformation-dependent biologic activity.

Scientific frontiers: emerging technologies for salivary diagnostics.

Saliva, a biofluid historically well-studied biochemically and physiologically, has entered the post-genomic 'omics' era, where its proteomic, genomic, and microbiome constituents have been comprehensively deciphered. The translational path of these salivary constituents has begun toward a variety of personalized individual medical applications, including early detection of cancer. Salivary diagnostics is a late-comer, but it is catching up where dedicated resources, like the Salivaomics Knowledge Base (SKB), now have taken center stage in the dissemination of the diagnostic potentials of salivary biomarkers and other translational and clinical utilities.

AAV5-mediated gene transfer to the parotid glands of non-human primates.

Salivary glands are potentially useful target sites for multiple clinical applications of gene transfer. Previously, we have shown that serotype 2 adeno-associated viral (AAV2) vectors lead to stable gene transfer in the parotid glands of rhesus macaques. As AAV5 vectors result in considerably greater transgene expression in murine salivary glands than do AAV2 vectors, herein we have examined the use of AAV5 vectors in macaques at two different doses (n = 3 per group; 10(10) or 3 x 10(11) particles per gland). AAV5 vector delivery, as with AAV2 vectors, led to no untoward clinical, hematological or serum chemistry responses in macaques. The extent of AAV5-mediated expression of rhesus erythropoietin (RhEpo) was dose-dependent and similar to that seen with an AAV2 vector. However, unlike results with the AAV2 vector, AAV5 vector-mediated RhEpo expression was transient. Maximal expression peaked at day 56, was reduced by approximately 80% on day 84 and thereafter remained near background levels until day 182 (end of experiment). Quantitative PCR studies of high-dose vector biodistribution at this last time point showed much lower AAV5 copy numbers in the targeted parotid gland (approximately 1.7%) than found with the same AAV2 vector dose. Molecular analysis of the conformation of vector DNA indicated a markedly lower level of concatamerization for the AAV5 vector compared with that of a similar AAV2 vector. In addition, cellular immunological studies suggest that host response differences may occur with AAV2 and AAV5 vector delivery at this mucosal site. The aggregate data indicate that results with AAV5 vectors in murine salivary glands apparently do not extend to macaque glands.