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1.  Scientific Frontiers 
Advances in Dental Research  2011;23(4):360-368.
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.
doi:10.1177/0022034511420433
PMCID: PMC3172997  PMID: 21917746
salivary biochemistry and physiology; proteome; biomarkers; early detection; genomics; microbiome
2.  Human Parathyroid Hormone Is Secreted Primarily into the Bloodstream After Rat Parotid Gland Gene Transfer 
Human Gene Therapy  2010;22(1):84-92.
Abstract
Hypoparathyroidism is a hormone deficiency syndrome that leads to low blood calcium levels and for which current replacement therapy is inadequate. Gene transfer to salivary glands leads to safe and abundant secretion of therapeutic protein into either saliva or the bloodstream. We previously reported the successful transduction of rat submandibular glands with an adenoviral vector encoding human parathyroid hormone (Ad.hPTH), but unfortunately most of the hPTH was secreted into saliva. Because submandibular and parotid glands are morphologically and functionally different, we hypothesized that hPTH sorting might be different in parotid glands. After 2 days, the pattern of hPTH secretion from transduced parotid glands of intact rats was reversed from that of transduced submandibular glands, that is, most transgenic hPTH was detected in serum (5 × 1010 viral particles per gland; the saliva-to-serum ratio of total hPTH secreted was 0.04). Vector copies were localized to the targeted parotid glands, with none detected in liver or spleen. Ad.hPTH next was administered to parotid glands of parathyroidectomized rats. Two days after delivery no hPTH was detectable in saliva, but high levels were found in serum, leading to normalization of serum calcium and a significant increase in the urinary phosphorus-to-creatinine ratio. This study demonstrates for the first time differential sorting of transgenic hPTH between submandibular and parotid glands, suggesting that hPTH may be a valuable model protein for understanding the molecular basis of transgenic secretory protein sorting in these exocrine glands. We also show the clinical potential of salivary gland hPTH gene therapy for patients with hypoparathyroidism.
Physiologically sufficient levels of calcium in the blood are important for a range of vital functions and hypocalcemia can lead to seizures, tetany, or heart failure. Parathyroid hormone (PTH) is central to maintaining adequate blood calcium concentration. In this study, Adriaansen et al. demonstrate that delivery of an adenoviral vector encoding human PTH to the parotid gland of hypocalcemic rats leads to a normalization of serum calcium levels.
doi:10.1089/hum.2010.097
PMCID: PMC3025188  PMID: 20977345
3.  Experience with experimental biological treatment and local gene therapy in Sjögren's syndrome: implications for exocrine pathogenesis and treatment 
Annals of the Rheumatic Diseases  2006;65(11):1406-1413.
Sjögren's syndrome is an autoimmune exocrinopathy, mainly affecting the lacrimal and salivary glands, and resulting in ocular and oral dryness (keratoconjunctivitis sicca and xerostomia). The aetiology and pathogenesis are largely unknown, and only palliative treatment is currently available. Data obtained from experimental animal and human studies using biological agents or gene therapeutics can offer insight into the disease process of Sjögren's syndrome. This article reviews the current literature on these approaches and assesses the lessons learnt about the pathogenesis of Sjögren's syndrome.
doi:10.1136/ard.2006.052761
PMCID: PMC1798364  PMID: 16880196
4.  Differential Sorting of Human Parathyroid Hormone After Transduction of Mouse and Rat Salivary Glands 
Human Gene Therapy  2008;19(10):1021-1028.
Abstract
Gene transfer to salivary glands leads to abundant secretion of transgenic protein into either saliva or the bloodstream. This indicates significant clinical potential, depending on the route of sorting. The aim of this study was to probe the sorting characteristics of human parathyroid hormone (hPTH) in two animal models for salivary gland gene transfer. PTH is a key hormone regulating calcium levels in the blood. A recombinant serotype 5 adenoviral vector carrying the hPTH cDNA was administered to the submandibular glands of mice and rats. Two days after delivery, high levels of hPTH were found in the serum of mice, leading to elevated serum calcium levels. Only low amounts of hPTH were found in the saliva. Two days after vector infusion into rats, a massive secretion of hPTH was measured in saliva, with little secretion into serum. Confocal microscopy showed hPTH in the glands, localized basolaterally in mice and apically in rats. Submandibular gland transduction was effective and the produced hPTH was biologically active in vivo. Whereas hPTH sorted toward the basolateral side in mice, in rats hPTH was secreted mainly at the apical side. These results indicate that the interaction between hPTH and the cell sorting machinery is different between mouse and rat salivary glands. Detailed studies in these two species should result in a better understanding of cellular control of transgenic secretory protein sorting in this tissue.
doi:10.1089/hum.2008.079
PMCID: PMC2705888  PMID: 18694295
5.  Distinct recognition of antibodies to centromere proteins in primary Sjögren's syndrome compared with limited scleroderma 
Annals of the Rheumatic Diseases  2006;65(8):1028-1032.
Background
Anticentromere antibodies are characteristically observed in scleroderma but have recently been reported in other autoimmune rheumatic disorders, including Sjögren's syndrome. It is not known whether distinct centromere proteins (CENP) are targeted in primary Sjögren's syndrome (pSS) and scleroderma.
Objective
To determine whether antibodies to CENP‐B and CENP‐C are present in these two disorders.
Methods
Sera from 45 patients with pSS and 33 with limited scleroderma were studied. All patients met classification criteria for pSS and scleroderma, respectively. Sera were used to immunoprecipitate in vitro translated CENP‐B and CENP‐C. The proportions recognising CENP‐B or CENP‐C were compared.
Results
10 of 45 patients (22%) with pSS and 18 of 33 (55%) with scleroderma had antibodies recognising CENPs (p = 0.004). Seven of 10 (70%) CENP positive patients with pSS recognised CENP‐C alone, compared with one of 18 (6%) CENP positive patients with scleroderma (odds ratio (OR) = 40 (95% confidence interval (CI), 3.5 to 450) (p = 0.003). In contrast, the majority (15 of 18 (83%)) of CENP positive scleroderma sera recognised both CENP‐B and CENP‐C, compared with none of 10 pSS sera (OR = 93 (95% CI, 4.4 to 1979) (p = 0.0001).
Conclusions
The pattern of CENP recognition differs markedly in pSS and limited scleroderma. While patients with pSS predominantly recognise CENP‐C alone, dual recognition of CENP‐B and CENP‐C is most frequent in scleroderma. These findings suggest that obtaining antibodies to specific centromere antigens is useful diagnostically, and imply that distinct mechanisms underlie the unique patterns of centromere autoreactivity in pSS and scleroderma.
doi:10.1136/ard.2005.046003
PMCID: PMC1798261  PMID: 16414973
anticentromere antibody; Sjögren's syndrome; scleroderma; immunoprecipitation
6.  Pak3 inhibits local actin filament formation to regulate global cell polarity 
HFSP Journal  2009;3(3):194-203.
Lamellipodia are broad actin-based structures that define the protruding edge of many motile animal cells. Here we identify a Drosophila homolog of the p21-activated kinases (Paks) as a novel inhibitor of Rac-mediated lamellipodial formation: Pak3 overexpression mimics a loss of Rac activity, while Pak3 RNAi-mediated silencing enhances lamellipodial dynamics. Strikingly, the depletion of Pak3 also polarizes the cellular distribution of actin filaments, is sufficient to induce nonmotile cells to migrate, and, in cells firmly attached to the substrate, gives rise to a wave of high actin filament density that encircles the cell periphery at a steady pace. To better understand these systems level phenomena, we developed a model of the cortical actin network as an active gel whose behavior is dominated by the rate of actin filament bundling and polymer synthesis. In the presence of filament treadmilling, this system generates a propagating density wave of actin filaments like that seen in Pak3 RNAi cells. This analysis reveals an intimate relationship between local regulation of actin filament dynamics and global cytoskeletal polarity, and suggests a role for negative regulators of lamellipodial formation, like Pak3, in the maintenance of a poised state, in which regulated directional cell movement can occur.
doi:10.2976/1.3100548
PMCID: PMC2714956  PMID: 19639041
7.  Effect of human vasoactive intestinal peptide gene transfer in a murine model of Sjögren's syndrome 
Annals of the Rheumatic Diseases  2005;65(2):195-200.
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.
doi:10.1136/ard.2005.038232
PMCID: PMC1798026  PMID: 15975969
vasoactive intestinal peptide; Sjögren's syndrome; gene transfer; adeno‐associated virus; autoimmune disease
9.  A functional genomic analysis of cell morphology using RNA interference 
Journal of Biology  2003;2(4):27.
Background
The diversity of metazoan cell shapes is influenced by the dynamic cytoskeletal network. With the advent of RNA-interference (RNAi) technology, it is now possible to screen systematically for genes controlling specific cell-biological processes, including those required to generate distinct morphologies.
Results
We adapted existing RNAi technology in Drosophila cell culture for use in high-throughput screens to enable a comprehensive genetic dissection of cell morphogenesis. To identify genes responsible for the characteristic shape of two morphologically distinct cell lines, we performed RNAi screens in each line with a set of double-stranded RNAs (dsRNAs) targeting 994 predicted cell shape regulators. Using automated fluorescence microscopy to visualize actin filaments, microtubules and DNA, we detected morphological phenotypes for 160 genes, one-third of which have not been previously characterized in vivo. Genes with similar phenotypes corresponded to known components of pathways controlling cytoskeletal organization and cell shape, leading us to propose similar functions for previously uncharacterized genes. Furthermore, we were able to uncover genes acting within a specific pathway using a co-RNAi screen to identify dsRNA suppressors of a cell shape change induced by Pten dsRNA.
Conclusions
Using RNAi, we identified genes that influence cytoskeletal organization and morphology in two distinct cell types. Some genes exhibited similar RNAi phenotypes in both cell types, while others appeared to have cell-type-specific functions, in part reflecting the different mechanisms used to generate a round or a flat cell morphology.
doi:10.1186/1475-4924-2-27
PMCID: PMC333409  PMID: 14527345
10.  Human Bak induces cell death in Schizosaccharomyces pombe with morphological changes similar to those with apoptosis in mammalian cells. 
Molecular and Cellular Biology  1997;17(5):2468-2474.
Apoptosis as a form of programmed cell death (PCD) in multicellular organisms is a well-established genetically controlled process that leads to elimination of unnecessary or damaged cells. Recently, PCD has also been described for unicellular organisms as a process for the socially advantageous regulation of cell survival. The human Bcl-2 family member Bak induces apoptosis in mammalian cells which is counteracted by the Bcl-x(L) protein. We show that Bak also kills the unicellular fission yeast Schizosaccharomyces pombe and that this is inhibited by coexpression of human Bcl-x(L). Moreover, the same critical BH3 domain of Bak that is required for induction of apoptosis in mammalian cells is also required for inducing death in yeast. This suggests that Bak kills mammalian and yeast cells by similar mechanisms. The phenotype of the Bak-induced death in yeast involves condensation and fragmentation of the chromatin as well as dissolution of the nuclear envelope, all of which are features of mammalian apoptosis. These data suggest that the evolutionarily conserved metazoan PCD pathway is also present in unicellular yeast.
PMCID: PMC232095  PMID: 9111315
11.  99mTc-pertechnetate uptake in parotid acinar cells by the Na+/K+/Cl- co-transport system. 
Journal of Clinical Investigation  1987;79(5):1310-1313.
99mTc-Pertechnetate (99mTcO4-) has widespread clinical use in the diagnosis and evaluation of dysfunctions in many different tissues. However, despite the broad clinical application of this radionuclide, very little is known about the mechanism by which 99mTcO4- enters a cell. We report evidence here that 99mTcO4- shares the Na+/K+/Cl- co-transport system localized to the basolateral membrane of rat parotid acinar cells. 99mTcO4- uptake by these cells was quite rapid (t1/2 approximately 30 s), was completely inhibited by the loop diuretics furosemide and bumetanide, and was markedly dependent on the presence of Na+, K+, and Cl- in the extracellular medium. Relative to uptake measured in the presence of physiological extracellular salt concentrations (Hanks' salts), 99mTcO4- uptake was inhibited 80% by sodium replacement and 50% by potassium replacement. When Cl- was replaced with the physiologically inert anion gluconate a threefold stimulation in 99mTcO4- uptake resulted. These observations provide strong evidence that 99mTcO4- can substitute for Cl- as a substrate for the Na+/K+/Cl- co-transporter and indicate that 99mTcO4- uptake by salivary glands (e.g., as seen with salivary scintiscans), and possibly by a variety of other tissues, reflects the functional activity of this co-transport mechanism.
PMCID: PMC424370  PMID: 3033020
12.  Isolation of milligram quantities of a group of histidine-rich polypeptides from human parotid saliva. 
Infection and Immunity  1984;44(3):688-694.
Freshly collected parotid saliva collected from human donors were shown by polyacrylamide gel electrophoresis to continuously secrete a group of low-molecular-weight cationic polypeptides. Up to 14 bands could be identified by Coomassie blue staining, and all bands migrated more rapidly than purified human leukemic lysozyme in cationic polyacrylamide gel electrophoresis. These peptides could be isolated as a group relatively free of other salivary components and recovered in high yields from concentrated parotid saliva by Sephadex G-25 chromatography. In sodium dodecyl sulfate gel electrophoresis, the histidine-rich polypeptide bands appeared as just two bands migrating at the tracking dye and ahead of insulin chain B. Amino acid analysis of the mixture revealed an average content of at least 48% cationic residues, of which half were histidine. When stained bands were eluted from electrophoretic gels, hydrolyzed, and subjected to amino acid analyses, they were found to be enriched in histidine. There was also a correlation of the electrophoretic mobility with the content of basic amino acids. Sephadex G-25 chromatography is a convenient, simple method for preparing milligram quantities of the histidine-rich polypeptides for chemical and biochemical studies.
Images
PMCID: PMC263670  PMID: 6724692
13.  Specific and nonspecific immune factors in dental plaque fluid and saliva from young and old populations. 
Infection and Immunity  1981;31(3):998-1002.
Separate samples of supragingival dental plaque overtly free of blood were centrifuged to obtain the free fluid phase (plaque fluid). Bound protein was eluted from the plaque bacteria and matrix by washing the plaque with a low-pH buffer. The plaque fluid, low pH eluate, and whole saliva were assayed for immunoglobulins A, G, and M, the third component of complement, lysozyme, lactoferrin, and lactoperoxidase. Concentrations of total protein and albumin were also determined. Antibody reactive with specific plaque bacteria was detected by indirect immunofluorescent microscopy. Specific and nonspecific immune proteins were present in plaque fluid from adult subjects at significantly greater concentrations than in their saliva, which suggests that these proteins are concentrated in dental plaque. The results indicate that both saliva and gingival exudate contribute to the immunological proteins found in the free fluid phase of dental plaque. The observation that immunoglobulin A antibody reactive with plaque bacteria could be detected in plaque fluid suggests that a wide variety of immunological reactions may occur in the dental plaque. These potential interactions between host, plaque bacteria, and their products could serve to influence the plaque flora and its ability to induce disease.
PMCID: PMC351417  PMID: 7228411

Results 1-13 (13)