Mobile oral heath technologies based on saliva.

In this review we have highlighted a few innovative microfluidic analytical technologies with mobile phone image processing tools for various bio-chemical tests performed using salivary biomarkers. Saliva-based assays for mobile monitoring with a smartphone sensor provide an excellent analytical technique which can be simple to perform. We describe several examples from the literature, utilizing different modalities of analysis, applied to several different applications of mobile health monitoring: cortisol monitoring, infectious disease testing, and drugs of abuse.

The autoimmunity-oral microbiome connection.

To date, there is a major effort in deciphering the role of complex microbial communities, especially the oral and gut microbiomes, in the pathogenesis of various diseases. Increasing evidence indicates a key role for the oral microbiome in autoimmune diseases. In this review article, we discuss links of the oral microbiota to a group of autoimmune diseases, that is, Sjögren's syndrome (SS), systemic lupus erythematosus (SLE), Crohn's disease (CD), and rheumatoid arthritis (RA). We particularly focus on factors that affect the balance between the immune system and the composition of microbiota leading to dysbiosis, loss of tolerance and subsequent autoimmune disease progression and maintenance.

Future opportunities for bioengineering research at the National Institutes of Health.

Bioengineering integrates physical, chemical, and mathematical sciences with engineering principles for the study of biology, medicine, dentistry, behavior, or health. It advances fundamental concepts, translates knowledge from molecular to organ system levels of understanding, and designs and fabricates innovative biologics, biomaterials, processes, medical and dental implants, devices, and bioinformatics for health promotion, disease prevention, diagnosis, treatment, and therapeutics to improve the health of all people. The National Institutes of Health in recent years has made numerous decisions to coordinate bioengineering activities across the various institutes and centers comprising the National Institutes of Health to increase efforts to support research and research training in bioengineering. This paper will focus on innovations from 1995 to the present that have catalyzed increased activities and opportunities in bioengineering across the National Institutes of Health and will highlight current activities related to tissue engineering at the National Institute of Dental and Craniofacial Research and the National Institute of Arthritis and Musculoskeletal and Skin Diseases.

Comparison of retention and release periods for implant overdenture attachments.

The aim of this study was to compare the retention and release periods of the Nobel Biocare bar and clip (NBC), Nobel Biocare ball (NB), Zest anchor (ZA), Zest magnet (ZM), and Sterngold ERA (SE) attachments on an implant-retained overdenture model. The attachments were tested using two permanently placed Brånemark implants on a test model that was attached to an Instron machine (cross-head speed 50.8 mm/minute). Each attachment had one part embedded in a denture-like housing, and the other part screwed into the implants. Dislodging tensile forces were applied to the housings in two directions simulating function: vertical and oblique. Eight tests were done in two directions with three samples of each attachment. The dislodging forces generated measurements of the peak load, break load, and displacement at peak load and break load. Release periods were calculated using displacements between the peak load and break load and the cross-head speed. Results showed the NBC to be significantly most retentive for the break load when subjected to vertical and oblique forces with mean values and standard deviations of 2104.5 +/- 506.7 g and 1958.1 +/- 165.4 g, respectively. Next most retentive was the SE, followed by the ZA and NB. The ZM was significantly least retentive (127.8 +/- 7.0 g and 143.5 +/- 19.7 g). For the release period, results showed the NBC to have significantly the fastest release period for vertical and oblique forces (1.86 x 10(-3) and 7.35 x 10(-4) minutes). The ZM significantly had the slowest release period for those forces (3.02 x 10(-2) and 2.35 x 10(-2) minutes). The data suggested that the NBC could be selected when a higher degree of retention and fast release period are desired. The next most retentive was the SE; ZM was the least retentive and had the slowest release period.

Regulation of salivary-gland-specific gene expression.

The results from in vivo transgenic and in vitro transfection studies designed to identify cis-element(s) and transfactor(s) governing the salivary proline-rich proteins (PRPs), amylase, and parotid secretory protein (PSP) gene expression are utilized as a paradigm to discuss the regulation of salivary-specific gene expression. Particular attention is given to the molecular mechanism(s) underlying the salivary PRP R15 gene regulation. In rodents, the PRPs are selectively expressed in the acinar cells of salivary glands, and are inducible by the beta-agonist isoproterenol and by dietary tannins. The results from a series of experiments using chimeric reporter constructs containing different lengths of the R15 distal enhancer region, their mutations, and various expressing constructs are analyzed and discussed. These data suggest that the inducible nuclear orphan receptor NGFI-B may participate in the regulation of salivary acinar-cell-specific and inducible expression of the rat R15 gene via three distinct distal NGFI-B sites. Taken together, a model for the induction of R15 gene expression by Ipr is proposed. However, the exact molecular basis of this NGFI-B-mediated transactivation of cAMP-regulated R15 expression remains to be established.

Temporal and spatial expression of laminin, collagen types IV and I and alpha 6/beta 1 integrin receptor in the developing rat parotid gland.

We have examined the temporal expression and cellular localization of the genes and proteins for the extracellular matrix (ECM) proteins laminin (B1, B2 and A chain), collagen types alpha 1 (IV) and alpha 1 (I) and the integrin receptor complex alpha 6/beta 1, during parotid gland postnatal development. Laminin B1 and B2 isoforms and collagens alpha 1 (IV) and alpha 1 (I) mRNA steady-state levels were highest at ages 0, 7 and 14 days after birth and declined to the adult (90 days) level at 21 days and older. Laminin A chain transcripts were not detected at any age. Collagen alpha 1 (IV) and laminin were localized in the basal membrane of the developing acinar and ductal cells, while collagen alpha 1 (I) was localized in the stroma surrounding the cells. The amounts of these ECM components were high at the early stages of development and lower at later times. The pattern of expression of the alpha 6/beta 1 integrin genes during development was similar to those of laminin and collagens alpha 1 (IV) and alpha 1 (I). Accumulations of mRNA were high at 0, 7 and 14 days after birth and lower at 21 days and older. High levels of beta 1 integrin were localized in the developing acinar and ductal cell membranes at early ages (7 days); lower amounts were present in the same distribution pattern at later stages of gland development.(ABSTRACT TRUNCATED AT 250 WORDS)

Characteristics of protooncogene expression in A5 cells.

Stimulation of beta-adrenergic receptors by isoproterenol or addition of 8-BrcAMP rapidly and transiently induces the expression of the protooncogenes, c-fos, and jun B, but not that of c-jun in A5 cells. These results indicate that different intracellular pathways may operate within the same cell for the induction of this group of early response genes. The inducibility of c-fos and jun B genes by either isoproterenol of 8-BrcAMP is transcriptionally regulated and accompanied by increases in their respective products. Furthermore, both c-fos and jun B mRNA levels are elevated at G0/G1 phase of the A5 cell cycle and are inducible by isoproterenol or 8-BrcAMP at the different phases of the cell cycle. These data further suggest a possible role of c-fos and jun B in A5 cell cycle.

Isoproterenol/tannin-dependent R15 expression in transgenic mice is mediated by an upstream parotid control region.

Transgenic mice were used to locate the cis-acting DNA elements that are essential for tissue-specific and inducible expression of the rat proline-rich protein gene, R15. Chimeric genes with up to 10 kb of R15 5'-flanking region fused to chloramphenicol acetyltransferase (CAT) or polyomaviral large T-antigen (PyLT) reporter genes were tested. Our results demonstrate that (1) the isoproterenol/tannin-inducible, parotid-specific transgene expression requires an upstream cis-regulatory domain, namely the parotid control region, which extends from -6 to -1.7 kb of the R15 gene; (2) this parotid control region functions with a heterologous promoter and is indispensable for achieving a reproducible chromosomal position-independent transgene expression; (3) deletion of the R15 5'-flanking region up to -1.7 kb results in a pleiotropic effect on the transgene expression, which includes ectopic (nonsalivary) reporter expression and lack of inducibility by either the beta-agonist isoproterenol or dietary tannin stimulation; (4) when the -10 to -6 kb region from the R15 gene is deleted in the construct, the inducible expression in the parotid glands of the transgenic mice decreases by over 30-fold, but position-independent and tissue-specific transgene expression is retained. Moreover, the mechanism of induction by either catecholamine isoproterenol or dietary tannin appears to be through a beta 1-adrenergic receptor-mediated pathway for both normal (non-transgenic) and transgenic animals.

Differential expression of early response genes, c-jun, c-fos, and jun B, in A5 cells.

We examined the expression of c-fos, c-jun, and jun B after activation of different signal transduction pathways in the A5 rat salivary epithelial cell line. Stimulation of beta-adrenergic receptors by isoproterenol, or addition of 8-bromoadenosine 3',5'-cyclic monophosphate, induces the expression of c-fos and jun B by a protein kinase A-mediated pathway. Phorbol 12-myristate 13-acetate (PMA) induces the expression of all three genes, but with different kinetics. While c-fos and jun B mRNA levels increase early (1 h) after stimulation and transiently, those of c-jun remain higher than control even after stimulation for 8 h and return to basal levels by 24 h. Inhibitors of protein kinase C block the effect of PMA on c-fos, c-jun, and jun B expression, indicating that these genes are also regulated by a protein kinase C-mediated mechanism in A5 cells. Increases in cytosolic Ca2+ by A23187 or ionomycin induce only the expression of c-fos gene. This induction is abolished when A5 cells are loaded with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid before treatment with the ionophores, or when serum is excluded from the incubation medium. Exclusion of serum from the medium does not change the effects of isoproterenol or PMA on c-fos, c-jun, or jun B. These results strongly suggest that serum factors act synergistically with Ca2+ to induce c-fos expression in A5 cells. The studies presented here indicate that different signal transduction pathways operate in A5 cells for the induction of c-fos, c-jun, and jun B genes.

Reciprocal expression of c-jun, proline-rich protein and amylase genes during rat parotid salivary gland development.

We have investigated the temporal expression and cellular localization of the c-jun proto-oncogene and two major rat parotid gland secretory protein genes, PRP (proline-rich protein) and amylase, during postnatal development. c-jun mRNA steady-state levels increased at days 1, 7 and 14 after birth and decreased to basal levels at 21 days and older. PRP mRNA was first detected at 14 days and abruptly increased to adult levels at day 21. Amylase transcripts were first seen at day 7 and progressively increased to adult levels by 28 days. In situ hybridization demonstrated c-jun mRNA accumulation in the differentiating acinar cells and the ducts. The c-jun mRNA accumulation with time corresponds with the proliferative activity reported to occur in these two cellular populations. PRP transcripts were present exclusively in the well differentiated acinar cells while the accumulation of amylase mRNA corresponded to the progressive commitment of parotid cells to acinar differentiation. Our data suggest that during the postnatal development of the rat parotid gland: (a) c-jun expression associates with parotid gland proliferation and precedes the expression of PRP and amylase genes, and (b) activation of PRP and amylase genes is not concomitant and apparently occurs only in differentiating acinar cells.

Regulation of ADP-ribosylation factor (ARF) expression. Cross-species conservation of the developmental and tissue-specific alternative polyadenylation of ARF 4 mRNA.

ADP-ribosylation factors (ARFs), approximately 20-kDa guanine nucleotide-binding proteins, are involved in protein trafficking and enhance cholera toxin ADP-ribosyltransferase activity. Expression of six ARF genes was examined in mammalian tissues; only ARF 4 mRNA was detected in rat testis in forms considerably shorter than those in other tissues. Testis-specific expression of short forms of ARF 4 mRNA was observed in several mammalian species. On Northern analysis of the developmental expression of rat ARF 4 mRNA, appearance of the shorter species was consistent with its involvement in a late stage of spermatogenesis. Sequences of products of rapid amplification of cDNA ends (RACE-polymerase chain reaction) of rat ARF 4 mRNA revealed that different mRNAs resulted from the use of three polyadenylation signals, one AUUAAA and two AAUAAA. Sequences of 3'-untranslated regions of rat and human ARF 4 mRNA were very similar with identical polyadenylation signals at similar positions. Of the ARF 4 mRNAs identified by RACE-PCR, with sizes of 1.1, 1.3, and 1.8 kb, the 1.1-kb mRNA was predominant in adult testis. By in situ hybridization, the 1.1-kb mRNA was identified primarily in mature sperm, consistent with the developmental studies. Shorter mRNAs, thought to be more stable, may compensate for cessation of transcription at late stages of spermatogenesis.

c-myc mRNA expression in minor salivary glands of patients with Sjögren's syndrome.

c-myc protooncogene is implicated in the pathogenesis of B cell lymphoid malignancies and high levels of c-myc mRNA expression are observed in activated blood mononuclear cells. Sjögren's syndrome (SS) is characterized by lymphocytic infiltrates of exocrine glands, remarkable B cell hyperreactivity and a strong predisposition to B cell neoplasia. In this study, c-myc protooncogene mRNA expression in 29 labial minor salivary gland biopsies from patients with primary SS and 15 controls was examined using in situ hybridization histochemistry. Two 40mer oligonucleotides from the 1st and the 2nd exon of the c-myc gene, labeled with 35S, were used as probes. To detect the origin of the cell hybridized with a c-myc probe, a combined immunochemistry in situ hybridization histochemistry technique was used. High c-myc mRNA expression was detected on acinar epithelial cells. c-myc did not correlate with c-fos and c-jun protein expression. Stronger c-myc mRNA expression was detected in labial salivary glands of patients with longer disease duration (p less than or equal to 0.002) and more intense T lymphocyte infiltrates (p less than 0.05) although these patients revealed no hypergammaglobulinemia. No correlation was observed between c-myc mRNA and B lymphocyte monoclonicity or lymphoma. In conclusion, strong c-myc mRNA expression was observed on epithelial cells of labial salivary glands from patients with primary SS. Our findings may indicate the presence of a reactivated virus hosted in these cells.

Effects of ionizing radiation and beta-adrenergic stimulation on the expression of early response genes in rat parotid glands.

There is little known about the regulation of gene expression in rat parotid glands after exposure to ionizing radiation. The present studies investigate the effects of in vivo ionizing radiation, with subsequent stimulation of beta-adrenergic receptors by isoproterenol, on parotid gland function and on the expression of the early response genes, c-fos, c-jun, and jun B. Ionizing radiation diminished parotid gland weight and saliva output. Treatment of irradiated rats with isoproterenol increased the gland weight to levels similar to those in nonirradiated rats. However, such treatment had no effect on saliva output as indicated by measurements of parotid salivary flow rate. Irradiation alone increased the expression of c-fos, c-jun, and jun B. The combination of irradiation and isoproterenol had an additional effect on the levels of c-fos and jun B mRNAs and proteins particularly at earlier experimental times (1 to 8 h). Isoproterenol alone induced high levels of c-fos and jun B mRNA but not of c-jun mRNA. However, c-jun mRNA was induced markedly by radiation and 8 h of isoproterenol treatment, indicating a combined effect on c-jun gene expression. These observations suggest that the expression of the proto-oncogenes c-fos, c-jun, and jun B is probably regulated through differential signal transduction pathways which may be activated by these external stimuli and may be associated with functional changes induced in the rat parotid gland by ionizing radiation and by ionizing radiation and isoproterenol.

Evidence that M3 muscarinic receptors in rat parotid gland couple to two second messenger systems.

The binding affinities of muscarinic antagonists were compared with their abilities to block carbachol (CCh)-mediated stimulation of Ca2+ mobilization and inhibition of isoproterenol-elicited adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in rat parotid cells. The binding of [3H]quinuclidinyl benzilate (QNB) to membranes was inhibited by antagonists with the following potencies (dissociation constant, nM): atropine (1.1) approximately 4-diphenylacetoxy-N-methylpiperidine methbromide (4-DAMP) (1.6) much greater than pirenzepine (136) greater than 11-[[2-[(diethylamino)methyl-1-piperidinyl]-acetyl]acetyl]-5,11- dihydro-6H-pyrido[2,3-b][1,4]-benzodiazepine-6-one (AF-DX 116) (5,293). AF-DX 116 blocked Ca2+ mobilization and inhibition of cAMP accumulation with low affinities [inhibitory concentration at 50% (IC50) = 3150 and 6,528 nM, respectively], whereas 4-DAMP blocked these responses with considerably higher affinities (IC50 = 4.3 and 11.4 nM, respectively). Schild plots of 4-DAMP and AF-DX 116 antagonism of CCh-stimulated inositol trisphosphate accumulation showed inhibitor constant (Ki) values of 0.85 and 1,585 nM, respectively, whereas Schild plots of 4-DAMP, AF-DX 116, and methoctramine antagonism of CCh-induced inhibition of cAMP accumulation showed Ki values of 1.3, 1,585, and 2,754 nM, respectively. Preincubation of cells with 0.1 mM 3-isobutyl-1-methylxanthine did not prevent the capacity of CCh to inhibit cAMP accumulation. Pertussis toxin blocked the CCh-elicited and Gi-mediated inhibition of cAMP formation. Northern blot analysis showed the presence of mRNA for the M3, but not for the M2, subtype in parotid gland. An immunochemical procedure using m1-m5 specific antibodies was performed in parotid membranes and showed that the m3 receptor accounts for 93% of precipitable receptors. These data suggest that M3 receptors in the rat parotid are coupled to both the stimulation of Ca2+ mobilization and the inhibition of cAMP accumulation.

Characteristics of c-fos and jun B gene expression in A5 cells after beta-adrenoreceptor stimulation and during the cell cycle.

The beta-adrenoreceptor agonist isoproterenol elevates cAMP concentrations in the A5 rat salivary epithelial cell line and rapidly and transiently induces the expression of c-fos and jun B at 30 and 60 min following continuous stimulation of these cells. The induction of both genes is mediated by cAMP. We show here that the inducibility of these genes by isoproterenol or 8-BrcAMP is transcriptionally regulated and short (5 min) incubations of A5 cells with either agent is sufficient to trigger the induction of c-fos and jun B. We also have investigated the expression and inducibility of these genes during the A5 cell cycle. Both c-fos and jun B mRNA are elevated at the early phase of the cell cycle and are detectable throughout the cycle. At different stages of the cell cycle in synchronous A5 cells, both genes are as highly induced by isoproterenol or 8-BrcAMP as in asynchronous A5 cells. These studies provide the first evidence for the transcriptional regulation of c-fos and jun B by beta-adrenergic receptor stimulation or cAMP in an epithelial cell line (A5) and demonstrate the coordinate expression and inducibility of these genes at the different stages of the A5 cell cycle.

Cellular characteristics of long-term cultured rat parotid acinar cells.

We have successfully maintained and biochemically characterized differentiated rat parotid acinar cells cultured for long periods (6 mo.). The cells were cultured on a reconstituted basement membrane matrix in a medium containing a variety of agents that promote cellular proliferation and differentiation. The cultured cells retain the characteristics of the parental parotid acinar cells. They exhibit both secretory granules and abundant cellular organelles required for protein synthesis and secretion. In situ hybridization and immunocytochemistry demonstrate high levels of proline-rich protein mRNA and protein, and lower levels of amylase mRNA and protein, in their cytoplasm. These findings suggest that rat parotid acinar cells can be maintained in a differentiated state in vitro for long periods, and can serve as a useful model system for studying the regulation of exocrine secretory processes.

Sequence and expression of the MnP4 gene encoding basic proline-rich protein in macaque salivary glands.

We report here the macaque MnP4 cDNA and genomic sequences which encode a basic proline-rich protein (PRP), which is synthesized in macaque parotid gland and submandibular gland. The locations of intron positions and the prototype of the tandem 20-amino-acid repeat motif with the sequence, PPPPGKPQGPPQQGGNKPQG, in MnP4, were compared to those in related genes encoding PRP and glutamic/glutamine-rich proteins (GRP) in humans and rodents. Exceedingly high homology of the first exon and 40-bp region immediately upstream of exon I is observed with other PRP genes of all species studied. In order to identify the regulatory elements involved in control of MnP4 gene expression, a rat submandibular gland-derived cell line (RSMT-A5) was transfected with MnP4-cat constructs that contained the promoter and 5'-flanking regions of the macaque MnP4 gene fused to the bacterial cat gene. Deletion analysis revealed that putative positive and negative regulatory elements reside between nucleotides (nt) -107 and +5, and nt -586 and -108, respectively. As part of this study, the promoter of the macaque MnP4 gene appears to be salivary gland specific. This salivary gland-specific gene expression attests to the complexity of transcriptional regulation in eukaryotes.

Genetic regulation of salivary proteins in rodents.

The presence of a protein in the cell is the result of a complex pathway that is known by the term gene expression. In this article we review the existing literature on the structure and expression of representative salivary gland genes and their regulated expression during development and upon extracellular stimulation. The expression of one of the "nuclear" protooncogenes, c-fos, in rat parotid glands is also discussed. Finally, we present some suggestions for future studies that will help to understand the mechanisms leading to gene regulation in rat salivary glands.

Two novel c-abl mRNAs are expressed in rat parotid salivary glands during in vivo beta-adrenergic receptor stimulation.

The c-abl proto-oncogene is transcribed in most cell lines and tissues into two mRNAs of 6.5 and 5.3 kb, which have different 5' ends and encode two 150 kDa proteins that are largely colinear, but have different N-termini. We show here that two unusually short and abundant c-abl-related mRNAs of 1.5 and 1.3 kb appear in rat parotid salivary glands, within 1 day of in vivo administration of the beta-adrenergic receptor agonist isoproterenol. These transcripts are not found in the submandibular salivary gland or in the heart and they are too short to encode the known c-abl proteins. RNA blot, S1 nuclease protection and primer extension analysis suggest that the isoproterenol inducible parotid gland mRNAs do not contain the kinase domain, but represent part of the C-terminal segment of the abl reading frame.