A Cluster-Randomized, Community-Based, Tribally Delivered Oral Health Promotion Trial in Navajo Head Start Children.

The authors tested the effectiveness of a community-based, tribally delivered oral health promotion (OHP) intervention (INT) at reducing caries increment in Navajo children attending Head Start. In a 3-y cluster-randomized trial, we developed an OHP INT with Navajo input that was delivered by trained Navajo lay health workers to children attending 52 Navajo Head Start classrooms (26 INT, 26 usual care [UC]). The INT was designed as a highly personalized set of oral health-focused interactions (5 for children and 4 for parents), along with 4 fluoride varnish applications delivered in Head Start during academic years of 2011 to 2012 and 2012 to 2013. The authors evaluated INT impact on decayed, missing, and filled tooth surfaces (dmfs) increment compared with UC. Other outcomes included caries prevalence and caregiver oral health-related knowledge and behaviors. Modified intention-to-treat and per-protocol analyses were conducted. The authors enrolled 1,016 caregiver-child dyads. Baseline mean dmfs/caries prevalence equaled 19.9/86.5% for the INT group and 22.8/90.1% for the UC group, respectively. INT adherence was 53% (i.e., ≥3 child OHP events, ≥1 caregiver OHP events, and ≥3 fluoride varnish). After 3 y, dmfs increased in both groups (+12.9 INT vs. +10.8 UC; P = 0.216), as did caries prevalence (86.5% to 96.6% INT vs. 90.1% to 98.2% UC; P = 0.808) in a modified intention-to-treat analysis of 897 caregiver-child dyads receiving 1 y of INT. Caregiver oral health knowledge scores improved in both groups (75.1% to 81.2% INT vs. 73.6% to 79.5% UC; P = 0.369). Caregiver oral health behavior scores improved more rapidly in the INT group versus the UC group (P = 0.006). The dmfs increment was smaller among adherent INT children (+8.9) than among UC children (+10.8; P = 0.028) in a per-protocol analysis. In conclusion, the severity of dental disease in Navajo Head Start children is extreme and difficult to improve. The authors argue that successful approaches to prevention may require even more highly personalized approaches shaped by cultural perspectives and attentive to the social determinants of oral health (ClinicalTrials.gov NCT01116739).

Association of parental health literacy with oral health of Navajo Nation preschoolers.

Health literacy is 'the capacity to obtain, process and understand basic health information and services needed to make appropriate health decisions'. Although numerous studies show a link between health literacy and clinical outcomes, little research has examined the association of health literacy with oral health. No large-scale studies have assessed these relationships among American Indians, a population at risk for limited health literacy and oral health problems. This analysis was conducted as part of a clinical trial aimed at reducing dental decay among preschoolers in the Navajo Nation Head Start program. Using baseline data for 1016 parent-child dyads, we examined the association of parental health literacy with parents' oral health knowledge, attitudes, and behavior, as well as indicators of parental and pediatric oral health. More limited health literacy was associated with lower levels of oral health knowledge, more negative oral health attitudes, and lower levels of adherence to recommended oral health behavior. Parents with more limited health literacy also had significantly worse oral health status (OHS) and reported their children to have significantly worse oral health-related quality of life. These results highlight the importance of oral health promotion interventions that are sensitive to the needs of participants with limited health literacy.

Venom production in long-term primary culture of secretory cells of the Bothrops jararaca venom gland.

There is an increasing interest of obtaining venom by other ways than from extracting it from snakes captured in the wild. A readily available source of this venom will be useful for all pharmacological and biotechnological studies, as well as providing an improved avenue for treatments of snakebites. Here, we show that secretory cells of venom gland can be a good in vitro apparatus to produce venom. We have maintained and morphologically characterized the secretory cells of the Bothrops jararaca venom gland cultured up to 21 days. The isolated cells assemble into acini that growth in size up to 21st day, instead of adhering to the substrate. Bothropasin, a venom metalloprotease, was localized in secretory vesicles by immunoelectron microscopy and venom was also detected in culture medium in a concentration as high as 63 microg/ml. These data show that the acini formed in culture are functionally viable; they can produce and secrete venom.

Synergistic suppression of apoptosis in salivary acinar cells by IGF1 and EGF.

Tissue homeostasis requires balancing cell proliferation and programmed cell death. IGF1 significantly suppressed etoposide-induced apoptosis, measured by caspase 3 activation and quantitation of cellular subG(1) DNA content, in rat parotid salivary acinar cells (C5). Transduction of C5 cells with an adenovirus expressing a constitutively activated mutant of Akt-suppressed etoposide-induced apoptosis, whereas a kinase-inactive mutant of Akt suppressed the protective effect of IGF1. IGF1 also suppressed apoptosis induced by taxol and brefeldin A. EGF was unable to suppress apoptosis induced by etoposide, but was able to synergize with IGF1 to further suppress caspase 3 activation and DNA cleavage after etoposide treatment. The catalytic activity of Akt was significantly higher following stimulation with both growth factors compared to stimulation with IGF1 or EGF alone. These results suggest that a threshold of activated Akt is required for suppression of apoptosis and the cooperative action of growth factors in regulating salivary gland homeostasis.

Neuroprotection of grafted neurons with a GDNF/caspase inhibitor cocktail.

Transplantation of fetal ventral mesencephalic (VM) tissue shows great promise as an experimental therapy for patients with Parkinson's disease. However, cell survival in brain tissue grafts is poor, with survival rates of only 5-15%. We have utilized a combination of the caspase inhibitor bocaspartyl (OMe)-fluoromethylketone (BOC-ASP-CH2F) and glial cell line-derived neurotrophic factor (GDNF) to enhance survival of grafted dopamine neurons. The VM tissue was dissected from embryonic day 13-15 rat fetuses, incubated in different doses of BOC-ASP-CH2F and GDNF, and transplanted to the anterior chamber of the eye of adult rats. Growth of the tissue was assessed through the translucent cornea. Doses of 50 and 100 micromolar of the general caspase inhibitor appeared to have detrimental effects on mesencephalic tissue, while 20 micromolar had beneficial effects on overall transplant growth. A combination of the caspase inhibitor and GDNF appeared to have more prominent effects on cell survival as well as dopaminergic fiber density than either agent by itself. The transplants doubled in size when they were treated with a combination of BOC-ASP-CH2F and GDNF, and cell death markers were significantly reduced at both 48 h and 4-6 days postgrafting. This is, to our knowledge, the first combined approach using apoptotic blockers with trophic factors, and demonstrates a viable strategy for protection of developing neurons, since several different aspects of graft function may be addressed simultaneously.

Activation of PKC is sufficient to induce an apoptotic program in salivary gland acinar cells.

Accumulating evidence suggests that specific isoforms of PKC may function to promote apoptosis. We show here that activation of the conventional and novel isoforms of PKC with 12-O-tetradecanoyl phorbol-13- ester (TPA) induces apoptosis in salivary acinar cells as indicated by DNA fragmentation and activation of caspase-3. TPA-induced DNA fragmentation, caspase-3 activation, and morphologic indicators of apoptosis, can be enhanced by pretreatment of cells with the calpain inhibitor, calpeptin, prior to the addition of TPA. Analysis of PKC isoform expression by immunoblot shows that TPA-induced downregulation of PKC alpha and PKC delta is delayed in cells pre-treated with calpeptin, and that this correlates with an increase of these isoforms in the membrane fraction of cells. TPA-induced apoptosis is accompanied by biphasic activation of the c-jun-N-terminal kinase (JNK) pathway and inactivation of the extracellular regulated kinase (ERK) pathway. Expression of constitutively activated PKC alpha or PKC delta, but not kinase negative mutants of these isoforms, or constitutively activated PKC epsilon, induces apoptosis in salivary acinar cells, suggesting a role for these isoforms in TPA-induced apoptosis. These studies demonstrate that activation of PKC is sufficient for initiation of an apoptotic program in salivary acinar cells. Cell Death and Differentiation (2000) 7, 1200 - 1209.

Protein kinase C delta is essential for etoposide-induced apoptosis in salivary gland acinar cells.

We have previously shown that parotid C5 salivary acinar cells undergo apoptosis in response to etoposide treatment as indicated by alterations in cell morphology, caspase-3 activation, DNA fragmentation, sustained activation of c-Jun N-terminal kinase, and inactivation of extracellular regulated kinases 1 and 2. Here we report that apoptosis results in the caspase-dependent cleavage of protein kinase C-delta (PKCdelta) to a 40-kDa fragment, the appearance of which correlates with a 9-fold increase in PKCdelta activity. To understand the function of activated PKCdelta in apoptosis, we have used the PKCdelta-specific inhibitor, rottlerin. Pretreatment of parotid C5 cells with rottlerin prior to the addition of etoposide blocks the appearance of the apoptotic morphology, the sustained activation of c-Jun N-terminal kinase, and inactivation of extracellular regulated kinases 1 and 2. Inhibition of PKCdelta also partially inhibits caspase-3 activation and DNA fragmentation. Immunoblot analysis shows that the PKCdelta cleavage product does not accumulate in parotid C5 cells treated with rottlerin and etoposide together, suggesting that the catalytic activity of PKCdelta may be required for cleavage. PKCalpha and PKCbeta1 activities also increase during etoposide-induced apoptosis. Inhibition of these two isoforms with Gö6976 slightly suppresses the apoptotic morphology, caspase-3 activation, and DNA fragmentation, but has no effect on the sustained activation of c-Jun N-terminal kinase or inactivation of extracellular regulated kinase 1 and 2. These data demonstrate that activation of PKCdelta is an integral and essential part of the apoptotic program in parotid C5 cells and that specific activated isoforms of PKC may have distinct functions in cell death.

Etoposide-induced activation of c-jun N-terminal kinase (JNK) correlates with drug-induced apoptosis in salivary gland acinar cells.

We have examined the ability of etoposide to induce apoptosis in two recently established rat salivary acinar cell lines. Etoposide induced apoptosis in the parotid C5 cell line as evidenced by the appearance of cytoplasmic blebbing and nuclear condensation, DNA fragmentation and cleavage of PARP. Etoposide also induced activation of c-jun N-terminal kinase (JNK) in parotid C5 cells by 4 h after treatment, with maximal activation at 8 - 10 h. Coincident with activation of JNK, the amount of activated ERK1 and ERK2 decreased in etoposide-treated parotid C5 cells. In contrast to the parotid C5 cells, the vast majority of submandibular C6 cells appeared to be resistant to etoposide-induced apoptosis. Likewise, activation of JNKs was not observed in etoposide-treated submandibular C6 cells, and the amount of activated ERK1 and ERK2 decreased only slightly. Etoposide treatment of either cell line had no effect upon the activation of p38. Treatment of the parotid C5 cells with Z-VAD-FMK, a caspase inhibitor, inhibited etoposide-induced activation of JNK and DNA fragmentation. These data suggest that etoposide may induce apoptosis in parotid C5 cells by activating JNKs and suppressing the activation of ERKs, thus creating an imbalance in these two signaling pathways.

Development and characterization of immortalized rat parotid and submandibular acinar cell lines.

The purpose of this investigation was to develop well-differentiated rat parotid and submandibular acinar cell lines. Acinar cells dissociated from rat parotid and submandibular glands were grown on Mitomycin C-treated 3T3 fibroblasts or Matrigel in primary culture and transfected by CaPO4 precipitation using a plasmid containing a replication-defective simian virus (SV40) genome. Cytokeratin analysis via indirect immunofluorescence and receptor mediated changes in intracellular calcium and cyclic AMP were assessed and used for the identification and selection of immortalized epithelial cells. Of the more than 60 clonal cell lines, four retained moderate to high levels of acinar differentiation through >60 passages. Ultrastructurally, there were tripartate junctional complexes and moderate amounts of rough endoplasmic reticulum and secretory granules. Functional studies indicated that beta-adrenoceptors, vasoactive intestinal peptide, and prostaglandin E1 were effective activators of intracellular cyclic AMP production in all cell lines. Alpha-adrenoceptors, muscarinic cholinoceptors, and P2U-purinoceptor agonists were effective in increasing intracellular inositol phosphate production and free calcium levels whereas substance P was ineffective. These data document the utility of the SV40 plasmid in immortalizing rat parotid and submandibular acinar cells that retain most of the features of acinar differentiation.

A rat parotid gland cell line, Par-C10, exhibits neurotransmitter-regulated transepithelial anion secretion.

Because of the lack of salivary gland cell lines suitable for Ussing chamber studies, a recently established rat parotid acinar cell line, Par-C10, was grown on permeable supports and evaluated for development of transcellular resistance, polarization, and changes in short-circuit current (Isc) in response to relevant receptor agonists. Par-C10 cultures reached confluence in 3-4 days and developed transcellular resistance values of >/=2,000 Omega . cm2. Morphological examination revealed that Par-C10 cells grew as polarized monolayers exhibiting tripartite junctional complexes and the acinar cell-specific characteristic of secretory canaliculi. Par-C10 Isc was increased in response to muscarinic cholinergic and alpha- and beta-adrenergic agonists on the basolateral aspect of the cultures and to ATP and UTP (through P2Y2 nucleotide receptors) applied apically. Ion replacement and inhibitor studies indicated that anion secretion was the primary factor in agonist-stimulated Isc. RT-PCR, which confirmed the presence of P2Y2 nucleotide receptor mRNA in Par-C10 cells, also revealed the presence of mRNA for the cystic fibrosis transmembrane conductance regulator and ClC-2 Cl- channel proteins. These findings establish Par-C10 cells as the first cell line of salivary gland origin useful in transcellular ion secretion studies in Ussing chambers.

Development and characterization of SV40 immortalized rat parotid acinar cell lines.

Rat parotid salivary gland acinar cells were transfected by CaPO4 precipitation using a plasmid containing a replication-defective simian virus (SV40) genome. Out of 30 clonal cell lines, 2 were shown to have moderate to high levels of cytodifferentiation and salivary gland acinar cell function. Functional studies with the two cell lines indicated that the beta-adrenergic agonist (isoproterenol), vasoactive intestinal peptide prostaglandin E1, and forskolin were effective activators of intracellular cyclic adenosine 3':5'-cyclic monophosphate production. Phenylephrine, carbamylcholine, and UTP were effective in increasing inositol phosphate production and intracellular free calcium levels, whereas substance P was without affect. Utilizing indirect immunofluorescence analysis, both cell lines were shown to express the SV40 large T antigen. Electron microscopic evaluation documented moderate to high levels of cytodifferentiation with the maintenance of tripartite junctional complexes, cellular polarization, and presence of moderate amounts of secretory granules and rough endoplasmic reticulum. The two cell lines had doubling times of 22 and 36 h, respectively.

Development and characterization of SV40 immortalized rat submandibular acinar cell lines.

Rat submandibular salivary gland acinar cells were transfected by CaPO4 precipitation using a plasmid containing a replication-defective simian virus (SV40) genome. Out of 27 clonal cell lines, two were shown to have moderate to high levels of cytodifferentiation and salivary gland acinar cell function. Functional studies with the two cell lines indicated that the beta-adrenergic agonist, isoproterenol, vasoactive intestinal peptide, and prostaglandin E1 were effective activators of intracellular cyclic AMP production. Epinephrine, norepinephrine, phenylephrine, acetylcholine, and P2U-purinoceptor agonists were effective in increasing inositol phosphate production and intracellular free calcium levels, whereas substance P was without effect. Utilizing indirect immunofluorescence analysis, both cell lines were shown to express glutamine/glutamic acid-rich proteins, a submandibular acinar cell specific secretory protein family. Electron microscopic evaluation documented the maintenance of tripartite junctional complexes, cellular polarization, and the presence of moderate amounts of secretory granules and rough endoplasmic reticulum. The two cell lines had doubling times of 25 h.

Characterization of synthesis and storage of TGF-alpha in rat parotid acinar and intercalated duct cells.

Although the expression and biological role of transforming growth factor-alpha (TGF-alpha) have been explored in a variety of normal cells in mammalian species, little is known about the storage of TGF-alpha in secretory cells of exocrine organs. Parotid glands from four rats were homogenized for RNA isolation followed by reverse transcription-polymerase chain reaction to determine the presence of TGF-alpha message. In situ hybridization using a hamster-specific TGF-alpha riboprobe was done on paraffin sections. Parotid gland and isolated acinar cells were processed for transmission electron microscopy (TEM) and postembedding immunogold labeled for TGF-alpha. Gold particles were counted on approximately 200 granules in 10 acinar cells and in 10 intercalated duct cells. Labeling density was calculated as the number of gold particles per square micrometer +/- SD. Statistical significance was calculated using one-way analysis of variance. Using multiple technologies, we have established that rat parotid acinar and intercalated duct cells synthesize TGF-alpha and store the precursor form of this cytokine in their secretory granules.

Effects of oxygen, insulin, and glucagon concentrations on rat submandibular acini in serum-free primary culture.

The objective of these studies was to develop serum-free culture conditions for dissociated acini from rat submandibular glands. Acini were isolated from the submandibular glands of 42-46 d old rats and cultured on reconstituted rat tail collagen containing laminin in 1:1 Ham's F12 and Dulbecco's media, supplemented with BSA, transferrin, insulin, T3, EGF, dexamethasone, retinoic acid, carbamylcholine, and trace elements, and gassed with 50% O2. The acini became partly embedded in the collagen gel and rapidly enlarged throughout the first 22 d of culture, maintaining modest seromucous acinar differentiation, as judged morphologically and by mucin secretion. Parallel cultures then were grown under 20, 35, 50, and 65% O2, and evaluated morphologically and by DNA content. Growth and retention of seromucous acinar characteristics were best with 35% O2, but lipid accumulation and cell death were unacceptably high. A spectrum of concentrations of insulin and glucagon then were tried. With 0.05 micrograms/ml insulin, cellular growth and organization were orderly, lipid accumulations were not excessive, and moderate differentiation was retained through 15 d of culture. With more than 0.1 microgram/ml insulin added to or subtracted from the optimum, the detrimental effects recurred. Addition of sufficient glucagon counteracted the effects of both optimum and excessive concentrations of insulin. We now have achieved an orderly growth of moderately differentiated rat submandibular acini for 15 d in serum-free primary culture.

Steroid hormone analysis in human saliva.

Despite the aforementioned complications, noninvasive saliva collection has provided the medical and research community with an excellent medium for the monitoring of plasma steroid levels. This noninvasive method has permitted the evaluation and assessment of a multitude of endocrine studies that would have been extremely difficult, if not impossible, using other more familiar methods.

Rat submandibular and parotid protein phosphorylation and exocytosis: effect of site-selective cAMP analogs.

A series of cAMP analogs that have different specificities for the two different binding sites on the regulatory subunit of type I and type II cAMP-dependent protein kinase (PKA) were used to determine whether selective activation of type I or type II PKA could link either or both isozyme forms of PKA with exocytosis and specific protein phosphorylation in salivary gland cells. Using dispersed rat submandibular or parotid cells, selective activation of either type I or type II resulted in a synergistic response for both rat submandibular mucin and parotid amylase secretion and the phosphorylation of a 26-kDa integral membrane phosphoprotein. These data suggest that the activation of either isozyme of PKA can elicit cellular exocytosis and specific protein phosphorylation in both of these cell types.

Purification and partial characterization of analogous 26-kDa rat submandibular and parotid gland integral membrane phosphoproteins that may have a role in exocytosis.

Rat submandibular and parotid gland exocytosis is primarily controlled by beta-adrenergic receptor stimulation. Although its precise role in the regulation of salivary gland exocytosis is not fully understood, protein phosphorylation, mediated by the activation of cAMP-dependent protein kinase, may be directly involved. Previous studies suggest that analogous 26-kDa integral membrane phosphoproteins may play a direct role in regulating exocytosis. Studies were here undertaken to purify and partially characterize both phosphoproteins. After endogenous phosphorylation with 32P, subcellular fraction and solubilization of the microsomal fraction in n-octyl beta-glucopyranoside, the 26-kDa integral membrane phosphoproteins were purified by high performance liquid chromatography (HPLC), followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electroelution of the proteins. Amino acid analysis indicated a significant number of serine amino acids: N-terminal sequence data demonstrated a high level of homology; and trypsin digestion followed by reversed-phase HPLC indicated the possibility of multiple phosphorylation sites.

Activation and distribution of rat parotid cAMP-dependent protein kinase following beta-adrenergic receptor stimulation in vitro.

The extent of activation of parotid protein kinase A (EC 2.7.1.37) isozymes was determined using dispersed cells and an 8-N3-[32P]-cAMP photoprobe. Cold-trap studies indicated that 40% of type I protein kinase A was activated following maximal beta-adrenergic receptor stimulation, whereas type II activation was less than 20%. Both cytosolic and microsomal type I activation occurred rapidly after stimulation and both remain activated throughout the entire secretory period. The dose-response relationship for the isotypes following beta-adrenergic receptor activation demonstrated a greater extent of type I activation at maximal concentration of agonist. Although protein kinase A may not be the only kinase involved in rat parotid amylase release, these findings add further evidence of a direct regulatory role for this kinase, with type I having potentially a greater role than type II.