Beáta Kerémi

Evidence for Bicarbonate Secretion by Ameloblasts in a Novel Cellular Model

Formation and growth of hydroxyapatite crystals during amelogenesis generate a large number of protons that must be neutralized, presumably by HCO3− ions transported from ameloblasts into the developing enamel matrix. Ameloblasts express a number of transporters and channels known to be involved in HCO3− transport in other epithelia. However, to date, there is no functional evidence for HCO3− transport in these cells. To address questions related to HCO3− export from ameloblasts, we have developed a polarized 2-dimensional culture system for HAT-7 cells, a rat cell line of ameloblast origin. HAT-7 cells were seeded onto Transwell permeable filters. Transepithelial resistance was measured as a function of time, and the expression of transporters and tight junction proteins was investigated by conventional and quantitative reverse transcription polymerase chain reaction. Intracellular pH regulation and HCO3− transport were assessed by microfluorometry. HAT-7 cells formed epithelial layers with measureable transepithelial resistance on Transwell permeable supports and expressed claudin-1, claudin-4, and claudin-8—key proteins for tight junction formation. Transport proteins previously described in maturation ameloblasts were also present in HAT-7 cells. Microfluorometry showed that the HAT-7 cells were polarized with a high apical membrane CO2 permeability and vigorous basolateral HCO3− uptake, which was sensitive to Na+ withdrawal, to the carbonic anhydrase inhibitor acetazolamide and to H2DIDS inhibition. Measurements of transepithelial HCO3− transport showed a marked increase in response to Ca2+- and cAMP-mobilizing stimuli. Collectively, 2-dimensional HAT-7 cell cultures on permeable supports 1) form tight junctions, 2) express typical tight junction proteins and electrolyte transporters, 3) are functionally polarized, and 4) can accumulate HCO3− ions from the basolateral side and secrete them at the apical membrane. These studies provide evidence for a regulated, vectorial, basolateral-to-apical bicarbonate transport in polarized HAT-7 cells. We therefore propose that the HAT-7 cell line is a useful functional model for studying electrolyte transport by ameloblasts.

Bacterial Lysine Decarboxylase Influences Human Dental Biofilm Lysine Content, Biofilm Accumulation, and Subclinical Gingival Inflammation

BACKGROUND Dental biofilms contain a protein that inhibits mammalian cell growth, possibly lysine decarboxylase from Eikenella corrodens. This enzyme decarboxylates lysine, an essential amino acid for dentally attached cell turnover in gingival sulci. Lysine depletion may stop this turnover, impairing the barrier to bacterial compounds. The aims of this study are to determine biofilm lysine and cadaverine contents before oral hygiene restriction (OHR) and their association with plaque index (PI) and gingival crevicular fluid (GCF) after OHR for 1 week. METHODS Laser-induced fluorescence after capillary electrophoresis was used to determine lysine and cadaverine contents in dental biofilm, tongue biofilm, and saliva before OHR and in dental biofilm after OHR. RESULTS Before OHR, lysine and cadaverine contents of dental biofilm were similar and 10-fold greater than in saliva or tongue biofilm. After 1 week of OHR, the biofilm content of cadaverine increased and that of lysine decreased, consistent with greater biofilm lysine decarboxylase activity. Regression indicated that PI and GCF exudation were positively related to biofilm lysine after OHR, unless biofilm lysine exceeded the minimal blood plasma content, in which case PI was further increased but GCF exudation was reduced. CONCLUSIONS After OHR, lysine decarboxylase activity seems to determine biofilm lysine content and biofilm accumulation. When biofilm lysine exceeds minimal blood plasma content after OHR, less GCF appeared despite more biofilm. Lysine appears important for biofilm accumulation and the epithelial barrier to bacterial proinflammatory agents. Inhibiting lysine decarboxylase may retard the increased GCF exudation required for microbial development and gingivitis.

Mesenchymal Stem Cells of Dental Origin-Their Potential for Antiinflammatory and Regenerative Actions in Brain and Gut Damage.

Alzheimer’s disease, Parkinson’s disease, traumatic brain and spinal cord injury and neuroinflammatory multiple sclerosis are diverse disorders of the central nervous system. However, they are all characterized by various levels of inappropriate inflammatory/immune response along with tissue destruction. In the gastrointestinal system, inflammatory bowel disease (IBD) is also a consequence of tissue destruction resulting from an uncontrolled inflammation. Interestingly, there are many similarities in the immunopathomechanisms of these CNS disorders and the various forms of IBD. Since it is very hard or impossible to cure them by conventional manner, novel therapeutic approaches such as the use of mesenchymal stem cells, are needed. Mesenchymal stem cells have already been isolated from various tissues including the dental pulp and periodontal ligament. Such cells possess transdifferentiating capabilities for different tissue specific cells to serve as new building blocks for regeneration. But more importantly, they are also potent immunomodulators inhibiting proinflammatory processes and stimulating anti-inflammatory mechanisms. The present review was prepared to compare the immunopathomechanisms of the above mentioned neurodegenerative, neurotraumatic and neuroinflammatory diseases with IBD. Additionally, we considered the potential use of mesenchymal stem cells, especially those from dental origin to treat such disorders. We conceive that such efforts will yield considerable advance in treatment options for central and peripheral disorders related to inflammatory degeneration.

Biofilm Lysine Decarboxylase, a New Therapeutic Target for Periodontal Inflammation

BACKGROUND Lysine, a nutritionally essential amino acid, enters the oral cavity in gingival crevicular fluid (GCF). During oral hygiene restriction (OHR), lysine decarboxylase (LDC) in dento-gingival biofilms converts lysine to cadaverine. Lysine depletion impairs the dental epithelial barrier to bacterial proinflammatory products. Antibodies to LDC from Eikenella corrodens (Ecor-LDC) inhibit LDC activity and retard gingival inflammation in beagle dogs. Whether E. corrodens is the major source of LDC in dental biofilms and whether the lysine analog tranexamic acid (TA) inhibits LDC activity, biofilm accumulation, and GCF exudation in a human gingivitis model were examined. METHODS Antibodies raised in goats to LDC-rich extracts from E. corrodens cell surfaces were used to inhibit Ecor-LDC and detect it in biofilm extracts using Western blots. Ecor-LDC activity was measured at pH 4.0 to 11.0 and its TA dissociation constant (Ki) at pH 7.0. Young adults used a 5% or 10% TA mouthwash three times daily during OHR for 1 week. RESULTS Ecor-LDC antibodies and TA inhibited biofilm LDC. Ki of TA for Ecor-LDC was 940 μM. TA reduced plaque index (PI) by downshifting the PI correlation with biofilm lysine content after OHR without TA. GCF was correspondingly suppressed. However, greater TA retention in saliva partially relieved GCF suppression but not biofilm lysine depletion. CONCLUSIONS TA slightly inhibits LDC but strongly reduces biofilm by inhibiting bacterial lysine uptake. Unfortunately, TA may impair dental epithelial attachments by also inhibiting lysine transporter uptake. Ecor-LDC inhibitors other than lysine analogs may maintain sufficient lysine levels and attachment integrity to prevent periodontal inflammation.

Role of nitric oxide in the regulation of blood flow in the rat submandibular gland during carotid artery occlusion

The possible involvement of nitric oxide in the preservation of blood flow to the rat submandibular gland after uni- or bilateral occlusion of the common carotid was studied. Glandular blood flow and mean blood pressure were monitored before, during and after carotid occlusion in the presence and absence of the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine-methyl-ester (L-NAME). To calculate vascular resistance, the local perfusion pressure distal to the point of occlusion was also measured. In normal rats, uni- or bilateral carotid occlusion resulted in an immediate decrease in ipsilateral glandular blood flow. After the cessation of carotid occlusion, hyperaemia was observed in the submandibular gland. Both local perfusion pressure and vascular resistance decreased during carotid occlusion. In the group pretreated with L-NAME, trends in blood-flow responses to uni- or bilateral occlusion were identical to those registered in the control groups, though the magnitude of the alterations was significantly less. The well-maintained glandular blood flow was due to functioning vascular anastomoses and compensating dilatation of glandular blood vessels. Nitric oxide had only a restrained effect on this compensatory mechanism.

Angiotensin Ii Type 1 (at1) Receptor Blockade Enhances the L‐NAME‐Induced Vasoconstriction in Rat Submandibular Gland

The vasoregulatory role of nitric oxide (NO) and angiotensin II type 1 (AT1) receptors in the circulation of the submandibular gland (SMG) of rats was studied. The glandular blood flow was determined by means of laser Doppler flowmetry and rubidium isotope technique. The data obtained by these two methods correlated well (r= 0.77; P < 0.01). The AT1 receptor antagonist candesartan (0.5 mg kg−1, I.V.) reduced the vascular resistance in the SMG by 37% (P < 0.05). By contrast, the NO synthase blocker L‐NAME (15 mg kg−1, I.V.) significantly increased vascular resistance in the SMG both in candesartan‐treated (P < 0.001) and non‐treated (P < 0.001) animals. The increase in resistance was greater (P < 0.05) after previous blockade of AT1 receptors. These findings suggest that the AT1 receptors have an important role in the vasoregulation of the SMG in the rat. As a result of AT1 blockade, NO‐dependent tone of glandular vessels may be enhanced significantly.

Stress and Salivary Glands

Salivary glands produce a bicarbonate-rich fluid containing digestive and protective proteins and other components to be delivered into the gastrointestinal tract. Its function is under strict control of the autonomic nervous system. Salivary electrolyte and fluid secretion are primarily controlled by parasympathetic activity, while protein secretion is primaily triggered by sympathetic stimulation. Stress activates the hypothalamic - pituitary - adrenal axis. The peripheral limb of this axis is the efferent sympathetic/adrenomedullary system. Stress reaction, even if it is sustained for long, does not cause obvious damage to salivary glands. However, stress induces dramatic changes in the constituents of secreted saliva. Since salivary protein secretion is strongly dependent on sympathetic control, changes in saliva can be utilized as sensitive stress indicators. Some of the secreted compounds are known for their protective effect in the mouth and the gut, while others may just pass through the glands from blood plasma because of their chemical nature and the presence of transcellular salivary transporting systems. Indeed, most compounds that appear in blood circulation can also be identified in saliva, although at different concentrations. This work overviews the presently recognized salivary stress biosensors, such as amylase, cortisol, heat shock proteins and other compounds. It also demonstrates that saliva is widely recognised as a diagnostic tool for early and sensitive discovery of salivary and systemic conditions and disorders. At present it may be too early to introduce most of these biomarkers in daily routine diagnostic applications, but advances in salivary biomarker standardisation should permit their wide-range utilization in the future including safe, reliable and non-invasive estimation of acute and chronic stress levels in patients.