Impact of diseases of the hard tissues of teeth on oral health-related quality of life of schoolchildren in area with a high concentration of fluoride in drinking water.

OBJECTIVE: To assess the impact of caries, Molar Incisor Hypomineralization (MIH), and fluorosis on the Oral Health-Related Quality of Life (OHRQoL) of schoolchildren aged 8-10 years living in area with different fluoride levels in the drinking water. SUBJECT AND METHODS: The prevalence of caries and fluorosis were assessed among 663 Mexican schoolchildren using the International Caries Detection and Assessment System (ICDAS II) and the Thylstrup and Fejerskov Index (TFI), respectively. MIH was recorded using the European Academy of Pediatric Dentistry (EAPD) criteria and OHRQoL using the Child Perceptions Questionnaire (CPQ8-10). Poisson regression models were used in data analysis. RESULTS: Schoolchildren presenting two of the three conditions (cavitated lesions and TFI≥4, cavitated lesions and MIH or TFI≥4 and MIH) experienced worse quality of life than children who did not [RR=4.18; (95% CI 3.83, 4.56)]. Children with all three conditions had worse quality of life than children who did not [RR=5.64; (95% CI 5.13, 6.20)]. CONCLUSIONS: Fluorosis, MIH, and caries have a negative impact on the OHRQoL of schoolchildren living in area with a high concentration of fluoride in their drinking water.

Marginalization and fluorosis its relationship with dental caries in rural children in Mexico: A cross-sectional study.

OBJECTIVE: Examine the association between marginalization and fluorosis with caries experience in Mexican rural children aged 8-12, in Oaxaca, Mexico. METHODS: Cross-sectional study of 283 rural schoolchildren selected from two locations with high and medium levels of marginalization where the water fluoride concentration ranged from 2.0 to 2.5 ppm/F. Caries was evaluated using the DMFT index and dental fluorosis with the Thylstrup-Fejerskov Index (TFI). Socioeconomic data were collected from participants' parents, with data on the children's characteristics collected from them via a questionnaire. RESULTS: The prevalence of caries was 72.4% (DMFT ≥1) in the permanent dentition. The prevalence of fluorosis was 98.0% (TFI ≥4=71.4%). 54.8% of the children brushed their teeth two or more times daily. In logistic regression children living in high levels of marginalization were more likely to present caries (OR=2.11, 95% CI 1.13 - 3.93) than children living in medium levels. Children with severe fluorosis (TFI ≥4) (OR=1.93, 95% CI 1.06 - 3.53) were more likely have caries than those with TFI ⟨3. CONCLUSION: Rural children with a high level of marginalization and fluorosis (TFI ≥4) were more likely to present caries. Poor oral hygiene and low dental service levels were found in both marginalized areas. Populations with medium/high marginalization are more susceptible to caries.

An inverse relationship between obesity and dental caries in Mexican schoolchildren: a cross-sectional study.

OBJECTIVE: The aim of the study was to examine the relationship between caries experience and obesity in Mexican schoolchildren aged 8-12 years. STUDY DESIGN: This is a cross-sectional study. METHODS: This study was conducted on 522 schoolchildren selected from public schools. The prevalence of caries was evaluated by applying the decayed, missing and filled teeth (DMFT) index and ascertaining the subjects' dental caries experience from the mean DMFT value. Socio-economic data were collected from the parents, with data on the children's characteristics collected from them via a questionnaire. Their weight and height were then measured and used to calculate their body mass index (BMI)-for-age Z-score, which was then adjusted by age and sex. RESULTS: The prevalence of caries was 79.9% (DMFT≥1) in permanent dentition. Of all children, 47.5% of them brushed their teeth two or more times per day, and the prevalence of overweight and obesity was 20.1% and 17.6%, respectively. The logistic regression model showed that children with obesity (a >2 Z-score on the BMI-for-age growth chart) were less likely to have dental caries (odds ratio [OR] = 0.53 [95% confidence interval {CI}: 0.31-0.89]; P = 0.017) than children without obesity, with schoolchildren who consume more sweets per day (OR = 1.65 [95% CI: 1.03-2.62]; P = 0.035) more likely to present caries than schoolchildren who consume fewer sweets per day. CONCLUSION: Children with obesity are less likely to present dental caries. Comprehensive strategies aimed at risk factors can be useful in controlling nutritional status and improving oral health.

Intragastric administration of Lactobacillus casei BL23 induces regulatory FoxP3+RORγt+ T cells subset in mice.

Many studies have highlighted the immunomodulatory properties of the probiotic strain Lactobacillus casei BL23. Recently, we demonstrated the ability of this strain to modulate the Th2-oriented immune response in a mouse model of cow's milk allergy based on the induction of a Th17-biased immune response. The probiotic function of L. casei has been also linked to gut-microbiota modifications which could been potentially involved in the immune regulation; however, its precise mechanism of action remains poorly understood. In this regard, recent studies suggest that gut microbiota induces a specific subset of CD4+FoxP3+ Treg cells that also express RORγt+, the specific transcription factor of Th17 cells. This new type of regulatory T cells, called type 3 Treg, displays suppressive function during intestinal inflammation, participating in inflammation control. We thus explored the ability of L. casei BL23 to specifically induce type 3 Treg cells, both in vitro and in vivo. Our results showed that intragastric administration of L. casei BL23 to mice induces local and systemic FoxP3+ RORγt+ type 3 Treg cells that could then participate in the beneficial effects of L. casei BL23 in different intestinal-related disorders.

Mitochondrial reactive oxygen species (ROS) as signaling molecules of intracellular pathways triggered by the cardiac renin-angiotensin II-aldosterone system (RAAS).

Mitochondria represent major sources of basal reactive oxygen species (ROS) production of the cardiomyocyte. The role of ROS as signaling molecules that mediate different intracellular pathways has gained increasing interest among physiologists in the last years. In our lab, we have been studying the participation of mitochondrial ROS in the intracellular pathways triggered by the renin-angiotensin II-aldosterone system (RAAS) in the myocardium during the past few years. We have demonstrated that acute activation of cardiac RAAS induces mitochondrial ATP-dependent potassium channel (mitoKATP) opening with the consequent enhanced production of mitochondrial ROS. These oxidant molecules, in turn, activate membrane transporters, as sodium/hydrogen exchanger (NHE-1) and sodium/bicarbonate cotransporter (NBC) via the stimulation of the ROS-sensitive MAPK cascade. The stimulation of such effectors leads to an increase in cardiac contractility. In addition, it is feasible to suggest that a sustained enhanced production of mitochondrial ROS induced by chronic cardiac RAAS, and hence, chronic NHE-1 and NBC stimulation, would also result in the development of cardiac hypertrophy.

Inactivation of the ybdD gene in Lactococcus lactis increases the amounts of exported proteins.

Random insertional mutagenesis performed on a Lactococcus lactis reporter strain led us to identify L. lactis ybdD as a protein-overproducing mutant. In different expression contexts, the ybdD mutant shows increased levels of exported proteins and therefore constitutes a new and attractive heterologous protein production host. This study also highlights the importance of unknown regulatory processes that play a role during protein secretion.

Construction and characterization of a Lactococcus lactis strain deficient in intracellular ClpP and extracellular HtrA proteases.

A Lactococcus lactis strain deficient in both its major proteases, intracellular (ClpP) and extracellular (HtrA), was constructed and characterized. This strain, hereafter called clpP-htrA, could be obtained only by conjugation between a clpP donor strain and an htrA recipient strain in the NZ9000 context, allowing heterologous gene expression under the control of the NICE (nisin-controlled expression) system. The clpP-htrA double mutant showed both higher stress tolerance (e.g. high temperature and ethanol resistance) and higher viability than single clpP or htrA mutant strains. In addition, the secretion rate of two heterologous proteins (staphylococcal nuclease Nuc and Nuc-E7) was also higher in clpP-htrA than in the wild-type strain. This strain should be a useful host for high-level production and quality of stable heterologous proteins.

An autocrine/paracrine mechanism triggered by myocardial stretch induces changes in contractility.

An autocrine/paracrine mechanism is triggered by stretching the myocardium. This mechanism involves release of angiotensin II, release/increased formation of endothelin, activation of the Na(+)/H(+) exchanger, increase in intracellular Na(+), and the increase in the Ca(2+) transient that underlies the slow force response to stretch. The autocrine/paracrine mechanism could explain how changes in afterload alter cardiac contractility.

Reverse mode of the Na+-Ca2+ exchange after myocardial stretch: underlying mechanism of the slow force response.

This study was designed to gain additional insight into the mechanism of the slow force response (SFR) to stretch of cardiac muscle. SFR and changes in intracellular Na(+) concentration ([Na(+)](i)) were assessed in cat papillary muscles stretched from 92% to approximately 98% of L(max). The SFR was 120+/-0.6% (n=5) of the rapid initial phase and coincided with an increase in [Na(+)](i). The SFR was markedly depressed by Na(+)-H(+) exchanger inhibition, AT(1) receptor blockade, nonselective endothelin-receptor blockade and selective ET(A)-receptor blockade, extracellular Na(+) removal, and inhibition of the reverse mode of the Na(+)-Ca(2+) exchange by KB-R7943. KB-R7943 prevented the SFR but not the increase in [Na(+)](i). Inhibition of endothelin-converting enzyme activity by phosphoramidon suppressed both the SFR and the increase in [Na(+)](i). The SFR and the increase in [Na(+)](i) after stretch were both present in muscles with their endothelium (vascular and endocardial) made functionally inactive by Triton X-100. In these muscles, phosphoramidon also suppressed the SFR and the increase in [Na(+)](i). The data provide evidence that the last step of the autocrine-paracrine mechanism leading to the SFR to stretch is Ca(2+) entry through the reverse mode of Na(+)-Ca(2+) exchange.

Frequency-dependent changes in calcium cycling and contractile activation in SERCA2a transgenic mice.

OBJECTIVE: This study was undertaken to investigate the mechanism of altered contractility in hearts from transgenic mice overexpressing the sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA2a). In particular, we sought to determine whether the reported increase in contractility is frequency-dependent, as might be expected if attributable to changes in SR Ca2+ loading. METHODS: Intracellular [Ca2+] and contractile force were measured at room temperature (22 degrees C) simultaneously in fura-2-loaded isometrically-contracting trabeculae dissected from the hearts of FVB/N control (n = 6) or SERCA2a transgenic (n = 6) mice. RESULTS: SERCA transgenics exhibit a positive force-frequency relationship, but this was flat in age- and strain-matched controls. SERCA transgenics exhibit a sizable increase in calcium transient amplitude relative to controls, with a concomitant increase in force generation at higher frequencies of stimulation. Amplitudes of Ca2+ transients (transgenics: 1.56 +/- 0.09 micromol/L, controls: 1.21 +/- 0.14) and twitches (transgenics: 21.71 +/- 0.91 mN/mm2, controls: 13.74 +/- 1.67) were significantly different at 2.0 Hz stimulation (P < 0.05). CONCLUSION: An increase in SERCA expression increases the ability of the sarcoplasmic reticulum to store calcium, such that more calcium is available to be released during each heartbeat at higher stimulation rates.

Preservation of myofilament calcium responsiveness underlies protection against myocardial stunning by ischemic preconditioning.

OBJECTIVE: Whereas diminution of infarct size by ischemic preconditioning (IP) is well-accepted, protection against stunning is controversial. Since stunning is characterized by decreased myofilament Ca2+ responsiveness, we investigated whether IP would preserve myofilament responsiveness in a model of stunning. METHODS: Rat hearts were retrogradely perfused with Krebs-Henseleit (K-H) solution for 20 min and then subjected to 20 min of no-flow global ischemia, followed by 20 min of reperfusion in the absence (stunning) or in the presence (IP) of a previous 5-min period of ischemia followed by 15 min of reperfusion. A group of hearts perfused under non-ischemic conditions served as control. Thin ventricular trabeculae were dissected from each of the experimental groups and loaded with fura-2 to measure intracellular calcium concentration ([Ca2+]i) and developed force. RESULTS: After 20 min of reperfusion, left ventricular developed pressure decreased in stunned hearts to 61 +/- 5% of control (P < 0.01), whereas recovery was complete in the IP hearts (97 +/- 4%). Steady-state [Ca2+]i-force relationships revealed a decreased maximal Ca(2+)-activated force in stunned hearts relative to control, but no change in the IP group. The Ca2+ required for 50% activation increased in stunning but not in IP. CONCLUSIONS: These results show that the decrease in myofilament responsiveness that characterizes stunning is prevented by ischemic preconditioning.

Mechanisms underlying the increase in force and Ca(2+) transient that follow stretch of cardiac muscle: a possible explanation of the Anrep effect.

Myocardial stretch produces an increase in developed force (DF) that occurs in two phases: the first (rapidly occurring) is generally attributed to an increase in myofilament calcium responsiveness and the second (gradually developing) to an increase in [Ca(2+)](i). Rat ventricular trabeculae were stretched from approximately 88% to approximately 98% of L(max), and the second force phase was analyzed. Intracellular pH, [Na(+)](i), and Ca(2+) transients were measured by epifluorescence with BCECF-AM, SBFI-AM, and fura-2, respectively. After stretch, DF increased by 1.94+/-0.2 g/mm(2) (P<0.01, n = 4), with the second phase accounting for 28+/-2% of the total increase (P<0.001, n = 4). During this phase, SBFI(340/380) ratio increased from 0.73+/-0.01 to 0.76+/-0.01 (P<0.05, n = 5) with an estimated [Na(+)](i) rise of approximately 6 mmol/L. [Ca(2+)](i) transient, expressed as fura-2(340/380) ratio, increased by 9.2+/-3.6% (P<0.05, n = 5). The increase in [Na(+)](i) was blocked by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA). The second phase in force and the increases in [Na(+)](i) and [Ca(2+)](i) transient were blunted by AT(1) or ET(A) blockade. Our data indicate that the second force phase and the increase in [Ca(2+)](i) transient after stretch result from activation of the Na(+)/H(+) exchanger (NHE) increasing [Na(+)](i) and leading to a secondary increase in [Ca(2+)](i) transient. This reflects an autocrine-paracrine mechanism whereby stretch triggers the release of angiotensin II, which in turn releases endothelin and activates the NHE through ET(A) receptors.

Origin of contractile dysfunction in heart failure: calcium cycling versus myofilaments.

BACKGROUND: Chronic congestive heart failure is a common, often lethal disorder of cardiac contractility. The fundamental pathophysiology of the contractile failure remains unclear, the focus being on abnormal Ca2+ cycling despite emerging evidence for depressed myofilament function. METHODS AND RESULTS: We measured intracellular Ca2+ concentration ([Ca2+]i) and contractile force in intact ventricular muscle from SHHF rats with spontaneous heart failure and from age-matched controls. At physiological concentrations of extracellular Ca2+ ([Ca2+]o), [Ca2+]i transients were equal in amplitude in the 2 groups, but [Ca2+]i peaked later in SHHF muscles. Twitch force peaked slowly and was equivalent or modestly decreased in amplitude relative to controls. Steady-state analysis revealed a much greater (53%) depression of maximal Ca2+-activated force in SHHF muscles, which, had other factors been equal, would have produced an equivalent suppression of twitch force. Phase-plane analysis reveals that the slowing of Ca2+ cycling prolongs the time available for Ca2+ to activate the myofilaments in failing muscle, partially compensating for the marked dysfunction of the contractile machinery. CONCLUSIONS: Our results indicate that myofilament activation is severely blunted in heart failure, but concomitant changes in [Ca2+]i kinetics minimize the contractile depression. These results challenge prevailing concepts regarding the pathophysiology of heart failure: the myofilaments emerge as central players, whereas changes in Ca2+ cycling are reinterpreted as compensatory rather than causative.

Altered cardiac excitation-contraction coupling in mutant mice with familial hypertrophic cardiomyopathy.

Excitation-contraction coupling in cardiac muscle of familial hypertrophic cardiomyopathy (FHC) remains poorly understood, despite the fact that the genetic alterations are well defined. We characterized calcium cycling and contractile activation in trabeculae from a mutant mouse model of FHC (Arg403Gln knockin, alpha-myosin heavy chain). Wild-type mice of the same strain and age ( approximately 20 weeks old) served as controls. During twitch contractions, peak intracellular Ca2+ ([Ca2+]i) was higher in mutant muscles than in the wild-type (P < 0.05), but force development was equivalent in the two groups. Ca2+ transient amplitude increased dramatically in both groups as stimulation rate increased from 0.2 to 4 Hz. Nevertheless, developed force fell at the higher stimulation rates in the mutants but not in controls (P < 0.05). The steady-state force-[Ca2+]i relationship was less steep in mutants (Hill coefficient, 2.94 +/- 0.27 vs. 5.28 +/- 0.64; P > 0.003), with no changes in the [Ca2+]i required for 50% activation or maximal Ca2+-activated force. Thus, calcium cycling and myofilament properties are both altered in FHC mutant mice: more Ca2+ is mobilized to generate force, but this does not suffice to maintain contractility at high stimulation rates.

Novel myofilament Ca2+-sensitizing property of xanthine oxidase inhibitors.

Antioxidants are known to mitigate the cardiac contractile dysfunction that follows brief periods of ischemia ("myocardial stunning"). Stunning decreases contractility at the level of the contractile proteins; therefore, we asked whether antioxidant treatment preserves myofilament Ca2+ responsiveness after global ischemia and reflow. Right ventricular trabeculae were dissected from rat hearts subjected either to 20 minutes ischemia and reperfusion in the absence of drugs (stunned group) or to the same protocol in the presence of allopurinol, an inhibitor of xanthine oxidase (XO), and mercaptopropionylglycine (MPG), a hydroxyl radical scavenger (antioxidant group). At 20 minutes of reflow, isovolumic developed pressure recovered completely in the antioxidant group, but in the stunned group it recovered by only 57%. [Ca2+]i and contractile force measurements in trabeculae revealed the expected depression of myofilament function in the stunned group, with no change in Ca2+ transients relative to nonischemic controls. In contrast, Ca2+ transients were smaller, but force was greater, in the antioxidant group relative to both the stunned group and to nonischemic controls. Steady-state [Ca2+]i-force relationships revealed a striking increase of maximal force and a modest shift of activation to a lower range of [Ca2+]i. The increase in maximal force was reproduced by allopurinol+MPG or by allopurinol alone under nonischemic conditions and also by oxypurinol (100 micromol/L), a potent inhibitor of XO. We conclude that allopurinol and oxypurinol sensitize the cardiac myofilaments to Ca2+. This Ca2+-sensitizing effect underlies the preservation of contractility observed with an allopurinol+MPG antioxidant cocktail in a model of stunned myocardium. These serendipitous findings identify allopurinol and oxypurinol as the lead compounds of a novel class of inotropic agents.

Calcium cycling and contractile activation in intact mouse cardiac muscle.

1. Excitation-contraction coupling in mouse cardiac muscle remains poorly characterized, despite the fact that the mouse is the mammalian species of choice for genetic manipulation. In this study, we characterized the relationship between internal calcium concentration ([Ca2+]i) and contraction in intact mouse ventricular muscle loaded with fura-2 salt at 20-22 degrees C. 2. Both Ca2+ transient amplitude and twitch force increased monotonically as external Ca2+ concentration ([Ca2+]o) was increased up to 8.0 mM, with no changes in diastolic levels or in the times to peak of either Ca2+ transients or force. The decay of Ca2+ transients was accelerated as [Ca2+]o increased, while relaxation was prolonged. Both Ca2+ transient amplitude and twitch force increased as stimulation rate increased from 0.2 to 4 Hz, but the increase in force was much greater than the underlying increase in [Ca2+]i. 3. The steady-state force-[Ca2+]i relationship revealed an [Ca2+]i required for 50 % of maximal activation (Ca50) of 0.95 +/- 0.08 microM, a Hill coefficient of 9.9 +/- 2.6, and a maximal Ca2+-activated force (Fmax) of 60 +/- 5 mN mm-2. 4. Unlike rat ventricular myocardium, mouse cardiac muscle resists supraphysiological [Ca2+]o. The strong positive force-frequency relationship in mouse cardiac muscle, with increases of force disproportionate to the increases in Ca2+ transients, suggests frequency-dependent 'sensitization' of the myofilaments. During steady-state activation, mouse muscle exhibits decreased Ca2+ responsiveness relative to other species, but high co-operativity. 5. These physiological features of mouse cardiac muscle merit consideration when interpreting the phenotypic consequences of genetic manipulations

Angiotensin II activates Na+-independent Cl--HCO3- exchange in ventricular myocardium.

The effect of angiotensin II (Ang II) on the activity of the cardiac Na+-independent Cl--HCO3- exchanger (anionic exchanger [AE]) was explored in cat papillary muscles. pHi was measured by epifluorescence with BCECF-AM. Ang II (500 nmol/L) induced a 5-(N-ethyl-N-isopropyl)amiloride-sensitive increase in pHi in the absence of external HCO3- (HEPES buffer), consistent with its stimulatory action on Na+-H+ exchange (NHE). This alkalinizing effect was not detected in the presence of a CO2-HCO3- buffer (pHi 7.07+/-0.02 and 7.08+/-0.02 before and after Ang II, respectively; n=17). Moreover, in Na+-free HCO3--buffered medium, in which neither NHE nor Na+-HCO3- cotransport are acting, Ang II decreased pHi, and this effect was canceled by previous treatment with SITS. These findings suggested that the Ang II-induced activation of NHE was masked, in the presence of the physiological buffer, by a HCO3--dependent acidifying mechanism, probably the AE. This hypothesis was confirmed on papillary muscles bathed with HCO3- buffer that were first exposed to 1 micromol/L S20787, a specific inhibitor of AE activity in cardiac tissue, and then to 500 nmol/L Ang II (n=4). Under this condition, Ang II increased pHi from 7.05+/-0.05 to 7.22+/-0.05 (P<.05). The effect of Ang II on AE activity was further explored by measuring the velocity of myocardial pHi recovery after the imposition of an intracellular alkali load in a HCO3--containing solution either with or without Ang II. The rate of myocardial pHi recovery was doubled in the presence of Ang II, suggesting a stimulatory effect on AE. The enhancement of the activity of this exchanger by Ang II was also detected when the AE activity was reversed by the removal of extracellular Cl- in a Na+-free solution. Under this condition, the rate of intracellular alkalinization increased from 0.053+/-0.016 to 0.108+/-0.026 pH unit/min (n=6, P<.05) in the presence of Ang II. This effect was canceled either by the presence of the AT1 receptor antagonist, losartan, or by the previous inhibition of protein kinase C with chelerythrine or calphostin C. The above results allow us to conclude that Ang II, in addition to its stimulatory effect on alkaline loading mechanisms, activates the AE in ventricular myocardium and that the latter effect is mediated by a protein kinase C-dependent regulatory pathway linked to the AT1 receptors.