Bacteroides forsythus has been described as a periodontopathogen and its presence in the subgingival plaque can lead to periodontal disease. Recently, a cysteine protease designated as prtH was ...isolated and characterized from B. forsythus ATCC 43037. The purpose of this study was to determine the prevalence and the association of the prtH gene of B. forsythus with periodontal disease. A total of 160 subgingival plaque samples were assayed with the polymerase chain reaction method using oligonucleotide primers targeting the prtH and the 16S rDNA genes of B. forsythus. Primers targeting the 16S rDNA gene of B. forsythus were used to determine the occurrence of the bacteria in the subgingival plaque samples at baseline. At baseline, B. forsythus was detected in 78 out of 86 (91%) diseased sites and 33 out of 74 (45%) healthy sites studied. Among the 86 diseased sites examined, 73 sites (85%) were colonized by the bacteria with the prtH genotype. In sites of the periodontally healthy, 7 out of 73 (10%) possessed B. forsythus with the prtH genotype. The results obtained suggested strong association of the prtH gene of B. forsythus with adult periodontitis. Although this bacterial species was detected from about half of the periodontally healthy samples, only a fraction of these subjects possess the bacteria strain with the prtH genetic subtype. We propose the use of the prtH gene as an alternative to the more widely used 16S rDNA gene of B. forsythus, for a more accurate determination of the prevalence of periodontal health and disease in epidemiological studies and clinical screening.
Individuals with type 2 diabetes are at increased risk of acquiring melioidosis, a disease caused by Burkholderia pseudomallei infection. Although up to half of melioidosis patients have underlying ...diabetes, the mechanisms involved in this increased susceptibility are unknown. We found that B. pseudomallei-infected PBMCs from dia-betic patients were impaired in IL-12p70 production, which resulted in decreased IFN-γ induction and poor bac-terial killing. The defect was specific to the IL-12-IFN- γ axis. Defective IL-12 production was also observed during Mycobacterium tuberculosis infection, in which diabetes is likewise known to be a strong risk factor. In contrast, IL-12 production in diabetic cells was not affected upon Salmonella enterica infection or in response to TLR2, -3, -4, and -5 ligands. Poor IL-12 production correlated with a deficiency in intracellular reduced glutathione (GSH) concentrations in diabetic patients. Addition of GSH or N-acetylcysteine to PBMCs selectively restored IL-12 and IFN- γ production and improved bacterial killing. Furthermore, the depletion of GSH in mice led to increased susceptibility to melioidosis, reduced production of IL-12p70, and poorer disease outcome. Our data thus establish a link between GSH deficiency in diabetes and increased susceptibility to melioidosis that may open up new therapeutic avenues to protect diabetic patients against some intracellular bacterial pathogens.
Catechol-
O-methyltransferase (COMT), an enzyme that metabolizes catecholamines, has recently been implicated in the modulation of pain. Our group demonstrated that human genetic variants of COMT are ...predictive for the development of Temporomandibular Joint Disorder (TMJD) and are associated with heightened experimental pain sensitivity Diatchenko, L, Slade, GD, Nackley, AG, Bhalang, K, Sigurdsson, A, Belfer, I, et al., Genetic basis for individual variations in pain perception and the development of a chronic pain condition, Hum Mol Genet 2005;14:135–43.. Variants associated with heightened pain sensitivity produce lower COMT activity. Here we report the mechanisms underlying COMT-dependent pain sensitivity. To characterize the means whereby elevated catecholamine levels, resulting from reduced COMT activity, modulate heightened pain sensitivity, we administered a COMT inhibitor to rats and measured behavioral responsiveness to mechanical and thermal stimuli. We show that depressed COMT activity results in enhanced mechanical and thermal pain sensitivity. This phenomenon is completely blocked by the nonselective β-adrenergic antagonist propranolol or by the combined administration of selective β
2- and β
3-adrenergic antagonists, while administration of β
1-adrenergic, α-adrenergic, or dopaminergic receptor antagonists fail to alter COMT-dependent pain sensitivity. These data provide the first direct evidence that low COMT activity leads to increased pain sensitivity via a β
2/3-adrenergic mechanism. These findings are of considerable clinical importance, suggesting that pain conditions resulting from low COMT activity and/or elevated catecholamine levels can be treated with pharmacological agents that block both β
2- and β
3-adrenergic receptors.
The beta sub(3)-adrenergic receptor ( beta sub(3)AR) is an essential regulator of metabolic and endocrine functions. A major cellular and clinically significant consequence of beta sub(3)AR ...activation is the substantial elevation in interleukin-6 (IL-6) levels. Although the beta sub(3)AR-dependent regulation of IL-6 expression is well established, the cellular pathways underlying this regulation have not been characterized. Using a novel method of homogenous reporters, we assessed the pattern of activation of 43 transcription factors in response to the specific beta sub(3)AR agonist CL316243 in adipocytes, cells that exhibit the highest expression of beta sub(3)ARs. We observed a unique and robust activation of the CRE-response element, suggesting that IL-6 transcription is regulated via the G sub(s)-protein/cAMP/protein kinase A (PKA) but not nuclear factor kappa B (NF-kappaB) pathway. However, pretreatment of adipocytes with pharmacologic inhibitors of PKA pathway failed to block beta sub(3)AR-mediated IL-6 up-regulation. Additionally, stimulation of adipocytes with the exchange protein directly activated by cAMP (Epac) agonist did not induce IL-6 expression. Instead, the beta sub(3)AR-mediated transcription of IL-6 required activation of both the p38 and PKC pathways. Western blot analysis further showed that transcription factors CREB and ATF-2 but not ATF-1 were activated in a p38- and PKC-dependent manner. Collectively, our results suggest that while stimulation of the beta sub(3)AR leads to a specific activation of CRE-dependent transcription, there are several independent cellular pathways that converge at the level of CRE-response element activation, and in the case of IL-6 this activation is mediated by p38 and PKC but not PKA pathways.
The β
3-adrenergic receptor (β
3AR) is an essential regulator of metabolic and endocrine functions. A major cellular and clinically significant consequence of β
3AR activation is the substantial ...elevation in interleukin-6 (IL-6) levels. Although the β
3AR-dependent regulation of IL-6 expression is well established, the cellular pathways underlying this regulation have not been characterized. Using a novel method of homogenous reporters, we assessed the pattern of activation of 43 transcription factors in response to the specific β
3AR agonist CL316243 in adipocytes, cells that exhibit the highest expression of β
3ARs. We observed a unique and robust activation of the CRE-response element, suggesting that IL-6 transcription is regulated
via the G
s-protein/cAMP/protein kinase A (PKA) but not nuclear factor kappa B (NF-κB) pathway. However, pretreatment of adipocytes with pharmacologic inhibitors of PKA pathway failed to block β
3AR-mediated IL-6 up-regulation. Additionally, stimulation of adipocytes with the exchange protein directly activated by cAMP (Epac) agonist did not induce IL-6 expression. Instead, the β
3AR-mediated transcription of IL-6 required activation of both the p38 and PKC pathways. Western blot analysis further showed that transcription factors CREB and ATF-2 but not ATF-1 were activated in a p38- and PKC-dependent manner. Collectively, our results suggest that while stimulation of the β
3AR leads to a specific activation of CRE-dependent transcription, there are several independent cellular pathways that converge at the level of CRE-response element activation, and in the case of IL-6 this activation is mediated by p38 and PKC but not PKA pathways.
Activation of the beta(2) adrenergic receptor (beta(2)AR) located on macrophages has been reported to possess anti-inflammatory properties, inhibiting nuclear factor kappaB (NF-kappaB) activation and ...cytokine production induced by pro-inflammatory stimuli. Here, we show that activation of the beta(2)AR in the absence of pro-inflammatory stimuli produced up to an 80- and 8-fold increase in IL-1beta and IL-6 transcripts, respectively, in the RAW 264.7 murine macrophage cell line. This increase in mRNA expression was accompanied by a significant increase in IL-1beta and IL-6 protein production. Pre-treatment of RAW cells with pharmacological inhibitors of protein kinase A (PKA) or NF-kappaB pathway failed to block this cytokine increase. Instead, the beta(2)AR-mediated increase in cytokines required activation of both the B-raf-ERK1/2 and p38 pathways. Treatment of RAW cells with the exchange protein directly activated by cAMP (EPAC) agonist also resulted in the up-regulation of IL-1beta and IL-6 transcripts. Examination of the main transcription factors downstream of the ERK1/2 and p38 signaling revealed that beta(2)AR activation resulted in the stimulation of CRE-, but not C/EBPbeta-, ETS-, or NF-kappaB-dependent transcription. Western blot analysis further showed that among the transcription factors which recognize the CRE-binding site, ATF-1 and ATF-2 but not CREB proteins were phosphorylated in an ERK1/2- and p38-dependent manner. Collectively, these results demonstrate that beta(2)ARs possess pro-inflammatory properties and that their activation leads to IL-1beta and IL-6 production through ERK1/2- and p38-dependent activation of ATF-1 and ATF-2 transcription factors.
Activation of the beta 2 adrenergic receptor ( beta 2AR) located on macrophages has been reported to possess anti-inflammatory properties, inhibiting nuclear factor mu B (NF- mu B) activation and ...cytokine production induced by pro-inflammatory stimuli. Here, we show that activation of the beta 2AR in the absence of pro-inflammatory stimuli produced up to an 80- and 8-fold increase in IL-1 beta and IL-6 transcripts, respectively, in the RAW 264.7 murine macrophage cell line. This increase in mRNA expression was accompanied by a significant increase in IL-1 beta and IL-6 protein production. Pre-treatment of RAW cells with pharmacological inhibitors of protein kinase A (PKA) or NF- mu B pathway failed to block this cytokine increase. Instead, the beta 2AR-mediated increase in cytokines required activation of both the B-raf-ERK1/2 and p38 pathways. Treatment of RAW cells with the exchange protein directly activated by cAMP (EPAC) agonist also resulted in the up-regulation of IL-1 beta and IL-6 transcripts. Examination of the main transcription factors downstream of the ERK1/2 and p38 signaling revealed that beta 2AR activation resulted in the stimulation of CRE-, but not C/EBP beta -, ETS-, or NF- mu B-dependent transcription. Western blot analysis further showed that among the transcription factors which recognize the CRE-binding site, ATF-1 and ATF-2 but not CREB proteins were phosphorylated in an ERK1/2- and p38-dependent manner. Collectively, these results demonstrate that beta 2ARs possess pro-inflammatory properties and that their activation leads to IL-1 beta and IL-6 production through ERK1/2- and p38-dependent activation of ATF-1 and ATF-2 transcription factors.
Activation of the β
2 adrenergic receptor (β
2AR) located on macrophages has been reported to possess anti-inflammatory properties, inhibiting nuclear factor κB (NF-κB) activation and cytokine ...production induced by pro-inflammatory stimuli. Here, we show that activation of the β
2AR in the absence of pro-inflammatory stimuli produced up to an 80- and 8-fold increase in IL-1β and IL-6 transcripts, respectively, in the RAW 264.7 murine macrophage cell line. This increase in mRNA expression was accompanied by a significant increase in IL-1β and IL-6 protein production. Pre-treatment of RAW cells with pharmacological inhibitors of protein kinase A (PKA) or NF-κB pathway failed to block this cytokine increase. Instead, the β
2AR-mediated increase in cytokines required activation of both the B-raf-ERK1/2 and p38 pathways. Treatment of RAW cells with the exchange protein directly activated by cAMP (EPAC) agonist also resulted in the up-regulation of IL-1β and IL-6 transcripts. Examination of the main transcription factors downstream of the ERK1/2 and p38 signaling revealed that β
2AR activation resulted in the stimulation of CRE-, but not C/EBPβ-, ETS-, or NF-κB-dependent transcription. Western blot analysis further showed that among the transcription factors which recognize the CRE-binding site, ATF-1 and ATF-2 but not CREB proteins were phosphorylated in an ERK1/2- and p38-dependent manner. Collectively, these results demonstrate that β
2ARs possess pro-inflammatory properties and that their activation leads to IL-1β and IL-6 production through ERK1/2- and p38-dependent activation of ATF-1 and ATF-2 transcription factors.
Aim: The aim of this study was to determine the prevalence and the structure of the leukotoxin promoter region of Actinobacillus actinomycetemcomitans in an ethnic Chinese population.
Method: ...Subgingival plaque samples were collected from 42 patients with moderate to advanced periodontitis and 50 periodontally healthy patients. A. actinomycetemcomitans was detected directly from the crude subgingival plaque by PCR using leukotoxin gene specific primers. The presence of A. actinomycetemcomitans was determined by a single 285 bp PCR amplicon.
Results: A. actinomycetemcomitans was found to be present in the subgingival plaque of 68 out of a total of 92 patients examined (74%). 29 out of the 42 periodontitis patients tested were carriers of A. actinomycetemcomitans (69%). Among the periodontally healthy patients studied, 39 out of 50 subjects possessed the bacteria (78%). PCR analysis of the promoter region of the ltx operon revealed that none of the 42 moderate to advanced periodontitis patients examined harboured A.actinomycetemcomitans strains with the JP2‐like promoter of the ltx operon, known to enhance leukotoxin expression. 2 out of the 27 advanced periodontitis patients clinically diagnosed as suffering from rapidly progressive periodontitis were found to be carriers of the mildly toxic strain of A. actinomycetemcomitans with the characteristic 652‐like promoter.
Conclusions: The high prevalence of A. actinomycetemcomitans, regardless of whether the subgingival samples were analysed from patients with healthy or diseased periodontium suggests that this bacterial species is part of the normal oral flora of ethnic Chinese. Our preliminary results also suggested that subjects who harboured the mildly toxic strain of A. actinomycetemcomitans were potentially susceptible to aggressive forms of periodontitis.
Zusammenfassung
Das Ziel dieser Studie war es, in einer ethnischen Population von Chinesen die Prävalenz von Actinobacillus actinomycetemcomitans und die Struktur der Leukotoxin‐Promoterregion zu bestimmen. Von 42 Patienten mit moderater bis fortgeschrittener Parodontitis und 50 parodontal gesunden Patienten wurden subgingivale Plaqueproben entnommen. A. actinomycetemcomitans wurde direkt in der unbehandelten subgingivalen Plaque durch PCR unter Verwendung eines Leukotoxingen‐spezifischen Primers nachgewiesen. Das Vorhandensein von A. actinomycetemcomitans wurde mittels eines einzigen 285 bp‐PCR‐Amplikons bestimmt. Es wurde A. actinomycetemcomitans bei 68 von 92 untersuchten Patienten (74%) vorgefunden. 29 von 42 getesteten Parodontitispatienten waren Träger von A. actinomycetemcomitans (69%). Unter den Studierten parodontal gesunden Patienten besaßen 39 von 50 Personen das Bakterium (78%). Die PCR‐Analyse der Promoterregion des ltx‐Operons zeigte, dass keiner der 42 untersuchten Patienten mit moderater bis fortgeschrittener Parodontitis den A. actinomycetemcomitans mit dem JP2‐ähnlichen Promoter des ltx‐Operons, welches die Leukotoxinexpression verstärkt, besaß. Bei 2 der 27 Patienten mit fortgeschrittener Parodontitis wurde klinisch eine rasch fortschreitende Parodontitis diagnostiziert und es wurde der mit geringer Toxizität versehene Stamm des A. actinomycetemcomitans mit dem charakteristischen 652‐ähnlichen Promoter vorgefunden. Bedingt durch die hohe Prävalenz von A. actinomycetemcomitans unabhängig davon, ob die Proben von Patienten mit gesundem oder erkranktem Parodontium stammen, lässt sich annehmen, dass diese Bakterienspezies bei ethnischen Chinesen ein Teil normalen Mundflora ist. Unsere vorläufigen Resultate lassen auch annehmen, dass Personen, die den mit geringer Toxizität versehenen Stamm des A. actinomycetemcomitans tragen eine potentielle Anfälligkeit für aggressive Formen der Parodontitis besitzen.
Résumé
Le but de l’étude présente a été de déterminer la fréquence globale et la structure de la région promoteur de leukotoxine de l’Actinobacillus actinomycetemcomitans (A.a.) dans une population chinoise. Des échantillons de plaque dentaire sous‐gingivale ont été prélevés chez 42 patients avec parodontite modérée à avancée et chez 50 patients sains. L’A.a. a été détecté directement dans la plaque sous‐gingivale par PCR en utilisant les sites spécifiques de gènes leukotoxines. La présence de l’A.a. a été déterminée par un amplicon PCR de 285 bp. L’A.a. a été décelé dans la plaque sous‐gingivale de 68 des 92 patients examinés (74%). 29 des 42 patients avec parodontite ont été reconnus comme porteurs d’A.a. (69%). Parmi les patients sains étudiés, 39 des 50 sujets étaient porteurs de la bactérie (78%). L’analyse PCR de la région promoteur de operon ltx a révélé que des 42 patients avec parodontite modéréà avancée aucun n’avaient de souche A.a. avec le promoteur ressemblant au JP2 de l’operon ltx, reconnu pour acroître la leukotoxine. 2 des 27 patients avec parodontit avancée souffraient d’une parodontite progressant rapidement et étaient porteurs d’une souche moyennement toxique d’A.a. avec la caractéristique du promoteur ressemblant au 652. La fréquence globale importante d’A.a., sans tenir compte si les échantillons sous‐gingivaux ont été analysés de patients avec un parodonte sain ou malade, suggère que ces espèces bactériennes font partie de la flore buccale normale de l’ethnie chinoise. Ces résultats indiquent également que les porteurs de la souche peu toxique d’A.a. seraient potentiellement susceptibles à des formes de parodontite agressive.
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