Continuous positive airway pressure for obstructive sleep apnea in children.

QUESTION: A 12-year-old child underwent adenotonsillectomy for treatment of obstructive sleep apnea (OSA) but continues to snore at night and struggles with attentiveness at school. The child's parent uses a continuous positive airway pressure (CPAP) machine at night and wonders whether the same therapy could be used in children. ANSWER: Unlike in adults, pediatric OSA is commonly related to adenotonsillar hypertrophy and is often amenable to treatment with adenotonsillectomy. As an alternative to surgery or in cases of postsurgical persistence of OSA, CPAP has shown effectiveness in improving both polysomnographic parameters and daytime neurobehavioural symptoms in children with OSA. Adherence to CPAP therapy is a challenge in children and requires parental education and special considerations such as a mask acclimatization period.

A cone-beam computed tomographic assessment of the proximity of the maxillary canine and posterior teeth to the maxillary sinus floor: Lessons from 4778 roots.

INTRODUCTION: When tooth roots protrude into the maxillary sinus, apical root resorption and tipping may occur during horizontal tooth movement across the sinus floor. Three-dimensional cone-beam computed tomography (CBCT) images may provide detailed information without distortion and overlap. We evaluated the relationships between the maxillary tooth root apices and the maxillary sinus floor using CBCT. METHODS: We evaluated 4778 roots from 76 men (aged 27.6 ± 10.4 [mean ± standard deviation] years; range, 18-69 years), and 225 women (aged 30.4 ± 12.0 years; range, 18-68 years). The positional relationships between the maxillary tooth root apices, including the canine, premolar (first and/or second), and molar (first and/or second), and the inferior wall of the maxillary sinus were comprehensively evaluated on 2 cross-sectional CBCT images (ie, the sagittal and coronal planes). These distances were measured in both images simultaneously. RESULTS: The sagittal plane distances were significantly larger than coronal plane distances, except for the distobuccal root of the first molar. Pearson correlation test revealed a significant negative correlation between the distance and the patient's age. CONCLUSIONS: Our study provides valuable information for planning orthodontic tooth movement through the maxillary sinus, which may help to predict the occurrence and severity of root resorption.

Alveolar bone morphology in patients with a unilateral palatally displaced maxillary lateral incisor: A cone-beam computed tomography study.

INTRODUCTION: Lingual displacement of the maxillary anterior teeth is 1 of the most common forms of malocclusion. The labial alveolar bone is thinner for the maxillary lateral incisor than for the central incisor and canine; however, the alveolar bone width at the actual position of the maxillary lateral incisor has not been examined. We investigated the morphologic characteristics of the alveolar bone around palatally displaced maxillary lateral incisors using cone-beam computed tomography and a split-mouth model. METHODS: Twenty-seven patients with a unilateral palatally displaced maxillary lateral incisor were included. Axial, sagittal, and horizontal measurements were recorded at 3 levels (ie, 25%, 50%, and 75% of the root length) using cone-beam computed tomography. All obtained data were statistically analyzed using paired t tests. RESULTS: The labial alveolar bone width at 25% of root length was significantly lesser on the affected side. At all 3 levels, the distance between a line tangential to the labial alveolar bone of the central incisor and canine and the position of the labial alveolar bone of the lateral incisor was significantly greater on the affected side. At 50% and 75% of root length, the horizontal distance between the posterior nasal spine and the labial alveolar bone of the lateral incisor was significantly lesser on the affected side. CONCLUSIONS: Palatal displacement of maxillary lateral incisors is significantly associated with decreased alveolar bone width at the apical level and asymmetry. However, a further elaborate investigation is necessary to determine the clinical relevance of the study.

Intermittent hypoxia and hypercapnia induces inhibitor of nuclear factor-κB kinase subunit ß-dependent atherosclerosis in pulmonary arteries.

Clinical studies have shown that obstructive sleep apnea (OSA) increases atherosclerosis risk. The inflammation, especially mediated by the macrophages via nuclear factor-κB (NF-κB), has been speculated to contribute to atherogenicity in OSA patients. Inhibitor of NF-κB kinase-ß (IKKß) is an essential element of the NF-κB pathway and is linked to atherosclerosis. We previously reported that atherosclerosis was accelerated in pulmonary artery (PA) but not in aorta when low-density lipoprotein receptor knockout (Ldlr-/-) mice were exposed to intermittent hypoxia/hypercapnia (IHH), a surrogate for recurrent upper-airway obstruction. Therefore, we hypothesized that IKKß-dependent NF-κB activation in monocytes and macrophages plays a role in IHH-induced PA atherosclerosis. To test this hypothesis, myeloid restricted IKKß deletion (IkkßΔMye) or control (IkkßF/F) mice were crossed with Ldlr-/- mice to generate double-knockout mice. Then, the mice were exposed to IHH or room air (Air) on high-fat diet for 8 or 16 wk. Lesions of PA and aorta were examined in IkkßΔMye;Ldlr-/- and IkkßF/F;Ldlr-/- male mice under IHH vs. Air. The results revealed that IKKß deletion abolished IHH-induced PA atherosclerosis after 8-wk exposure but not after 16-wk exposure (8 wk: IkkßF/F;Ldlr-/-, IHH 13.5 ± 1.4 vs. Air 5.7 ± 0.7%, P < 0.01; IkkßΔMye;Ldlr-/-, IHH 7.4 ± 1.9% vs. Air 4.6 ± 1.3%, P = 0.24). Both IKKß deletion and IHH had no effects on atherosclerosis in the aorta. Our findings demonstrate that IKKß-dependent NF-κB activity in myeloid-lineage cells plays a critical role in IHH-induced PA atherosclerosis at the early stage.

Exploring miRNA-mRNA regulatory network in cardiac pathology in Na+/H+ exchanger isoform 1 transgenic mice.

Numerous studies have demonstrated that Na+/H+ exchanger isoform 1 (NHE1) is elevated in myocardial diseases and its effect is detrimental. To better understand the involvement of NHE1, we have previously studied cardiac-specific NHE1 transgenic mice and shown that these mice develop cardiac hypertrophy, interstitial fibrosis, and cardiac dysfunction. The purpose of current study was to identify microRNAs and their mRNA targets involved in NHE1-mediated cardiac injury. An unbiased high-throughput sequencing study was performed on both microRNAs and mRNAs. RNA sequencing showed that differentially expressed genes were enriched in hypertrophic cardiomyopathy pathway by Kyoto Encyclopedia of Genes and Genomes annotation in NHE1 transgenic hearts. These genes were classified as contraction defects (e.g., Myl2, Myh6, Mybpc3, and Actb), impaired intracellular Ca2+ homeostasis (e.g., SERCA2a, Ryr2, Rcan1, and CaMKII delta), and signaling molecules for hypertrophic cardiomyopathy (e.g., Itga/b, IGF-1, Tgfb2/3, and Prkaa1/2). microRNA sequencing revealed that 15 microRNAs were differentially expressed (2-fold, P < 0.05). Six of them (miR-1, miR-208a-3p, miR-199a-5p, miR-21-5p, miR-146a-5p, and miR-30c-5p) were reported to be related to cardiac pathological functions. The integrative analysis of microRNA and RNA sequencing data identified several crucial microRNAs including miR-30c-5p, miR-199a-5p, miR-21-5p, and miR-34a-5p as well as 10 of their mRNA targets that may affect the heart via NFAT hypertrophy and cardiac hypertrophy signaling. Furthermore, important microRNAs and mRNA targets were validated by quantitative PCR. Our study comprehensively characterizes the expression patterns of microRNAs and mRNAs, establishes functional microRNA-mRNA pairs, elucidates the potential signaling pathways, and provides novel insights on the mechanisms underlying NHE1-medicated cardiac injury.

Intermittent Hypoxia and Hypercapnia Accelerate Atherosclerosis, Partially via Trimethylamine-Oxide.

Obstructive sleep apnea (OSA) is a common disorder characterized by intermittent hypoxia and hypercapnia (IHC) during sleep. OSA has been shown to be a risk factor for atherosclerosis, but the relation of IHC to the induction or progression of atherosclerosis is not well understood. To dissect the mechanisms involved, we compared atherosclerotic lesion formation in two mouse models, i.e., apolipoprotein E (ApoE) and low density lipoprotein receptor (Ldlr)-deficient mice, with or without IHC exposure. Ten-week-old ApoE-/- or Ldlr-/- mice were fed a high-fat diet for 4 or 8 weeks while being exposed to IHC for 10 hours/day or room air (RA) for 24 hours/day. En face lesions of the aorta, aortic arch, and pulmonary artery (PA) were examined. Moreover, 3,3-dimethyl-1-butanol (DMB), an inhibitor of microbial trimethylamine (TMA) production, was used to determine the contribution of TMA-oxide (TMAO) to IHC-induced atherosclerosis. Eight weeks of IHC exposure expedited the formation of atherosclerosis in both the PA and aortic arch of ApoE-/- mice, but only in the PA of Ldlr-/- mice (ApoE-/- PA 8 wk, IHC 35.4 ± 1.9% versus RA 8.0 ± 2.8%, P < 0.01). The atherosclerotic lesions evolved faster and to a more severe extent in ApoE-/- mice as compared with Ldlr-/- mice (PA IHC 8 wk, ApoE-/- 35.4 ± 1.9% versus Ldlr-/- 8.2 ± 1.5%, P < 0.01). DMB significantly attenuated but did not totally eliminate IHC-induced PA atherosclerosis. Our findings suggest that IHC, a hallmark of OSA, accelerates the progression of atherosclerosis in the aorta and especially in the PA. This process is partly inhibited by DMB, demonstrating that microbial metabolites may serve as therapeutic targets for OSA-induced atherosclerosis.

Synaptic dysfunction connects autism spectrum disorder and sleep disturbances: A perspective from studies in model organisms.

Sleep disturbances (SD) accompany many neurodevelopmental disorders, suggesting SD is a transdiagnostic process that can account for behavioral deficits and influence underlying neuropathogenesis. Autism Spectrum Disorder (ASD) comprises a complex set of neurodevelopmental conditions characterized by challenges in social interaction, communication, and restricted, repetitive behaviors. Diagnosis of ASD is based primarily on behavioral criteria, and there are no drugs that target core symptoms. Among the co-occurring conditions associated with ASD, SD are one of the most prevalent. SD often arises before the onset of other ASD symptoms. Sleep interventions improve not only sleep but also daytime behaviors in children with ASD. Here, we examine sleep phenotypes in multiple model systems relevant to ASD, e.g., mice, zebrafish, fruit flies and worms. Given the functions of sleep in promoting brain connectivity, neural plasticity, emotional regulation and social behavior, all of which are of critical importance in ASD pathogenesis, we propose that synaptic dysfunction is a major mechanism that connects ASD and SD. Common molecular targets in this interplay that are involved in synaptic function might be a novel avenue for therapy of individuals with ASD experiencing SD. Such therapy would be expected to improve not only sleep but also other ASD symptoms.

Deaths associated with pandemic (H1N1) 2009 among children, Japan, 2009-2010.

To clarify the cause of deaths associated with pandemic (H1N1) 2009 among children in Japan, we retrospectively studied 41 patients <20 years of age who had died of pandemic (H1N1) 2009 through March 31, 2010. Data were collected through interviews with attending physicians and chart reviews. Median age of patients was 59 months; one third had a preexisting condition. Cause of death was categorized as unexpected cardiopulmonary arrest for 15 patients, encephalopathy for 15, and respiratory failure for 6. Preexisting respiratory or neurologic disorders were more frequent in patients with respiratory failure and less frequent in patients with unexpected cardiopulmonary arrest. The leading causes of death among children with pandemic (H1N1) 2009 in Japan were encephalopathy and unexpected cardiopulmonary arrest. Deaths associated with respiratory failure were infrequent and occurred primarily among children with preexisting conditions. Vaccine use and public education are necessary for reducing influenza-associated illness and death.

Unilateral maxillary central incisor root resorption after orthodontic treatment for Angle Class II, division 1 malocclusion with significant maxillary midline deviation: A possible correlation with root proximity to the incisive canal.

Root resorption can be caused by several factors, including contact with the cortical bone. Here we report a case involving a 21-year-old female with Angle Class II, division 1 malocclusion who exhibited significant root resorption in the maxillary right central incisor after orthodontic treatment. The patient presented with significant left-sided deviation of the maxillary incisors due to lingual dislocation of the left lateral incisor and a Class II molar relationship. Cephalometric analysis demonstrated a Class I skeletal relationship (A pointnasion-B point, 2.5°) and proclined maxillary anterior teeth (upper incisor to sella-nasion plane angle, 113.4°). The primary treatment objectives were the achievement of stable occlusion with midline agreement between the maxillary and mandibular dentitions and appropriate maxillary anterior tooth axes and molar relationship. A panoramic radiograph obtained after active treatment showed significant root resorption in the maxillary right central incisor; therefore, we performed cone-beam computed tomography, which confirmed root resorption along the cortical bone around the incisive canal. The findings from this case, where different degrees of root resorption were observed despite comparable degrees of orthodontic movement in the bilateral maxillary central incisors, suggest that the incisive canal could be an inducing factor for root resorption. However, further investigation is necessary to confirm this assumption.

Cone-beam computed tomography-based diagnosis and treatment simulation for a patient with a protrusive profile and a gummy smile.

For patients with bimaxillary protrusion, significant retraction and intrusion of the anterior teeth are sometimes essential to improve the facial profile. However, severe root resorption of the maxillary incisors occasionally occurs after treatment because of various factors. For instance, it has been reported that approximation or invasion of the incisive canal by the anterior tooth roots during retraction may cause apical root damage. Thus, determination of the position of the maxillary incisors is key for orthodontic diagnosis and treatment planning in such cases. Cone-beam computed tomography (CBCT) may be useful for simulating the post-treatment position of the maxillary incisors and surrounding structures in order to ensure safe teeth movement. Here, we present a case of Class II malocclusion with bimaxillary protrusion, wherein apical root damage due to treatment was minimized by pretreatment evaluation of the anatomical structures and simulation of the maxillary central incisor movement using CBCT. Considerable retraction and intrusion of the maxillary incisors, which resulted in a significant improvement in the facial profile and smile, were achieved without severe root resorption. Our findings suggest that CBCT-based diagnosis and treatment simulation may facilitate safe and dynamic orthodontic tooth movement, particularly in patients requiring maximum anterior tooth retraction.

Intermittent hypoxia induces murine macrophage foam cell formation by IKK-ß-dependent NF-κB pathway activation.

Obstructive sleep apnea (OSA) is a common sleep disorder characterized by intermittent hypoxia (IH). Clinical studies have previously shown that OSA is an independent risk factor for atherosclerosis. Atherogenicity in OSA patients has been assumed to be associated with the NF-κB pathways. Although foam cells are considered to be a hallmark of atherosclerosis, how IH as in OSA affects their development has not been fully understood. Therefore, we hypothesized that IH induces macrophage foam cell formation through NF-κB pathway activation. To test this hypothesis, peritoneal macrophages collected from myeloid-restricted IKK-ß-deleted mice were incubated with native LDL and exposed to either IH or normoxia. After exposure, NF-κB pathway activity and intracellular cholesterol were measured. In control macrophages, IH significantly increased NF-κB pathway activity by 93% compared with normoxia (P < 0.05). However, such response to IH was diminished by IKK-ß deletion (increased by +31% compared with normoxia; P = 0.64), suggesting that IKK-ß is critical for IH-induced NF-κB pathway activation. Likewise, in control macrophages, total cholesterol was increased in IH compared with normoxia (65.7 ± 3.8 µg/mg cellular protein and 53.2 ± 1.2, respectively; P < 0.05). However, this IH-induced foam cell formation was disappeared when IKK-ß was deleted (52.2 ± 1.2 µg/mg cellular protein for IH and 46.3 ± 1.7 for normoxia; P = 0.55). This IH-mediated effect still existed in macrophages without LDL receptor. Taken together, our findings show that IH activates the IKK-ß-dependent NF-κB pathway and that this, in turn, induces foam cell formation in murine macrophages.

Significant Efficacy of Tramadol/Acetaminophen in Elderly Patients with Chronic Low Back Pain Uncontrolled by NSAIDs: An Observational Study.

Chronic low back pain (LBP) is a common condition and is generally treated using non-steroidal anti-inflammatory drug (NSAID); however, chronic NSAID use can decrease renal function. Tramadol, a weak opioid agonist, may improve chronic LBP and disability, while avoiding adverse effects such as gastrointestinal and renal toxicity. However, few studies have evaluated the short-term efficacy of opioids in Asian patients with chronic LBP. In this study, 24 patients with chronic LBP unresponsive to NSAIDs (10 men, 14 women; mean age, 65.1 ± 12.1 years) were prescribed tramadol/acetaminophen (37.5 mg/325 mg; four tablets daily) for 1 month. Then, the following parameters were assessed at baseline and after 1 week and 1 month of treatment: leg pain and LBP (Visual Analog Score [VAS]); activity of daily life (Roland-Morris Disability Questionnaire [RDQ]); and disability (Oswestry Disability Index [ODI]). Leg pain resolved within 1 week (p = 0.00093); however, LBP was relieved only at 1 month (p = 0.00034). The mean RDQ (p = 0.015) and ODI (p = 0.0032) scores were improved at 1 month. A total 41.6% of patients reported nausea and floating sensation beginning tramadol/acetaminophen treatment, and 12.5% (four patients) discontinued treatment as a result. LBP did not improve in 25% of patients administered tramadol/acetaminophen. Because this was an observational study, rather than a comparative study, further investigation is needed to evaluate the long-term efficacy of tramadol/acetaminophen in elderly patients with chronic LBP unresponsive to NSAIDs.

Infiltration of trace metal ions in the oral mucosa of a rat analyzed using SRXRF, XAFS, and ICP-MS.

Although the accumulation and distribution of metals from metallic orthodontic appliances in the oral mucosa have been studied extensively, they remain unclear because their concentration is quite low. In this study, metal specimens (Ni, Ni-Ti, and Co-Cr) were sutured in the unilateral oral mucosa of rats, and the distribution of the eluted elements in the mucosal tissue was estimated using inductively coupled plasma mass spectrometry (ICP-MS) and synchrotron radiation X-ray fluorescence analysis (SR-XRF). While the infiltrations of Ni, Co, and Cr into the oral mucosal connective tissue were observed with SR-XRF, significant increases were only found in Ni from the pure Ni group and Cr from the Co-Cr group. Furthermore, Ni and Co were estimated as hydrated ions while Cr was estimated in oxide form through X-ray absorption fine structure (XAFS) analysis.

Severe form of encephalopathy associated with 2009 pandemic influenza A (H1N1) in Japan.

BACKGROUND: Every year, an estimated 200-500 children in Japan develop influenza-associated encephalopathy (IAE), and 10-30% of these children die. OBJECTIVE: To clarify the clinical features of a severe form of acute encephalopathy seen with 2009 pandemic influenza A (H1N1). STUDY DESIGN: This retrospective survey examined 20 children with acute encephalopathy associated with the 2009 pandemic influenza A (H1N1) who died or were in a prolonged deep coma with a flat electroencephalogram tracing and loss of spontaneous respiration. We obtained demographic, clinical, laboratory, and neuroimaging data through interviews with the attending physicians and chart reviews. RESULTS: Subjects were 13 boys and seven girls. Their median age was 45 (range 11-200) months. Five patients had one or more pre-existing conditions. Acute encephalopathy developed within 2 days after influenza onset in 16 patients. As the initial neurological symptom, delirious behavior was seen in six children, and brief seizures in six. Eighteen patients were comatose within 6h of the onset of encephalopathy. Marked brain edema on computed tomography (CT) was seen in all but one patient. Brainstem lesions on CT were recognized in 12 patients. Sixteen patients died 0-45 (median 2.5) days after the onset of acute encephalopathy, and the others remained in deep comas without spontaneous respiration. CONCLUSIONS: The clinical course of the patients was characterized by an onset with mild neurological symptoms and rapid deterioration of consciousness into coma. Head CT revealed marked cerebral edema, often associated with brainstem lesions.

Intermittent hypoxia and hypercapnia induce pulmonary artery atherosclerosis and ventricular dysfunction in low density lipoprotein receptor deficient mice.

Patients with obstructive sleep apnea, who experience episodic hypoxia and hypercapnia during sleep, often demonstrate increased inflammation, oxidative stress, and dyslipidemia. We hypothesized that sleep apnea patients would be predisposed to the development of atherosclerosis. To dissect the mechanisms involved, we developed an animal model in mice whereby we expose mice to intermittent hypoxia/hypercapnia (IHH) in normobaric environments. Two- to three-month-old low-density lipoprotein receptor deficient (Ldlr(-/-)) mice were fed a high-fat diet for 8 or 16 wk while being exposed to IHH for either 10 h/day or 24 h/day. Plasma lipid levels, pulmonary artery and aortic atherosclerotic lesions, and cardiac function were then assayed. Surprisingly, atherosclerosis in the aorta of IHH mice was similar compared with controls. However, in IHH mice, atherosclerosis was markedly increased in the trunk and proximal branches of the pulmonary artery of exposed mice; even though plasma cholesterol and triglycerides were lower than in controls. Hemodynamic analysis revealed that right ventricular maximum pressure and isovolumic relaxation constant were significantly increased in IHH exposed mice and left ventricular % fractional shortening was reduced. In conclusion, 1) Intermittent hypoxia/hypercapnia remarkably accelerated atherosclerotic lesions in the pulmonary artery of Ldlr(-/-) mice and 2) increased lesion formation in the pulmonary artery was associated with right and left ventricular dysfunction. These findings raise the possibility that patients with obstructive sleep apnea may be susceptible to atherosclerotic disease in the pulmonary vasculature, an observation that has not been previously recognized.

Validation of pediatric index of mortality 2 (PIM2) in a single pediatric intensive care unit in Japan.

OBJECTIVE: The Pediatric Index of Mortality 2 (PIM2), one of the key mortality prediction models for children in intensive care units, has not been validated in Japan. The purpose of this study was to validate the performance of PIM2 in a population of patients admitted to one pediatric intensive care unit (PICU) in Japan. METHODS: This was a prospective cohort study involving consecutive patients admitted to the largest multidisciplinary PICU in Japan between 1 January 2008 and 31 December 2010. There were no interventions. RESULTS: A total of 2,536 patients were included in this study of whom 67 (2.6%) died. Discrimination between survival and death assessed by the area under the receiver operating characteristic curve was 0.92 [95% confidence interval (CI) 0.89-0.96]. Calibration across the five risk intervals according to the Hosmer-Lemeshow goodness-of-fit test showed a chi-square value of 4.8 (df = 5, p = 0.44). The standardized mortality ratio for the whole population was 0.77 (95% CI 0.59-0.96). CONCLUSIONS: At the largest PICU center in Japan, the PIM2 was found to have excellent discriminatory power and good calibration, although it over-predicted deaths. Based on these results, PIM2 can be used as a good prediction model for pediatric mortality, which is a tool used to assess the overall quality of care in a PICU.

Detection of submicroscopic disease in the bone marrow and unaffected testis of a child with T-cell acute lymphoblastic leukemia who experienced "isolated" testicular relapse.

Testicular relapse has an impact on the prognosis of boys with acute lymphoblastic leukemia (ALL). Because isolated testicular relapse often precedes hematological relapse, systemic therapy is required in addition to local therapy. However, a rationale for the use of a combination of systemic chemotherapy and local therapy is unclear. A 12-year-old boy with T-ALL suffered from isolated testicular relapse at 27 months after diagnosis. He was successfully treated with systemic chemotherapy with orchiectomy and prophylactic irradiation to the contralateral testis. We retrospectively estimated the minimal residual disease in the bone marrow (BM) and the testis by detection of clone-specific T-cell receptor rearrangement of leukemic cells. We detected leukemic cells in the affected testis at relapse, as well as in the BM at initial diagnosis. In addition, we confirmed submicroscopic disease in the unaffected testis and the BM at relapse. We conclude that molecular analysis could reveal the submicroscopic disease in the patient with apparently isolated testicular relapse. This finding may provide a rationale for intensified systemic treatment of patients with isolated testicular relapse.

A novel tumor necrosis factor alpha-responsive CCAAT/enhancer binding protein site regulates expression of the cartilage-derived retinoic acid-sensitive protein gene in cartilage.

OBJECTIVE: Inflammatory processes in rheumatoid arthritis are primarily regulated by the cytokines tumor necrosis factor alpha (TNFalpha) and interleukin-1beta (IL-1beta). Previous studies in our laboratory have shown that IL-1beta represses expression of the cartilage characteristic genes, cartilage-derived retinoic acid-sensitive protein (cd-rap) and type II collagen (COL2A1); this mechanism of repression involves activation of a CCAAT/enhancer binding protein (c/EBP) site within promoter regions. The aim of this study was to investigate novel TNFalpha-mediated mechanisms that regulate the expression of cd-rap. METHODS: Rat chondrosarcoma cells were transiently transfected with complementary DNA constructs encoding cd-rap, in the presence of TNFalpha. The expression of c/EBPbeta, SOX9, and p300 in rat chondrosarcoma cells and primary human articular chondrocytes after treatment with TNFalpha was examined by reverse transcription-polymerase chain reaction and Western blotting. The effect of TNFalpha on endogenous binding of c/EBPbeta or SOX9 to the cd-rap promoter was examined by chromatin immunoprecipitation assays. RESULTS: We identified a new c/EBP binding site in the cd-rap promoter (from position -1059 bp to position -1046 bp). Binding of c/EBP to this site was regulated by TNFalpha but not IL-1beta, resulting in down-regulation of cd-rap expression. This effect was reversed by mutational inactivation of the c/EBP motif. In addition, the activation potential of SOX9 and CREB binding protein/p300 on the cd-rap promoter was enhanced after mutation of the new c/EBP binding site, indicating that blockage of this site would increase transcription. CONCLUSION: TNFalpha regulates the expression and/or DNA-binding potential of key positive-acting and negative-acting transcription factors that control the expression of the cartilage matrix gene, cd-rap.