Denisova. Calcium-dependent regulation of cholinergic cell phenotype in the hypothalamus in vitro. J Neurophysiol 88: 1352-1362, 2002; 10.1152/jn.00827.2001. Glutamate is a major fast excitatory neurotransmitter in the CNS including the hypothalamus. Our previous experiments in hypothalamic neuronal cultures showed that a long-term decrease in glutamate excitation upregulates ACh excitatory transmission. Data suggested that in the absence of glutamate activity in the hypothalamus in vitro, ACh becomes the major excitatory neurotransmitter and supports the excitation/inhibition balance. Here, using neuronal cultures, fura-2 Ca 2ϩ digital imaging, and immunocytochemistry, we studied the mechanisms of regulation of cholinergic properties in hypothalamic neurons. No ACh-dependent activity and a low number (0.5%) of cholinergic neurons were detected in control hypothalamic cultures. A chronic (2 wk) inactivation of N-methyl-D-aspartate (NMDA) ionotropic glutamate receptors, Ltype voltage-gated Ca 2ϩ channels, calmodulin, Ca 2ϩ /calmodulin-dependent protein kinases II/IV (CaMK II/IV), or protein kinase C (PKC) increased the number of cholinergic neurons (to 15-24%) and induced ACh activity (in 40 -60% of cells). Additionally, ACh activity and an increased number of cholinergic neurons were detected in hypothalamic cultures 2 wk after a short-term (30 min) pretreatment with bis-(o-aminophenoxy)-N,N,NЈ,NЈ-tetraacetic acid tetrakis(acetoxy-methyl) ester (BAPTA AM; 2.5 M), a membrane permeable Ca 2ϩ -chelating agent that blocks cytoplasmic Ca 2ϩ fluctuations. An increase in the number of cholinergic neurons following a chronic NMDA receptor blockade was likely due to the induction of cholinergic phenotypic properties in postmitotic noncholinergic neurons, as determined using 5-bromo-2Ј-deoxyuridine (BrdU) labeling. In contrast, a chronic inactivation of non-NMDA glutamate receptors or cGMP-dependent protein kinase had little effect on the expression of ACh properties. The data suggest that Ca 2ϩ , at normal intracellular concentrations, tonically suppresses the development of cholinergic properties in hypothalamic neurons. However, a decrease in Ca 2ϩ influx into cells (through NMDA receptors or L-type Ca 2ϩ channels), inactivation of intracellular Ca 2ϩ fluctuations, or downregulation of Ca 2ϩ -dependent signal transduction pathways (CaMK II/IV and PKC) remove the tonic inhibition and trigger the development of cholinergic phenotype in some hypothalamic neurons. An increase in excitatory ACh transmission may represent a novel form of neuronal plasticity that regulates the activity and excitability of neurons during a decrease in glutamate excitation. This type of plasticity has apparent region-specific character and is not expressed in the cortex in vitro; neither increase in ACh activity nor change in the number of cholinergic neurons were detected in cortical cultures under all experimental conditions.

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Previous antibiotic use is correlated with increased bacterial resistance in the case of otitis externa. Highly resistant bacteria were associated with an increased rate of treatment failure. Culture plays an essential role in the management of refractory otorrhea.

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Background: Tobacco use is the primary risk factor for head and neck squamous cell carcinoma (HNSCC), the sixth most common malignancy worldwide. Aberrant activation of the Wnt pathway is a key event in tumorigenesis, but the precise mechanisms leading to up-regulation of Wnt signaling in HNSCC have not been defined. Wnt inhibitory factor 1 (WIF1) is significantly down-regulated in many tumors but its expression status in oropharyngeal HNSCC is unknown. Aims: To characterize WIF1 expression in oropharyngeal normal and tumor samples and delineate the mechanisms leading to alterations in WIF1 expression. Methods: Paraffin-embedded tissue samples from fifty patients with HNSCC identified from the annotated tumor registry at the University of Oklahoma were obtained. Additionally, fifty paraffin-embedded tissues were obtained from smoker and non-smoker individuals undergoing tonsillectomy at the University of Oklahoma for non-cancerous diagnosis. Histological diagnoses were reviewed and samples from non-cancer individuals were matched for age and sex. Smoking habits were registered in pack-years. WIF1 protein expression was determined by immunohistochemistry. Genomic DNA isolated from normal and tumor samples were treated with bisulfite, and methylation-specific PCR and bisulfite sequence analysis were performed. Results: WIF1 expression was very high in normal tonsil epithelium obtained from non-smokers. However, WIF1 expression was reduced in smokers compared to non-smokers (p=0.01). WIF1 expression was frequently and significantly reduced in tumors compared to normal adjacent epithelium (p<0.001). Methylation-specific PCR and bisulfite sequence analysis revealed that WIF1 promoter methylation is common in oropharyngeal HNSCC and occurs in some normal epithelium samples obtained from smokers. Conclusions: WIF1 promoter hypermethylation is frequent in oropharyngeal cancer and contributes to WIF1 down-regulation. Importantly, by studying samples obtained from non-cancer patients, we show clearly and for the first time that WIF1 promoter methylation is an early event associated with tobacco-use. Taken together, this study establishes an important role for WIF1 in oropharyngeal HNSCC and suggests that WIF1 promoter methylation in normal epithelium is a biomarker of risk for tobacco-associated cancer. Grant support: This work was supported by the Oklahoma Center for the Advancement of Science & Technology. LQ holds a Presbyterian Health Foundation Endowed Chair in Otorhinolaryngology. Citation Format: Lurdes VF Queimado, Ryan Raju, Liu Cheng, Ilangovan Ramachandran, Eva Brabcova, Matt Naifeh, Greg Krempl. Down-regulation of Wnt inhibitory factor 1 is an early event in oropharyngeal cancer associated with tobacco-use. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4299. doi:10.1158/1538-7445.AM2013-4299