Novel scoring system and algorithm for classifying chronic rhinosinusitis: the JESREC Study.

BACKGROUND: Chronic rhinosinusitis (CRS) can be classified into CRS with nasal polyps (CRSwNP) and CRS without nasal polyps (CRSsNP). CRSwNP displays more intense eosinophilic infiltration and the presence of Th2 cytokines. Mucosal eosinophilia is associated with more severe symptoms and often requires multiple surgeries because of recurrence; however, even in eosinophilic CRS (ECRS), clinical course is variable. In this study, we wanted to set objective clinical criteria for the diagnosis of refractory CRS. METHODS: This was a retrospective study conducted by 15 institutions participating in the Japanese Epidemiological Survey of Refractory Eosinophilic Chronic Rhinosinusitis (JESREC). We evaluated patients with CRS treated with endoscopic sinus surgery (ESS), and risk of recurrence was estimated using Cox proportional hazard models. Multiple logistic regression models and receiver operating characteristics curves were constructed to create the diagnostic criterion for ECRS. RESULTS: We analyzed 1716 patients treated with ESS. To diagnose ECRS, the JESREC scoring system assessed unilateral or bilateral disease, the presence of nasal polyps, blood eosinophilia, and dominant shadow of ethmoid sinuses in computed tomography (CT) scans. The cutoff value of the score was 11 points (sensitivity: 83%, specificity: 66%). Blood eosinophilia (>5%), ethmoid sinus disease detected by CT scan, bronchial asthma, aspirin, and nonsteroidal anti-inflammatory drugs intolerance were associated significantly with recurrence. CONCLUSION: We subdivided CRSwNP in non-ECRS, mild, moderate, and severe ECRS according to our algorithm. This classification was significantly correlated with prognosis. It is notable that this algorithm may give useful information to clinicians in the refractoriness of CRS before ESS or biopsy.

The role of IL-33 and its receptor ST2 in human nasal epithelium with allergic rhinitis.

BACKGROUND: Interleukin (IL)-33 is a novel member of the IL-1 cytokine family and a ligand for the orphan IL-1 family receptor ST2. The IL-33 induces T helper 2-type inflammatory responses and is considered to play a crucial rule in allergic inflammations, such as asthma and atopic dermatitis. However, the role of IL-33 and its receptor ST2 in allergic rhinitis remains unknown. OBJECTIVE: We investigated expression of IL-33 and ST2 in the nasal epithelium of patients with allergic rhinitis and the mechanisms of the production of cytokines/chemokines induced by treatment with IL-33 using normal human nasal epithelial cells (HNECs) in vitro. METHODS: Expression of IL-33 and ST2 in normal and allergic rhinitis nasal mucosa was evaluated by reverse transcription- and real-time polymerase chain reactions and immunohistochemical methods. The IL-33 in serum, and IL-8 and GM-CSF were measured by ELISA. For in vitro experiments, HNECs in primary culture were used. RESULTS: The IL-33 levels in the sera of patients with allergic rhinitis were significantly higher than that in normal controls. Expression of IL-33 and ST2 was significantly elevated in the epithelium from patients with allergic rhinitis. The IL-33 mRNA in HNECs in vitro was significantly induced by treatment with IFN-γ and the toll-like receptor 9 ligand ODN2006. The IL-33-induced production of IL-8 and GM-CSF from HNECs in vitro was significantly suppressed by corticosteroid treatment and distinct signal transduction inhibitors of ERK, p38 MAPK, JNK, NF-κB and epidermal growth factor receptor. CONCLUSIONS AND CLINICAL RELEVANCE: The IL-33 and its receptor ST2 play important roles in allergic rhinitis. The IL-33-mediated inflammatory responses via ST2 are regulated by distinct signalling pathways in HNECs and the IL-33/ST2 pathway may provide new therapeutic targets for allergic rhinitis.

Platelet-activating factor (PAF) increases NO production in human endothelial cells-real-time monitoring by DAR-4M AM.

BACKGROUND: Platelet-activating factor (PAF) is a potent inflammatory lipid mediator that increases vascular permeability and vasodilation. Several studies have addressed the effect of PAF on nitric oxide (NO) production from microvessels in vivo. OBJECTIVE: The aim of present study was to evaluate the effect of PAF on NO production in primary cultured human vascular endothelial cells. METHODS: Human umbilical vein endothelial cells (HUVECs) were loaded with diaminorhodamine-4M acetoxymethyl ester (DAR-4MAM), and the cells were stimulated with PAF. Intracellular NO production was monitored as increase in fluorescence intensity. Also, NO production was visualized at cellular levels using DAR-4M AM and fluorescence imaging. RESULTS: Significant increases in NO production in HUVECs were soon after the PAF stimulation, reaching a plateau after 10 min of the stimulation. The increase of NO production at 10 min after the stimulation was statistically significant (p<0.05) for 0.01-10 microM PAF. PAF-induced NO production was abolished by pretreatment of HUVECs with a NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA) or PAF receptor antagonist BN 52021. LysoPAF, the inactive metabolite of PAF, did not exert a significant effect on intracellular NO levels. CONCLUSIONS: These results provide direct evidence that PAF cause intracellular NO production via activation of PAF receptors in human vascular endothelial cells.

Inhibition of phosphatidylinositol-3 kinase/Akt or mitogen-activated protein kinase signaling sensitizes endothelial cells to TNF-alpha cytotoxicity.

Bovine carotid artery endothelial (BAE) cells are resistant to tumor necrosis factor-alpha (TNF), like most other cells. We examined if mitogen-activated protein (MAP) kinase and phosphatidylinositol-3 (PI3) kinase/Akt pathways are involved in this effect. In BAE cells, TNF activates MAP kinase in a MAP kinase kinase 1 (MEK1) manner and Akt in PI3-kinase-dependent manner. Pretreatment with either the MEK1 inhibitor U0126 or PI3-kinase inhibitor LY294002 sensitized BAE cells to TNF-induced apoptosis. Neither U0126 nor LY294002 pretreatment affected TNF-induced activation of NF-kappaB, suggesting that the MAP kinase or PI3-kinase/Akt-mediated anti-apoptotic effect induced by TNF was not relevant to NF-kappaB activation. Both MAP kinase and PI3-kinase/Akt -mediated signaling could prevent cytochrome c release and mitochondrial transmembrane potential (Deltapsi) decrease. PI3-kinase/Akt signaling attenuated caspase-8 activity, whereas MAP kinase signaling impaired caspase-9 activity. These results suggest that TNF-induced MAP kinase and PI3-kinase/Akt signaling play important roles in protecting BAE cells from TNF cytotoxicity.

Induction of stathmin mRNA during liver regeneration.

Stathmin is a 19 kDa phosphoprotein, and is proposed to play a role in signal transduction in response to various extracellular stimuli that promote cellular growth and/or differentiation. We examined stathmin mRNA expression during development and liver regeneration in mice. Stathmin mRNA expression declined during the post-natal period and was undetected in adult liver. 36 h after partial hepatectomy, stathmin mRNA was rapidly induced and remained at elevated levels for at least 10 days. In situ hybridization experiments confirmed that stathmin mRNA expression occurred in hepatocytes. These results indicate that the stathmin gene expression appears to be repressed during the post-natal liver development, and is de-repressed by liver regeneration, which suggests that stathmin may be a good molecular marker of liver plasticity.

Prognostic factors of nasopharynx tumors investigated by MR imaging and the value of MR imaging in the newly published TNM staging.

PURPOSE: To examine the usefulness of MR imaging for predicting local control of nasopharyngeal carcinoma (NPC) and the value of MR imaging in the newly published fifth edition of the TNM classification. METHODS AND MATERIALS: We studied 29 patients with NPC with MR imaging and CT before and after treatment. Staging was done according to the fourth and newly published fifth editions of the International Union Against Cancer (UICC) staging system. The radiotherapy protocol was designed to deliver 66 to 68 Gy to the primary tumor and clinically involved nodes. RESULTS: MR proved better than CT at identifying obliteration of the pharyngobasilar fascia, invasion of the sinus of Morgagni, through which the cartilaginous portion of the eustachian tube and the levator veli palatini muscle pass, invasion of the skull base, and metastases to lymph nodes in the carotid and retropharyngeal spaces. All seven patients without invasion of the pharyngobasilar fascia had local control. The local control rates of patients with invasion of the skull base were not good (60 to 73%). There was no apparent relationship between tumor volume determined by T1-weighted MR images and local control when the tumor volume was more than 20 cc. The newly published N staging system appears to successfully identify the high-risk group for distant metastasis as N3. In our series, four of five patients with N3 disease developed distant metastases. CONCLUSION: Deep infiltration of the tumor is a more important prognostic factor in NPC than tumor volume. Since the newly published T staging system requires a search for tumor invasion into soft tissue such as parapharyngeal space and bony structures, MR imaging may be indispensable for the newly published NPC staging system.

Retinobenzoic acids. 4. Conformation of aromatic amides with retinoidal activity. Importance of trans-amide structure for the activity.

N-Methylation of two retinoidal amide compounds, 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbamoyl]benz oic acid (3, Am80) and 4-[[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2- naphthalenyl)carbonyl]amino]benzoic acid (5, Am580), resulted in the disappearance of their potent differentiation-inducing activity on human promyelocytic leukemia cell line HL-60. Studies with 1H NMR and UV spectroscopy indicated that large conformational differences exist between the active secondary amides and the inactive N-methyl amides. From a comparison of the spectroscopic results of these amides with those of stilbene derivatives, the conformations of the active amides are expected to resemble that of (E)-stilbene, whereas the inactive amides resemble the Z isomer: 3 (Am80) and 5 (Am580) have a trans-amide bond and their whole structures are elongated, while the N-methylated compounds [4 (Am90) and 6 (Am590)] have a cis-amide bond, resulting in the folding of the two benzene rings. These structures in the crystals were related to those in solution by 13C NMR spectroscopic comparison between the two phases (solid and solution).

Retinobenzoic acids. 1. Structure-activity relationships of aromatic amides with retinoidal activity.

Two types of aromatic amides, terephthalic monoanilides and (arylcarboxamido)benzoic acids, have been shown to possess potent retinoidal activities and can be classified as retinoids. The structure-activity relationships of these amides are discussed on the basis of differentiation-inducing activity on human promyelocytic leukemia cells HL-60. In generic formula 4 (X = NHCO or CONH), the necessary factors to elicit the retinoidal activities are a medium-sized alkyl group (isopropyl, tert-butyl, etc.) at the meta position and a carboxyl group at the para position of the other benzene ring. The bonding of the amide structure can be reversed, this moiety apparently having the role of locating the two benzene rings at suitable positions with respect to each other. Substitution at the ring position ortho to the amide group or N-methylation of the amide group caused loss of activity, presumably owing to the resultant change of conformation. It is clear that the mutual orientation of the benzylic methyl group(s) and the carboxyl group and their distance apart are essential factors determining the retinoidal activity. Among the synthesized compounds, 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbamoyl]benz oic acid (Am80) and 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido] benzoic acid (Am580) were several times more active than retinoic acid in the assay. They are structurally related to retinoic acid, as is clear from the biological activity of the hybrid compounds (M2 and R2).

Retinobenzoic acids. 3. Structure-activity relationships of retinoidal azobenzene-4-carboxylic acids and stilbene-4-carboxylic acids.

Alkyl-substituted azobenzene-4-carboxylic acids are potent differentiation inducers of human promyelocytic leukemia cell line HL-60 to mature granulocytes. Their structure-activity relationships are very similar to those of other retinoidal benzoic acids which are generally represented by 4 and named retinobenzoic acids. The structure-activity relationships of azobenzenecarboxylic acids can also be applied to the known retinoid TTNPB (3). Thus, (E)-4-[2-(3,4-diisopropylphenyl)-1-propenyl]benzoic acid (St30 (28] and (E)-4-[2-(3-tert-butylphenyl)ethenyl]benzoic acid (St40 (29], the acyclic alkyl analogues of TTNPB, are nearly as active as retinoic acid. Among the oxidatively derived compounds (Az90, Ep series and Ox series) of azobenzene- or stilbenecarboxylic acids, Az90 (71) and Ep80 (61) have strong activities. However, all the bishydroxylated derivatives of TTNPB are inactive, while a diketo analogue Ox580 (69) has only weak potency. The activities of conformationally restricted compounds of TTNPB offer some information on the stereochemistry of the active form of these retinoidal compounds.

Differential localization of SCG10 and p19/stathmin messenger RNAs in adult rat brain indicates distinct roles for these growth-associated proteins.

SCG10 is a developmentally regulated, growth-associated protein (GAP) that was isolated as a neuronal marker of the neural crest. It was recently found that SCG10 shares an amino acid sequence similarity with a phosphoprotein named stathmin or p19 of which phosphorylation is induced by nerve growth factor and vasoactive intestinal peptide in PC12 cells and striatal neurons, respectively. While expression of SCG10 messenger RNA dramatically decreases during postnatal development, significant levels of expression still persist into adulthood. To examine possible roles of SCG10 in the adult brain, we examined the distribution of messenger RNAs encoding SCG10 and p19/stathmin as well as GAP-43 in adult rat brain sections by northern blot, RNase protection and in situ hybridization. SCG10 transcripts are found at high levels in long-distance projecting neurons and neurons with extensive dendritic arbors, while p19/stathmin messenger RNA was weakly distributed over most brain areas. Both messenger RNAs are expressed in neuronal subpopulations but not in glia, although the overall distribution of the transcripts of these two structurally related genes is distinct. The spatial and temporal expression profiles of SCG10 messenger RNA is comparable to that of GAP-43, another neuronal GAP, in the developing nervous system, however the expression of SCG10 messenger RNA in the adult brain is distinct from that of GAP-43, especially in the hippocampus and brain stem, where the dentate granule cells and sensory and motor neurons of brainstem express SCG10 but not GAP-43. These results suggest that SCG10 may have a unique role in the neuronal growth-response of subsets of mature neurons, and that SCG10 plays a stathmin-like function at nerve terminals, to which it may be rapidly transported by means of membrane attachment due to a hydrophobic domain present in SCG10 but not in p19/stathmin. This suggests that SCG10 may play a role in structural plasticity in the adult brain.

Simple and Patlak models for myocardial blood flow measurements with nitrogen-13-ammonia and PET in humans.

UNLABELLED: The Simple and Patlak models for estimating myocardial blood flow with 13N-ammonia have become attractive for clinical applications with PET because of their simplicity and ease of implementation. However, these models are sensitive to factors such as the data acquisition times and data integration times, which can cause errors in the estimation of myocardial blood flow, as demonstrated in this study. Limiting the application of these models to specific conditions can minimize the errors. METHODS: Dynamic PET images of the uptake of 13N-ammonia in the heart were obtained in seven humans under rest and dipyridamole stress. Myocardial blood flow was estimated using the Simple and Patlak models for different data acquisition times and data integration times. Blood flow values were compared to flow values computed with the two-compartment model as a reference. RESULTS: Blood flow values calculated with the Simple and Patlak models during the first 2 min of data acquisition were closely correlated to the two-compartment model values. Longer acquisition times resulted in significant underestimation of blood flow for the Simple model. Long integration times of greater than 60 sec also resulted in significant underestimation of blood flow for both models. CONCLUSION: The Simple and Patlak models produce estimates of myocardial blood flow that are well correlated with the two-compartment model estimated blood flows for the integration time of 60 sec from 60 to 120 sec postinjection. Because of the errors associated with longer data acquisition times and longer integration times, use of these models should be limited to a well-documented data acquisition paradigm.

Simplified PET quantitation of myocardial glucose utilization.

UNLABELLED: The purpose of this study was to validate experimentally a simple method to quantify tissue glucose utilization with the brain reference index (BRI) using 14C-deoxyglucose and assess its clinical feasibility for myocardial PET. METHODS: To validate the BRI method, glucose utilization in myocardial and skeletal muscle was studied in rats with 14C-deoxyglucose after increasing doses of oral glucose loading. To assess clinical feasibility of the method, the BRI was applied to nine patients undergoing myocardial PET and compared to rMGU measured by the deoxyglucose model of Sokoloff et al. and by Patlak graphical analysis. The normal range of myocardial FDG uptake expressed as the BRI was estimated with four normal volunteers. RESULTS: In skeletal muscle, a dose-dependent increase of glucose utilization was observed during oral glucose loading with doses up to 4 mg/g. In the myocardium, glucose utilization increased with a glucose loading dose of up to 1 mg/g without increasing further at greater glucose doses. Ratios of maximal glucose utilization in glucose-loaded rats to 19-hr fasted rats (controls), expressed as the BRI for left and right ventricular myocardium and skeletal muscle were 4.16, 3.74 and 7.39, respectively. Glucose utilization of right ventricular myocardium was approximately 70% of left ventricular myocardium for all glucose-loaded conditions. For patients, the BRI correlated with rMGU; four of these patients had a constant plasma glucose concentration. CONCLUSION: Myocardial BRI is a sensitive indicator of rMGU that does not require dynamic data acquisition or constant plasma glucose concentrations.

Prognostic utility of myocardial blood flow assessed by N-13 ammonia positron emission tomography in patients with idiopathic dilated cardiomyopathy.

Previous studies in patients with idiopathic dilated cardiomyopathy (IDC) have suggested that myocardial perfusion is impaired and spatially heterogeneous in such cases. Our objective was to identify any association between an abnormality in myocardial perfusion and the prognosis of patients with IDC. We collected data on N-13 ammonia positron emission tomography (PET) studies performed in 26 patients with IDC (9 nonsurvivors, 17 survivors) and in 8 normal control subjects. Regional myocardial blood flow (rMBF) was quantified using N-13 ammonia positron emission tomography and the Simple flow model. The spatial heterogeneity of myocardial perfusion was assessed by calculating the coefficient of variance of rMBF. Mean rMBF of the survivors was significantly lower (0.54 +/- 0.13 ml/min/g) than that of control subjects (0.66 +/- 0.06 ml/min/g) (p = 0.03 vs control), but did not differ significantly between nonsurvivors (0.58 +/- 0.15 ml/min/g) and control subjects. The coefficient of variance of rMBF was significantly higher in nonsurvivors than in either survivors or control subjects (0.24 +/- 0.08 vs 0.15 +/- 0.08, p = 0.007, and 0.16 +/- 0.05, p = 0.03, respectively). The probability of 3-year survival (Kaplan-Meier method) was 33.0% in subjects whose coefficient of variance of rMBF was above the median compared with 90.0% in subjects whose coefficient of variance of rMBF was below the median (p = 0.01). The probability of 3-year survival did not differ among subjects whose mean rMBF was above versus below the median (61.5% vs 62.9%, respectively). The results suggest that the prognosis of patients with IDC is associated with the spatial heterogeneity of myocardial perfusion, not with initial absolute rMBF.

Nuclear BAG-1 localization and the risk of recurrence after radiation therapy in laryngeal carcinomas.

BAG-1 is a multifunctional chaperone modulator may contribute to p53-mediated cell cycle arrest. We attempted to investigate whether BAG-1 expression is correlated with prognosis of laryngeal carcinoma patients after radiotherapy. Immunohistochemical analyses revealed BAG-1 expression was present in all laryngeal carcinomas examined, and its expression pattern varied, i.e. cytoplasmic, nuclear and both these staining types. Patients whose tumors predominantly express nuclear BAG-1 have a significantly poor failure-free survival rate after radiotherapy. We thus propose that nuclear BAG-1 localization is a prediction of unfavorable outcome should radiation therapy be undertaken for laryngeal carcinoma patients.

High incidence of Alloiococcus otitis in otitis media with effusion.

BACKGROUND: The etiology of otitis media with effusion (OME) is unclear. Although the majority of effusions show inflammation, culture methods yield positive results for bacteria in only 20 to 30% of cases. METHODS: The polymerase chain reaction was used for detection of three upper respiratory tract pathogens, Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae, and a fairly recently described bacterium, Alloiococcus otitis (A. otitidis), that is solely found in OME. The study included 67 middle ear effusions that were collected from 48 pediatric OME patients during ventilation tube placement. RESULTS: PCR tested positive for 57 (85.1%) of the middle ear effusions. Thirty-one (46.3%) A. otitis-, 12 (17.9%) H. influenzae-, 25 (37.3%) M. catarrhalis- and 14 (20.9%) S. pneumoniae-positive effusions were obtained. All four study organisms showed similar distribution in effusions of various duration (P = 0.72) and in different effusion types (P = 0.59). Only the proportion of M. catarrhalis-positive effusions was lowered by recent antimicrobial therapy (P < 0.05). Although the study organisms had equal distributions among singly and multiply positive specimens (P = 0.90), A. otitis was detected significantly more often with one of the three other species (15 of 19, 78.9%) than the other species with each other (4 of 19, 21.1%, P < 0.001). CONCLUSIONS: The findings suggest a bacterial etiology for OME. Association of A. otitis with the three other species implies that this organism might have the capability of augmenting bacterial colonization in the middle ear.

Reduced expression of the molecular markers of dopaminergic neuronal atrophy in the aging rat brain.

Expression of mRNAs encoding the dopamine transporter (DAT) and tyrosine hydroxylase (TH) in dopaminergic neurons of the substantia nigra (SN) was examined in young and aged Fischer 344 rats by in situ hybridization. Quantitative analysis revealed a statistically significant decline in both DAT and TH mRNA expression in 24-month-old rats in comparison to 6-month-old rats. In addition, it was noted that DAT mRNA expression tended to decrease by 18 months, while TH mRNA reduction did not occur until 24 months. The age-related loss of DAT and TH mRNA expressions was accompanied by diminished expression of mRNA for a neuronal growth-associated protein GAP-43, but not for SCG10 or alpha 1-tubulin. The loss of GAP-43 mRNA became evident when both DAT and TH gene expression declined with advanced age. Our findings indicate that DAT may be a marker of atrophy in dopamine neurons during normal aging.

SCG10 mRNA localization in the hippocampus: comparison with other mRNAs encoding neuronal growth-associated proteins (nGAPs).

SCG10 is a nerve growth factor (NGF)-inducible, neuron-specific protein whose expression is tightly correlated with axonal and/or dendritic growth. We have recently shown that the mRNA encoding SCG10 is expressed at significant levels in certain subsets of neurons in the adult rat brain, while its expression is undetectable or negligible in other non-neuronal tissues. Here we show that regional SCG10 mRNA expression in the adult mouse brain is comparable to that in the rat, however, in the hippocampus its expression profile is distinct. In the mouse, SCG10 mRNA is expressed at high levels in pyramidal cells of CA3-CA4 sub-fields of Ammon's horn and at low levels in the CA1-CA2 sub-fields, while it is found rather uniformly throughout the pyramidal cell layer of the rat hippocampus. SCG10 mRNA is not detectable in the dentate gyrus of the mouse hippocampus, although it is expressed in the rat dentate gyrus. Comparison with other mRNAs encoding neuronal growth-associated proteins (nGAPs) such as GAP-43, MAP2, alpha 1-tubulin and stathmin suggests that dentate granule cells express a different repertoire of neuronal growth-associated genes in mouse and rat.

Involvement of glycine site associated with the NMDA receptor in hippocampal long-term potentiation and acquisition of spatial memory in rats.

The effects of 7-chlorokynurenic acid (7-Cl-Kyn), a selective antagonist at the glycine site associated with the N-methyl-D-aspartate (NMDA) receptor, on hippocampal long-term potentiation (LTP) and behavioral performances in a spatial learning task were investigated. Extracellular recordings of evoked potential (population spike) were made in rat hippocampal slices. Perfusion of 7-Cl-Kyn (10(-5) M) inhibited the induction of LTP following a tetanic stimulation (51 or 101 pulses at 100 Hz) both in the Schaffer/commissural-CA1 pyramidal cell synapses and in the perforant path-dentate granule cell synapses. Acquisition of a spatial memory in the Morris water maze was examined using rats chronically cannulated for application of drugs. The intact and vehicle-injected rats learned easily to escape onto a hidden platform with short latencies, while the rats given an injection of 7-Cl-Kyn (10(-8) mol/brain, i.c.v.) prior to every session took a longer time and a longer path to escape even after all 5 sessions of trials. Injection of 7-Cl-Kyn did not affect the swimming speed, an index of swimming ability. This is the first report providing direct evidence that endogenous glycine supports the processes of learning and memory.

Purkinje cells express neuronal nitric oxide synthase after methylmercury administration.

To study the effects of chemical injury on the cerebellar nitric oxide synthase (NOS), we administered methylmercury chloride subcutaneously to mice, 10 mg/kg/day for 9 days. In the methylmercury-treated cerebellum. Purkinje cells were positive both for NADPH-diaphorase and for neuronal NOS. Calcium-dependent NOS activity was increased to 160% of the controls. The present study suggests the ability of Purkinje cells to produce NO through the expression of neuronal NOS.

Effect of Alloiococcus otitidis and three pathogens of otitis media in production of interleukin-12 by human monocyte cell line.

Alloiococcus otitidis is detected in middle ear effusion of otitis media with effusion (OME). Only a limited number of studies are available concerning the immunological profile of A. otitidis. We have studied the ability of A. otitidis and three other representative pathogens of otitis media to stimulate the production of interleukin-12 (IL-12) from a monocytic cell line THP-1. Viable A. otitidis induced the production of IL-12 in THP-1 cells but IL-12 production was reduced if glutaraldehyde-fixed bacteria were used as stimulants. When viable bacteria were physically separated from THP-1 cells during the stimulation period, remarkable reductions of IL-12 secretion were shown after challenge with gram-positive bacteria A. otitidis and S. pneumoniae. When stimulated with soluble extracts of A. otitidis, THP-1 secreted IL-12 in a dose-dependent manner. The subfraction with a molecular mass over 100 kDa showed a strong ability to induce IL-12 production. Our results show that A. otitidis has immunostimulatory capacity with regard to IL-12 production. We also show that soluble antigen(s) of A. otitidis can modulate the immune response in OME.