The potential antidiabetic properties of green and purple tea [Camellia sinensis (L.) O Kuntze], purple tea ellagitannins, and urolithins.

ETHNOPHARMACOLOGICAL RELEVANCE: Tea (Camellia sinensis) has been consumed for centuries as traditional medicine for various diseases, including diabetes. The mechanism of action of many traditional medicines, including tea, often requires elucidation. Purple tea is a natural mutant of Camellia sinensis, grown in China and Kenya, and is rich in anthocyanins and ellagitannins. AIM OF THE STUDY: Here we aimed to determine whether commercial green and purple teas are a source of ellagitannins and whether green and purple teas, purple tea ellagitannins and their metabolites urolithins have antidiabetic activity. MATERIALS AND METHODS: Targeted UPLC-MS/MS was employed to quantify the ellagitannins corilagin, strictinin and tellimagrandin I, in commercial teas. The inhibitory effect of commercial green and purple teas and purple tea ellagitannins was evaluated on α-glucosidase and α-amylase. The bioavailable urolithins were then investigated for additional antidiabetic effects, by evaluating their effect on cellular glucose uptake and lipid accumulation. RESULTS: Corilagin, strictinin and tellimagrandin I (ellagitannins) were identified as potent inhibitors of α-amylase and α-glucosidase, with Ki values significantly lower (p < 0.05) than acarbose. Commercial green-purple teas were identified as ellagitannin sources, with especially high concentrations of corilagin. These commercial purple teas, containing ellagitannins, were identified as potent α-glucosidase inhibitors with IC50 values significantly lower (p < 0.05) than green teas and acarbose. Urolithin A and urolithin B were as effective (p> 0.05) as metformin in increasing glucose uptake in adipocytes, muscle cells and hepatocytes. In addition, similar (p > 0.05) to metformin, both urolithin A and urolithin B reduced lipid accumulation in adipocytes and hepatocytes. CONCLUSIONS: This study identified green-purple teas as an affordable widely available natural source with antidiabetic properties. Furthermore, additional antidiabetic effects of purple tea ellagitannins (corilagin, strictinin and tellimagrandin I) and urolithins were identified.

Underlying mechanisms of cytotoxicity in HepG2 hepatocarcinoma cells exposed to arsenic, cadmium and mercury individually and in combination.

BACKGROUND: Toxicity data regarding combinational exposure of humans to arsenic, cadmium and mercury is scarce. Although hepatotoxicity has been reported, limited information is available on their mechanistic underpinnings. The cytotoxic mechanisms of these metals were determined in HepG2 hepatocarcinoma cell lines after individual and combinational exposure. METHODS: HepG2 cells were exposed to heavy metals (sodium arsenite, cadmium chloride, and mercury chloride) individually or in combination for 24 h, after which cell density, mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS), reduced glutathione (GSH), adenosine triphosphate (ATP) and caspase-3/7 activity was assessed. RESULTS AND DISCUSSION: Cadmium (IC50 = 0.43 mg/L) and the combination (0.45 mg/L, arsenic reference) were most cytotoxic, followed by arsenic (6.71 mg/L) and mercury (28.23 mg/L). Depolarisation of the ΔΨm and reductions in ROS, GSH and ATP levels occurred. Arsenic, cadmium and the combination increased caspase-3/7 activity, while mercury reduced it. CONCLUSION: The combination produced a greater, albeit mechanistically similar, cytotoxicity compared to individual metals. Cytotoxicity was dependent on altered mitochondrial integrity, redox-status, and bioenergetics. Although the combination's cytotoxicity was associated with caspase-3/7 activity, this was not true for mercury. Heavy metal interactions should be assessed to elucidate molecular underpinnings of cytotoxicity.

[Surgical treatment of talocalcaneal coalition : Experience with 80 cases of pediatric or adolescent patients]. / Operative Behandlung der Coalitio talocalcanearis : Erfahrungen aus 80 Fällen kindlich-jugendlicher Patienten.

INTRODUCTION: There is still a controversial discussion on the treatment of talocalcaneal coalition with and without planovalgus deformity. From 2002-2014 80 cases of talocalcaneal coalition in children and adolescents under 18 years of age were surgically treated by J. Hamel. The objective of this study is a retrospective analysis, especially of unfavourable results. METHODS: Patients with minimum follow up of 12 months were included (35.7 months on average). In 31 cases treatment consisted of resection and fat-grafting (group 1), in 26 additional cases tarsal osteotomy was added because of planovalgus-deformity (group 2), and in 23 cases primary fusion of the talocalcaneal joint was performed (group 3). RESULTS: In group 1 one patient was lost to follow-up early with a severe peroneal spasticity and unknown further course. Two further patients still had remarkable pain after 15 months, and another patient had to undergo secondary fusion because of persistent pain. In group 2 secondary fusion was undertaken in two cases and offered in four other cases. In group 3 two patients still suffered from moderate tarsal pain, although complete fusion occurred; one of these patients underwent bilateral treatment. All other patients were pain free or nearly pain free on the last follow-up visit, with marked improvement in comparison to the preoperative situation. DISCUSSION: Surgical treatment of talocalcaneal coalition is successful in most cases in the short to medium follow-up, but unfavourable results are not uncommon and may require secondary fusion. Differential indication between resection, additional deformity correction, or primary fusion seems to be most important.

A randomized, 5-arm dose finding study with a mite allergoid SCIT in allergic rhinoconjunctivitis patients.

BACKGROUND: The safety and tolerability of a mite allergoid subcutaneous allergen immunotherapy (SCIT) product was previously established. The aim of this study (EudraCT number: 2011-000393-61) was to find the optimally safe and effective allergoid dose by evaluating several dosages in patients with house dust mite (HDM)-induced allergic rhinoconjunctivitis (ARC) using a titrated nasal provocation test (TNPT). METHODS: In total, 290 adult ARC patients (148 females; 142 males) with established HDM allergy and with a positive TNPT were randomized to receive placebo or mite allergoid SCIT 6667, 20 000, 50 000 or 100 000 AUeq/ml for 12 months. Patients were updosed weekly, followed by monthly maintenance dosing. The primary study endpoint comprised the clinical response to TNPT after 12 months of treatment. Secondary endpoints included response to TNPT after 6 months, PNIF measurements, symptom and medication scores during the last 8 weeks of treatment, serum immunoglobulins and safety assessments. RESULTS: After 12 months, a dose-response was observed showing statistically significant improvements in the TNPT with SCIT concentrations of ≥20 000 AUeq/ml, while no significantly different outcomes were reached after 6 months. Specific serum IgG and IgG4 levels were dose dependently increased. In the highest dose group, more treatment-emergent adverse events were observed compared with the lower dose groups. CONCLUSION: In this mite allergoid SCIT dose finding study in HDM-induced ARC, concentrations of ≥20 000 AUeq/ml showed both immunological effects and clinical efficacy in the TNPT compared with placebo. The risk-benefit ratio favours 20 000 AUeq/ml and 50 000 AUeq/ml strengths for further clinical development.

[Osteosynthesis of talonavicular fusion with a claw plate and compression screw]. / Kombinierte Schrauben-Clawplate-Osteosynthese zur Stabilisierung der talonavikularen Arthrodese.

OBJECTIVE: A new method of osteosynthetic stabilization of talonavicular fusion is presented. INDICATIONS: Idiopathic and posttraumatic talonavicular arthritis, talonavicular destruction in rheumatoid arthritis, adult acquired flatfoot deformity, cavovarus deformity, talonavicular degenerative disease in tarsal coalition. CONTRAINDICATION: Major hindfoot deformity or instability, severe osteopenic conditions of tarsal bones. SURGICAL TECHNIQUE: Talonavicular fusion is stabilized with a medioplantar 6.5-mm lag screw in combination with a dorsolateral 3.5-mm claw plate (Charlotte Claw compression plate; Fa. Wright Medical Technology, Memphis, USA) by a dorsal surgical access. POSTOPERATIVE MANAGEMENT: Nonweight-bearing in a cast or walker for 6 weeks; after radiologic control increasing weight-bearing is allowed. RESULTS: The technique was used in 44 patients, among them 31 cases of isolated talonavicular fusion. Complete bony healing was observed in 42 cases after medium follow-up time of 13.3 months. All 19 cases of isolated talonavicular fusion without posterior tibial tendon dysfunction healed uneventfully; two cases of non-union were observed in 12 patients with posterior tibial tendon dysfunction.

Monosodium Urate and Tumor Necrosis Factor-α Increase Apoptosis in Human Chondrocyte Cultures.

Monosodium urate and tumor necrosis factor-α, are two potent mediators of separate inflammatory response pathways in arthritic joints where inflammation may be accompanied by the loss of chondrocyte vitality via apoptosis. To address this possibility in vitro, chondrocyte cultures were employed to determine the extent to which monosodium urate and recombinant TNF-α altered the frequency of apoptotic chondrocytes. Apoptosis as a function of the activation of p38 kinase, C-Jun-terminal kinase, signal transducer and activator of transcription-3 and/or the activity of xanthine oxidase was also studied. Using normal human chondrocytes, monosodium urate or recombinant tumor necrosis factor-α increased the frequency of apoptosis and activity of xanthine oxidase. However, the xanthine oxidase-specific inhibitor, febuxostat, failed to blunt this response. Monosodium urate, tumor necrosis factor-α or the Janus kinase inhibitor, AG-490, increased the frequency of apoptotic nuclei in macroaggregate pellet cultures initiated from juvenile human chondrocytes, but not in pellet cultures derived from mesenchymal stem cells. In OA chondrocytes, activation of p38, C-Jun-NH2-kinase and signal transducer and activator of transcription-3 preceded apoptosis. Activation of signal transducer and activator of transcription-3 also was seen in pellet cultures initiated from juvenile chondrocytes and MSCs incubated with MSU, recombinant tumor necrosis factor-α or febuxostat, but apoptosis was increased only in the pellet cultures derived from juvenile chondrocytes. Although AG-490 or the combination of AG-490 and febuxostat inhibited signal transducer and activator of transcription-3 activation, apoptosis was unaffected. These results showed that recombinant tumor necrosis factor-α, monosodium urate and AG-490 increased apoptosis in normal human chondrocytes, OA chondrocytes and human juvenile chondrocyte pellet cultures, but not in chondrocyte pellet cultures initiated from MSCs. The increased frequency of apoptotic chondrocytes in response to recombinant tumor necrosis factor-α or monosodium urate was not dependent on either activation of STAT3 or the activity of XO.

Low oxygen tension during incubation periods of chondrocyte expansion is sufficient to enhance postexpansion chondrogenesis.

To determine whether low oxygen (O(2)) tension during expansion affects the matrix density, as well as quantity, of cartilage formed, and to determine whether application of low O(2) tension during incubation periods alone is sufficient to modulate chondrogenic expression, rabbit chondrocytes expanded at either 21% O(2) or 5% O(2) were analyzed for glycosaminoglycan (GAG) and DNA content, total collagen, and gene expression during expansion and postexpansion aggregate cultures. When cultured as aggregates at 21% O(2), chondrocytes expanded at 5% O(2) produced cartilage aggregates that contained more total GAG, GAG per wet weight, GAG per DNA, and total collagen than chondrocytes expanded at 21% O(2). Less of an effect on GAG and collagen content was observed when aggregate culture was performed at 5% O(2). Real-time polymerase chain reaction analysis of COL2A1 expression showed upregulated levels of type IIA (an early marker) and IIB (a late marker) during expansion and elevated levels of type IIB during aggregate culture in chondrocytes expanded in low O(2). The application of low O(2) tension during incubation periods of chondrocyte expansion enhances the ultimate cartilage matrix density and quantity, and this enhancement can be achieved through the use of an O(2) control incubator.

An internet-based abstinence reinforcement smoking cessation intervention in rural smokers.

The implementation of cigarette smoking abstinence reinforcement programs may be hindered by the time intensive burden placed on patients and treatment providers. The use of remote monitoring and reinforcement of smoking abstinence may enhance the accessibility and acceptability of this intervention, particularly in rural areas where transportation can be unreliable and treatment providers distant. This study determined the effectiveness of an Internet-based abstinence reinforcement intervention in initiating and maintaining smoking abstinence in rural smokers. Sixty-eight smokers were enrolled to evaluate the efficacy of an Internet-based smoking cessation program. During the 6-week intervention period, all participants were asked to record 2 videos of breath carbon monoxide (CO) samples daily. Participants also typed the value of their CO readings into web-based software that provided feedback and reinforcement based on their smoking status. Participants (n=35) in the Abstinence Contingent (AC) group received monetary incentives contingent on recent smoking abstinence (i.e., CO of 4 parts per million or below). Participants (n=33) in the Yoked Control (YC) group received monetary incentives independent of smoking status. Participants in the AC group were significantly more likely than the YC group to post negative CO samples on the study website (OR=4.56; 95% CI=2.18-9.52). Participants assigned to AC were also significantly more likely to achieve some level of continuous abstinence over the 6-week intervention compared to those assigned to YC. These results demonstrate the feasibility and short-term efficacy of delivering reinforcement for smoking abstinence over the Internet to rural populations.

The anti-tumour properties and biodistribution (as determined by the radiolabeled equivalent) of Au-compounds intended as potential chemotherapeutics.

The anti-tumour activity of the Au (I) phosphine complex [Au(dppe(2)]Cl was first discovered in the mid 1980s although promising results were obtained it did not pass clinical studies because of its toxicity to organs such as the liver and heart. The aim of this study was to determine whether the two novel gold compounds (MM5 and MM6), selected for this study, have higher selectivity for cancer cells with less toxicity towards normal cells than [Au(dppe)(2)]Cl, and also to determine whether they have improved bio distribution compared to [Au(dppe)(2)]Cl. The Au-compounds as potential chemotherapeutic drugs were evaluated by using radioactive tracers in the in vitro and in vivo studies. Results obtained from these experiments showed that the uptake of these experimental compounds was dependent on their octanol/water partition coefficient. However; the inhibition of cell growth did not correlate with the uptake of these compounds by the cells that were tested. In terms of the total uptake it was found that the compounds that were less lipophilic (MM5, MM6) were taken up less efficiently in cells than those that are more lipophilic. Therefore hydrophilic drugs are expected to have a limited biodistribution compared to lipophilic drugs. This might imply a more selective tumour uptake.

Arbuscular mycorrhiza alter the concentration of essential oils in oregano (Origanum sp., Lamiaceae).

The effect of root colonization by Glomus mosseae on the qualitative and quantitative pattern of essential oils (EO) was determined in three oregano genotypes (Origanum sp.). To exclude a simple P-mediated effect through mycorrhization the effect of P application to plants on the EO accumulation was also tested. In two genotypes the leaf biomass was increased through mycorrhization. Root colonization by the arbuscular mycorrhizal fungus (AMF) did not have any significant effect on the EO composition in oregano; however, in two genotypes the EO concentration significantly increased. As EO levels in P-treated plants were not enhanced, we conclude that the EO increase observed in mycorrhizal oregano plants is not due to an improved P status in mycorrhizal plants, but depends directly on the AMF-oregano plant association.

Source apportionment of molecular markers and organic aerosol. 3. Food cooking emissions.

The chemical mass balance model is applied to a large dataset of organic molecular marker concentrations to apportion ambient organic aerosol to food cooking emissions in Pittsburgh, Pennsylvania. Ambient concentrations of key cooking markers such as palmitoleic acid, oleic acid, and cholesterol are well correlated, which implies the existence of well-defined source profiles. However, significant inconsistencies exist between the ambient data and published source profiles. Most notably, the ambient ratio of palmitoleic-acid-to-oleic-acid is more than a factor of 10 greater than essentially all published source profiles. This problem is not unique to Pittsburgh. The reason for this discrepancy is not known but it means that both acids cannot be fit simultaneously by CMB. CMB analysis is performed using three different combinations of food cooking source profiles and molecular markers. Although all three solutions have high statistical quality, the amount of OC apportioned to food cooking emissions varies by a factor of 9. Differences in fitting species and source profile marker-to-organic-carbon ratios cause most of the large systematic biases between the different solutions. The best CMB model includes two alkanoic acids as fitting species in addition to other cooking markers, which helps constrain the source contribution estimates. It also includes two meat cooking source profiles to account for the variability in the ambient data. This model apportions 320+/-140 ng-C m(-3) or 10% of the study average ambient organic carbon to food cooking emissions. Although these results illustrate the significant challenges created by source profile variability, the strong correlations in the ambient dataset underscore the significant promise that molecular markers hold for source apportionment analysis.

A comparison of propofol and remifentanil for sedation and limitation of movement during periretrobulbar block.

STUDY OBJECTIVES: To compare clinical conditions in patients sedated with propofol or remifentanil during combined peri-bulbar and retrobulbar block (PRBB) for cataract surgery. DESIGN: Prospective, randomized, double-blind study. SETTING: Private clinic. PATIENTS: 106 ASA physical status I and II patients scheduled for cataract surgery. INTERVENTIONS: Patients were randomized to receive either 0.5 mg/kg propofol (Group P) or 0.3 microg/kg remifentanil (Group R) as an intravenous (IV) bolus 1 minute prior to PRBB. At the same time, patients in both groups also received 0.5 to 1 mg midazolam IV. Movement of the hands, arms, head, and eyes were counted during each stage of the procedure by an observer who was blinded to the sedation used. Heart rate (HR), blood pressure (BP), respiratory rate (RR), expiratory CO(2) (PECO(2)), and hemoglobin oxygen saturation (SaO(2)) were recorded every minute for 10 minutes after the PRBB. Anesthetic complications, recall, and the pain experienced with the block and surgery were compared between the two groups. Means and variance of the results were compared with one-way analysis of variance and Fisher's exact test. MEASUREMENTS AND MAIN RESULTS: Movements of the hands, arms, and head were significantly greater in Group P during all stages of the block. Almost no movements were recorded in the remifentanil group. Immediately after the PRBB (1 to 6 min), HRs were higher in Group P (73 +/- 11 bpm vs. 67 +/- 10 bpm; p = 0.0075), whereas the RRs were slower in Group R for the period 1 to 5 minutes after the PRBB (16 +/- 5 breaths/min vs.14 +/- 4 breaths/min; p = 0.0206). At these times, the mean PECO(2) was higher in Group R (36 +/- 7 mmHgvs. 32 +/- 9 mmHg; p = 0.0125). Nineteen patients in the propofol group sneezed during the medial peribulbar injection compared with none in the remifentanil group. Anesthetic and surgical complications were unremarkable and similar for the two groups. CONCLUSIONS: Respiratory depression with remifentanil was mild and not clinically significant. Remifentanil sedation for this application was superior to sedation with propofol.

Efficacy of bactericidal/permeability-increasing protein in experimental otitis media with effusion in rats: a new therapy for mucosal infections.

Otitis media with effusion (OME) is characterized by the presence of fluid in the middle ear without signs or symptoms of acute infection and by persistent changes in the middle ear mucosa. These are mainly induced by gram-negative bacterial infection and dysfunction of the eustachian tube (ET). Gram-negative bacteria (GNB) contain lipopolysaccharide (LPS) in their outer membrane that is responsible for inflammatory reactions in the middle ear. In this study we investigated the therapeutic effect of a recombinant LPS-binding protein, bactericidal/permeability-increasing protein (rBPI21), on the repair of mucosal damage in rats with experimentally induced OME. OME was induced by obstruction of the eustachian tube in combination with LPS injection. Twelve weeks after OME induction, secretory cells in the tympanic orifice of the middle ear were increased from an average of 14 +/- 2 to 31 +/- 5, ciliated cells were decreased from 24 +/- 4 to 6 +/- 4, and the number of macrophages in the subepithelial layer increased from 13 +/- 4 to 27 +/- 3. A single dose of rBPI21 was administered directly into the middle ear cavity 2 weeks after the induction of OME. Histologic examination of the middle ear mucosa at 4 and 12 weeks after OME induction showed that mucosal changes were restored by rBPI21 treatment. These results demonstrate that the middle ear mucosa recovers from inflammatory changes associated with OME after treatment with rBPI21. This suggests that rBPI21 may be useful in the treatment of OME and of mucosal infections of the respiratory tract.

Inhibition of endotoxin effects on cultured human middle ear epithelium by bactericidal permeability-increasing protein.

HYPOTHESIS/BACKGROUND: Endotoxin can induce morphologic changes to middle ear epithelium, which can disturb the mucociliary clearance system (MCS) and lead to otitis media with effusion (OME). The bactericidal/permeability-increasing (BPI) protein is a major component of neutrophil granules and binds with high affinity to endotoxin. In this study, the capacity to inhibit the effects of endotoxin by rBPI21, a recombinant amino-terminal analog derived from BPI, was investigated on cultured human middle ear epithelium using light microscopy and scanning- and transmission electron microscopy. METHODS: Human middle ear epithelium was air-exposed cultured on a collagenous underlayer with different additions of endotoxin and rBPI21 to the culture medium. The tissue specimens were inspected after 4 weeks for the number of ciliated and secretory cells, thickness of the mucosal layer, and cell size. RESULTS: The morphologic changes induced by endotoxin were increased thickness of the mucosal layer and increased number of secretory cells. These changes were significantly diminished or even absent when endotoxin was added with rBPI21 to the culture medium. CONCLUSION: rBPI21 can inhibit morphologic changes in the middle ear epithelium due to endotoxin. Hence, the authors believe that rBPI21 can be a new therapeutic agent in the treatment of OME.

Bactericidal/permeability-increasing protein prevents mucosal damage in an experimental rat model of chronic otitis media with effusion.

In this study, the efficacy of bactericidal/permeability-increasing protein (BPI) was assessed in a rat model of chronic otitis media with effusion. BPI injection prevented disturbance of the mucociliary clearance system of the middle ear. Hence, it is postulated that BPI can be a new therapy for chronic otitis media with effusion.

Topical anesthesia versus retrobulbar block for cataract surgery: the patients' perspective.

STUDY OBJECTIVES: To compare patients' perception of topical anesthesia (TA) with combined peribulbar and retrobulbar block (PRBB) for cataract surgery. DESIGN: Prospective, randomized, controlled, cross-over observational study. SETTING: Private clinic. PATIENTS: 98 ASA physical status I and II patients presenting for bilateral cataract surgery 1 week apart. INTERVENTIONS: Patients were prospectively randomized to receive either TA for surgery to one eye, followed by PRBB for surgery to the other eye 1 week later, or to receive PRBB first, followed by TA for the second operation the following week. Surgery, PRBB, and TA were standard for all cases. Interviews were conducted the day following surgery by an unbiased observer unaware of the technique used. Surgical pain was estimated on a visual analog scale of 0 to 10, and the surgeon judged the difficulty of surgery based on patient compliance and cooperation on a scale of 0 to 5. Means and variance of results were compared with analysis of variance. MEASUREMENTS AND MAIN RESULTS: Mean age was 71.45 +/- 9.76 years (mean +/- SD). Seventy patients (71.43%) preferred PRBB while 10 patients (10.20%) preferred TA (p = 0.0001). Eighteen patients (18.37%) reported no difference between the two techniques. Ninety-six patients (97.96%) were not aware of the PRBB being injected. Duration of surgery was similar for TA (11.92 +/- 3.43 min) and PRBB (10.78 +/- 3.00 min; p = 0.06). Surgery was more difficult during TA (p = 0.0004). Pain was worse during TA (p = 0.0001). Surgical and anaesthetic complications were unremarkable for both techniques. CONCLUSIONS: Patients who experienced both TA and PRBB preferred PRBB.

Mobilizing demand for contraception in rural Gambia.

A community trial was conducted in rural Gambia in order to determine whether a community-based intervention designed to mobilize latent demand for contraception would increase use of modern contraceptives, even in the absence of improved availability of family planning services. Analysis of trial data indicates that the demand-mobilization intervention had a statistically significant positive effect on nonusers' adoption of modern contraception and that coterminous implementation of an intervention designed to improve access to services offered no additional benefit. The program component found to have the greatest impact was the "kabilo approach," in which village women provide basic health and family planning counseling to other women in their extended families. These results suggest that the principal barriers to increased contraceptive use in rural Gambia are psychosocial and that these barriers can be overcome through village-based interventions designed to provide socially appropriate counseling to potential contraceptive users.

Endotoxin and tumor necrosis factor-alpha in middle ear effusions in relation to upper airway infection.

OBJECTIVES/HYPOTHESIS: This study was performed to elucidate the role of endotoxin and tumor necrosis factor-alpha (TNF-alpha) in the middle ear effusions (MEEs) of children with otitis media with effusion (OME) in relation to the chronicity of the disease and the presence of upper respiratory tract infection (URTI). STUDY DESIGN: In a retrospective study 140 MEEs were collected from 101 children between 2 and 12 years of age, and evaluated for the cytokine TNF-alpha and the lipopolysaccharide endotoxin. The amounts were quantified and correlated with the type of MEE, OME duration, and the presence of URTI. METHODS: Endotoxin levels were measured using a limulus amebocyte lysate assay and TNF-alpha levels were measured with an enzyme-linked immunosorbent assay (ELISA). Means of the different variables were compared using the one-way ANOVA least significance difference test with P<.05. RESULTS: In MEEs classified as mucopurulent (22.8%) both endotoxin and TNF-alpha levels (11.9+/-3 ng/mg total protein and 61.1+/-21 pg/mg total protein, respectively) were significantly higher compared with serous- (23.6%) or mucoid- (53.6%) type effusions. Fifty-five percent of the children who were classified as having chronic OME also had significantly higher amounts of endotoxin and TNF-alpha. The majority of the children (61%) had no URTI, although children with URTI (36%) did also have significantly higher levels of endotoxin and TNF-alpha in their middle ears. CONCLUSIONS: These results indicate that there is a strong correlation between the endotoxin and the TNF-alpha concentration in the middle ear and the type of MEE, the presence of URTI, and the chronicity of the disease.

Effect of endotoxin on cultured human middle ear epithelium.

The effect of endotoxin was investigated on air-exposed cultured human middle ear epithelium. Concentrations of 0, 1 and 100 ng/ml endotoxin were used. Complete differentiation of the cells was not reached at 12 days. After 21 days, endotoxin had induced an increased proliferation of the epithelial layer. Furthermore, an increase in the number of secretory cells and in the amount and length of microvilli was observed at this time. There were no significant morphological differences between the high and the low endotoxin concentrations, which supports our hypothesis that endotoxin induces an all-or-nothing reaction. These findings are in agreement with our previous results on serially submerged cultured rat middle ear epithelium. From these results we conclude that endotoxin is an important factor in the disturbance of the morphology of the middle ear epithelium, which may lead to chronic otitis media with effusion. In addition, our tissue culture method proved to be a good model for further studies on human middle ear mucosa.

Structural changes in the rat middle ear mucosa due to endotoxin and eustachian tube obstruction.

The middle ears of 48 rats were used to examine the effects of endotoxin injection, eustachian tube obstruction or a combination of eustachian tube obstruction and endotoxin injection. Animals were killed after 1, 2, 4, or 12 weeks and the middle ears processed for light and scanning electron microscopy. Compared to the normal middle ear mucosa, the epithelial layer was more pseudostratified, cuboidal or cylindrical after endotoxin injection or obstruction of the eustachian tube. In the early phase, numerous ciliated cells occurred in areas originally almost devoid of these cells. At 3 months, degeneration of ciliated cells was observed. The combination of eustachian tube obstruction and endotoxin injection also induced a more pseudostratified, cuboidal or cylindrical epithelium with an increased number of goblet cells. However, an early decrease occurred in the number of ciliated cells in the tympanic orifice of the eustachian tube. Furthermore, inflammatory cells, mainly PMNs, macrophages and lymphocytes, invaded the subepithelial layer after eustachian tube obstruction and endotoxin injection. These structural changes resulted in an impairment of the mucociliary transport system for clearance of the middle ear cavity. For this reason we believe that both endotoxin and eustachian tube obstruction or dysfunction play an important role in inducing persistent mucosal changes in the middle ear cavity, thereby prolonging otitis media with effusion.