Turbulence suppression by cardiac-cycle-inspired driving of pipe flow.

Flows through pipes and channels are, in practice, almost always turbulent, and the multiscale eddying motion is responsible for a major part of the encountered friction losses and pumping costs1. Conversely, for pulsatile flows, in particular for aortic blood flow, turbulence levels remain low despite relatively large peak velocities. For aortic blood flow, high turbulence levels are intolerable as they would damage the shear-sensitive endothelial cell layer2-5. Here we show that turbulence in ordinary pipe flow is diminished if the flow is driven in a pulsatile mode that incorporates all the key features of the cardiac waveform. At Reynolds numbers comparable to those of aortic blood flow, turbulence is largely inhibited, whereas at much higher speeds, the turbulent drag is reduced by more than 25%. This specific operation mode is more efficient when compared with steady driving, which is the present situation for virtually all fluid transport processes ranging from heating circuits to water, gas and oil pipelines.

Increased matrix metalloproteinases expression in tuberous sclerosis complex: modulation by microRNA 146a and 147b in vitro.

AIM: Matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs) control proteolysis within the extracellular matrix (ECM) of the brain. Dysfunction of this enzymatic system due to brain inflammation can disrupt the blood-brain barrier (BBB) and has been implicated in the pathogenesis of epilepsy. However, this has not been extensively studied in the epileptogenic human brain. METHODS: We investigated the expression and cellular localization of major MMPs (MMP2, MMP3, MMP9 and MMP14) and TIMPs (TIMP1, TIMP2, TIMP3 and TIMP4) using quantitative real-time polymerase chain reaction (RT-PCR) and immunohistochemistry in resected epileptogenic brain tissue from patients with tuberous sclerosis complex (TSC), a severe neurodevelopmental disorder characterized by intractable epilepsy and prominent neuroinflammation. Furthermore, we determined whether anti-inflammatory microRNAs, miR146a and miR147b, which can regulate gene expression at the transcriptional level, could attenuate dysregulated MMP and TIMP expression in TSC tuber-derived astroglial cultures. RESULTS: We demonstrated higher mRNA and protein expression of MMPs and TIMPs in TSC tubers compared to control and perituberal brain tissue, particularly in dysmorphic neurons and giant cells, as well as in reactive astrocytes, which was associated with BBB dysfunction. More importantly, IL-1ß-induced dysregulation of MMP3, TIMP2, TIMP3 and TIMP4 could be rescued by miR146a and miR147b in tuber-derived TSC cultures. CONCLUSIONS: This study provides evidence of dysregulation of the MMP/TIMP proteolytic system in TSC, which is associated with BBB dysfunction. As dysregulated MMP and TIMP expression can be ameliorated in vitro by miR146a and miR147b, these miRNAs deserve further investigation as a novel therapeutic approach.

Purinergic receptors P2Y12R and P2X7R: potential targets for PET imaging of microglia phenotypes in multiple sclerosis.

BACKGROUND: Microglia are major players in the pathogenesis of multiple sclerosis (MS) and may play a dual role in disease progression. The activation status of microglia in vivo is highly dynamic and occurs as a continuum, with the pro-inflammatory and anti-inflammatory phenotypes on either end of this spectrum. Little is known about in vivo dynamics of microglia phenotypes in MS due to the lack of diagnostic tools. Positron emission tomography (PET) imaging is a powerful non-invasive technique that allows real-time imaging of microglia activation phenotypes in the central nervous system, depending on the availability of selective PET tracers. Our objective is to investigate and characterize the expression of the purinergic receptors P2Y12R and P2X7R as potential targets for PET tracer development and subsequent PET imaging in order to evaluate the dynamics of microglia status in vivo. METHODS: We used immunohistochemical analysis to explore the expression of P2Y12R and P2X7R in experimental autoimmune encephalomyelitis (EAE) post-mortem tissues and different stages of well-characterized MS lesions. We evaluated by quantitative real-time polymerase chain reaction the expression of P2Y12R and P2X7R in human polarized microglia, and we performed autoradiography binding assay with radiolabeled P2Y12R and P2X7R antagonists using MS and rat EAE tissues. RESULTS: Here, we demonstrate that P2X7R is associated with a pro-inflammatory phenotype of human microglia in vitro, and is highly expressed in microglia in MS lesions as well as during the peak of EAE. In contrast, P2Y12R was associated with an anti-inflammatory phenotype in human microglia in vitro and was expressed at lower levels in active inflammatory MS lesions compared to normal-appearing white matter (NAWM) and similarly in EAE, while its expression increased in the remission phase of EAE. Binding of radiolabeled tracers specific for P2Y12R and P2X7R on ex vivo tissues validated the value of these receptors as PET imaging targets for microglia phenotypes in vivo. CONCLUSION: Our results suggest that P2Y12R and P2X7R are excellent targets for PET imaging to discriminate distinct microglia phenotypes in MS. Ultimately, this may provide insight into the role of microglia in disease progression and monitor novel treatment strategies to alter microglia phenotype.

Streamwise-localized solutions at the onset of turbulence in pipe flow.

Although the equations governing fluid flow are well known, there are no analytical expressions that describe the complexity of turbulent motion. A recent proposition is that in analogy to low dimensional chaotic systems, turbulence is organized around unstable solutions of the governing equations which provide the building blocks of the disordered dynamics. We report the discovery of periodic solutions which just like intermittent turbulence are spatially localized and show that turbulent transients arise from one such solution branch.

Liver X receptor alpha ensures blood-brain barrier function by suppressing SNAI2.

In Alzheimer's disease (AD) more than 50% of the patients are affected by capillary cerebral amyloid-angiopathy (capCAA), which is characterized by localized hypoxia, neuro-inflammation and loss of blood-brain barrier (BBB) function. Moreover, AD patients with or without capCAA display increased vessel number, indicating a reactivation of the angiogenic program. The molecular mechanism(s) responsible for BBB dysfunction and angiogenesis in capCAA is still unclear, preventing a full understanding of disease pathophysiology. The Liver X receptor (LXR) family, consisting of LXRα and LXRß, was reported to inhibit angiogenesis and particularly LXRα was shown to secure BBB stability, suggesting a major role in vascular function. In this study, we unravel the regulatory mechanism exerted by LXRα to preserve BBB integrity in human brain endothelial cells (BECs) and investigate its role during pathological conditions. We report that LXRα ensures BECs identity via constitutive inhibition of the transcription factor SNAI2. Accordingly, deletion of brain endothelial LXRα is associated with impaired DLL4-NOTCH signalling, a critical signalling pathway involved in vessel sprouting. A similar response was observed when BECs were exposed to hypoxia, with concomitant LXRα decrease and SNAI2 increase. In support of our cell-based observations, we report a general increase in vascular SNAI2 in the occipital cortex of AD patients with and without capCAA. Importantly, SNAI2 strongly associated with vascular amyloid-beta deposition and angiopoietin-like 4, a marker for hypoxia. In hypoxic capCAA vessels, the expression of LXRα may decrease leading to an increased expression of SNAI2, and consequently BECs de-differentiation and sprouting. Our findings indicate that LXRα is essential for BECs identity, thereby securing BBB stability and preventing aberrant angiogenesis. These results uncover a novel molecular pathway essential for BBB identity and vascular homeostasis providing new insights on the vascular pathology affecting AD patients.

Edge state in pipe flow experiments.

Recent numerical studies suggest that in pipe and related shear flows, the region of phase space separating laminar from turbulent motion is organized by a chaotic attractor, called an edge state, which mediates the transition process. We here confirm the existence of the edge state in laboratory experiments. We observe that it governs the dynamics during the decay of turbulence underlining its potential relevance for turbulence control. In addition we unveil two unstable traveling wave solutions underlying the experimental flow fields. This observation corroborates earlier suggestions that unstable solutions organize turbulence and its stability border.

Relaminarization by Steady Modification of the Streamwise Velocity Profile in a Pipe.

We show that a rather simple, steady modification of the streamwise velocity profile in a pipe can lead to a complete collapse of turbulence and the flow fully relaminarizes. Two different devices, a stationary obstacle (inset) and a device which injects fluid through an annular gap close to the wall, are used to control the flow. Both devices modify the streamwise velocity profile such that the flow in the center of the pipe is decelerated and the flow in the near wall region is accelerated. We present measurements with stereoscopic particle image velocimetry to investigate and capture the development of the relaminarizing flow downstream these devices and the specific circumstances responsible for relaminarization. We find total relaminarization up to Reynolds numbers of 6000, where the skin friction in the far downstream distance is reduced by a factor of 3.4 due to relaminarization. In a smooth straight pipe the flow remains completely laminar downstream of the control. Furthermore, we show that transient (temporary) relaminarization in a spatially confined region right downstream the devices occurs also at much higher Reynolds numbers, accompanied by a significant local skin friction drag reduction. The underlying physical mechanism of relaminarization is attributed to a weakening of the near-wall turbulence production cycle.

Differences in sulfation patterns of heparan sulfate derived from human bone marrow and umbilical vein endothelial cells.

OBJECTIVE: Heparan sulfates (HS), the polysaccharide side chains of HS proteoglycans, differ in structure and composition of sulfated domains among various tissue types, resulting in selective protein binding. HS proteoglycans on bone marrow endothelial cells (BMEC) could contribute to tissue specificity of the bone marrow endothelium and play a role in the presentation of chemokines such as stromal cell-derived factor-1 (SDF-1) and adhesion of hematopoietic progenitor cells after stem cell transplantations. We characterized differences in HS structure and SDF-1 binding between BMEC and human umbilical vein endothelial cells (HUVEC). MATERIALS AND METHODS: Expression of HS proteoglycans on human bone marrow microvessels was investigated by immunohistochemical staining. Comparison of three human BMEC cell lines with HUVEC and an HUVEC cell line was studied by flow cytometry using antibodies against different epitopes of the HS polysaccharide chain. HS proteoglycans were biochemically characterized after isolation from metabolically labeled cultures of the BMEC cell line 4LHBMEC and HUVEC. Binding of radiolabeled SDF-1 to 4LHBMEC and HUVEC and competition with heparins were investigated. RESULTS: Bone marrow microvessels constitutively expressed HS proteoglycans. Flow cytometric experiments showed differences in HS chain composition between BMEC and HUVEC. Biochemical characterization revealed more O-sulfation of the N-sulfated domains present in cell-associated HS glycosaminoglycans in 4LHBMEC compared to HUVEC. Binding experiments showed that 4LHBMEC bound more 125[I]-SDF-1 per cell than HUVEC. This could be inhibited largely by heparin and O-sulfated heparin and to a lesser extent by N-sulfated heparin. CONCLUSIONS: Cellular HS from BMEC differs in composition from HUVEC. We postulate that the presence of highly sulfated domains in the HS chains from BMEC contributes to tissue specificity of bone marrow endothelium in which HS may be involved in SDF-1 presentation and adhesion of hematopoietic progenitor cells.

Evaluation of three physiologically based pharmacokinetic (PBPK) modeling tools for emergency risk assessment after acute dichloromethane exposure.

INTRODUCTION: Physiologically based pharmacokinetic (PBPK) models may be useful in emergency risk assessment, after acute exposure to chemicals, such as dichloromethane (DCM). We evaluated the applicability of three PBPK models for human risk assessment following a single exposure to DCM: one model is specifically developed for DCM (Bos) and the two others are semi-generic ones (Mumtaz and Jongeneelen). MATERIALS AND METHODS: We assessed the accuracy of the models' predictions by simulating exposure data from a previous healthy volunteer study, in which six subjects had been exposed to DCM for 1h. The time-course of both the blood DCM concentration and percentage of carboxyhemoglobin (HbCO) were simulated. RESULTS: With all models, the shape of the simulated time course resembled the shape of the experimental data. For the end of the exposure, the predicted DCM blood concentration ranged between 1.52-4.19mg/L with the Bos model, 1.42-4.04mg/L with the Mumtaz model, and 1.81-4.31mg/L with the Jongeneelen model compared to 0.27-5.44mg/L in the experimental data. % HbCO could be predicted only with the Bos model. The maximum predicted % HbCO ranged between 3.1 and 4.2% compared to 0.4-2.3% in the experimental data. The % HbCO predictions were more in line with the experimental data after adjustment of the Bos model for the endogenous HbCO levels. CONCLUSIONS: The Bos Mumtaz and Jongeneelen PBPK models were able to simulate experimental DCM blood concentrations reasonably well. The Bos model appears to be useful for calculating HbCO concentrations in emergency risk assessment.

Increased alpha3-fucosylation of alpha(1)-acid glycoprotein in patients with congenital disorder of glycosylation type IA (CDG-Ia).

Increased fucosylation of the type (sialyl) Lewis(x) was detected on the acute-phase plasma protein alpha(1)-acid glycoprotein (AGP) in patients with the congenital disorder of glycosylation type IA. This is remarkable, because in these patients the biosynthesis of guanosine 5'-diphosphate (GDP)-D-mannose is strongly decreased, and GDP-D-mannose is the direct precursor for GDP-L-fucose, the substrate for fucosyltransferases. The concomitantly occurring increased branching of the glycans of AGP and the increased fucosyltransferase activity in plasma suggest that a chronic hepatic inflammatory reaction has induced the increase in fucosylation.

Scanning electron microscopic analysis of the stapled microvascular anastomosis in the rabbit.

BACKGROUND: Despite the high percentages of experimental and clinical patency rates achieved using so-called mechanical anastomotic devices (Unilink; 3M, St. Paul, MN; vascular staples) they remain little known and occasionally used. METHODS: The VCS Auto Suture microstapler technique for microvascular anastomosis was tested experimentally and compared with the conventional "gold standard" 10/0 end-to-end microvascular technique. Thirty carotid arteries on one side of 30 rabbits were stapled using nonpenetrating 0.9-mm (small) VCS Auto Suture microclips, and the other 30 carotid arteries on the other side were sutured in a conventional way with 10/0 monofilament nylon. A 100% patency rate was achieved on both sides. Biopsy was performed in five groups of rabbits at different time intervals postoperatively, and the specimens were examined under scanning electron microscopy. RESULTS: All 60 anastomoses were patent. Histomorphologic examination of the anastomotic site revealed no major differences between sutured and stapled groups. CONCLUSIONS: Stapled microvascular anastomosis technique is fast and reliable.

Efficacy and tolerability of slow release urapidil (ebrantil) in hypertensive patients with non-insulin dependent diabetes mellitus (NIDDM).

Urapidil, a new alpha-blocker that peripherally antagonizes postsynaptic alpha 1 receptors and centrally causes a reduction in sympathetic tone as agonist of the serotoninergic receptors, was assessed for its antihypertensive effect and its role on glucose and lipid metabolism and insulin sensitivity in diabetic hypertensive patients. Thirty-three non-insulin dependent diabetes mellitus (NIDDM) patients with diastolic blood pressure (BP) of 95-115 mm Hg were treated with either 30 or 60 mg urapidil twice a day, with a gradual increment up to a maximum of 90 mg b.i.d., in order to reduce diastolic pressure to < 90 mm Hg or by at least 10% in the sitting position. A significant reduction in systolic and diastolic arterial pressure, not accompanied by an increased heart rate, was achieved after 12 weeks of treatment. Lipid and carbohydrate homeostasis and glycemic control, as assessed by HbA1C levels, were not affected. The fasting insulin concentration before the glucose load remained similar, but there was a trend toward reduction in peak insulin concentration, and the ratio of insulin change to glucose change between fasting levels and peak levels was significantly lowered by treatment, suggesting improved insulin sensitivity. In conclusion, urapidil is an effective antihypertensive agent in NIDDM patients with essential hypertension, with a neutral effect on lipids and carbohydrates and a possible beneficial effect on insulin resistance.

Silicones in the rehabilitation of burns: a review and overview.

This article gives an overview of the use of silicones in the treatment and prevention of hypertrophic (burn related) scars. Of all non-invasive treatment modalities the use of continuous pressure and occlusive contact media, e.g. silicones, seem to be generally accepted as the only ones that are able to manage hypertrophic scarring without significant side-effects. A summary of the current opinions of the assumed working mechanisms of pressure as well as silicones is given. The use of silicones, either alone or in combination with pressure, is discussed. The recent development of custom made silicone devices has led to combinations of both modalities. Some of these, including the inflatable silicone insert systems (ISIS), are shown and discussed.

Progress in larynx-sparing surgery for glottic cancer through tracheal transplantation.

The current surgical treatment for unilateral, advanced glottic cancer is a total laryngectomy. Usually, the noninvolved hemilarynx needs resection because the resulting laryngeal defect cannot be reconstructed after adequate tumor resection. Experimental findings suggest that segments of autologous trachea may restore extended laryngeal defects. The authors used tracheal transplantation to save laryngeal function after the removal of advanced glottic cancer. In this case series review, 10 patients were treated during a 1.5-year period, with an average follow-up of 8 months. Evaluated factors included survival of the tracheal transplant and functional outcome with regard to the onset and quality of the airway, speech, and deglutition. The authors showed that segments of cervical trachea may restore extended laryngeal defects after initial revascularization by a radial forearm fascial flap. The fascial flap served as a vascular carrier for the transplanted trachea. Follow-up showed the stability of the reconstruction. Compared with a total laryngectomy, a striking improvement in patient comfort and function was noticed. Transplantation of the trachea is a technique that may save laryngeal function after the treatment of advanced-stage glottic cancer. These findings may improve laryngeal preservation strategies in treating laryngeal cancer.

Prefabrication of composite tissue for improved tracheal reconstruction.

Tracheal repair tissues were evaluated experimentally to provide an evidence-based choice for decision-making in clinical tracheal reconstruction. Tracheal reconstructive tissue was characterized as providing for vascularization, support, and/or lining. A tissue equivalent was developed in the rabbit for each of the individual tissues. The individual tissues consisted of nonepithelialized soft tissue (vascularized fascia), epithelialized tissue (vascularized fascia grafted with buccal mucosa), and supportive tissue (ear cartilage). The 3 reconstructive tissues were evaluated in 30 rabbits after repair of an anterior laryngotracheal defect. Morphometric and histologic analysis was applied to the reconstructions. After a 1-month follow-up period, defects repaired with nonepithelialized soft tissue showed healing by secondary intention and a wound that was contracted to 44% of the initial surface area of the defect. Mucosa-lined soft tissue flaps and cartilage grafts showed a less than 10% wound contraction. Compared to cartilage grafts, mucosalined soft tissue (vascularized fascia grafted with buccal mucosa) seemed preferable for clinical use, because it showed healing by primary intention. A mucosa-lined radial forearm fascia flap was used successfully in cases of restenosis after tracheal resection. One deficiency of the mucosa-lined soft tissue was the absence of supportive tissue. In cases of extensive stenosis, it might be useful to obtain additional expansion of the airway lumen by creating a convexity at the site of reconstruction. In a second set of experiments, we attempted to improve the mucosa-lined soft tissue concept by adding elastic cartilage. A composite tissue consisting of vascularized fascia, buccal mucosa, and auricular cartilage was developed. Heterotopic prefabrication of the composite tissue was essential for survival of the cartilaginous component. Additional airway lumen expansion could be obtained after heterotopic flap prefabrication. After experimental evaluation, the concept of the prefabricated composite tissue was successfully applied in a clinical case of long-segment stenosis. Experimental and clinical evidence suggests that the combination of buccal mucosa and fascia form an optimized tissue combination for tracheal reconstruction. This combination can be improved by adding strips of autologous ear cartilage.

Interpreting microbiopsies in cervical smears. A cytohistologic approach.

OBJECTIVE: To assess interobserver variation in the diagnosis of thick tissue specimens (microbiopsies) in cytology smears and histologic sections taken from them, to evaluate the applicability of MIB-1 in histologic sections from microbiopsies and to evaluate whether processing microbiopsies in inconclusive smears has additional diagnostic value. STUDY DESIGN: Cytologic smears were selected in which there were diagnostic disagreements between pathologists and cytologists and microbiopsies were present. Interobserver variation among three pathologists and three cytologists in the diagnosis of these microbiopsies was investigated. The smears were processed for histologic sections, and interobserver variation between pathologist diagnoses were analyzed. An additional histologic slide stained for MIB-1 was used for consensus diagnosis. The consensus diagnosis was compared with available follow-up and its sensitivity and specificity determined. The value of applying the microbiopsy technique in slides diagnosed as inadequate or atypical squamous cells of undetermined significance (ASCUS) was analysed. RESULTS: From a series of 62,334 cervical smears, 49 with microbiopsies were selected. It was possible to derive histologic slides from 38 cases. Interobserver variability in the diagnosis of microbiopsies and histologic sections from them was moderate--kappa = .44 (SE = .06) and kappa = .44 (SE = .09), respectively. In the consensus meeting for all cases, a conclusive diagnosis was reached. The Pearson correlation coefficient between the consensus diagnosis and MIB-1 staining was r = .62. The sensitivity of the consensus diagnosis for the follow-up diagnosis was 71% and the specificity 60%. Diagnosis on approximately 50% of slides diagnosed as inadequate or ASCUS could be made. CONCLUSION: The histotechnical workup of microbiopsies is not difficult; however, their diagnosis can be a problem. Adequate diagnostic criteria are not available. Aided by MIB-1 staining, histologic sections from microbiopsies can be diagnosed, and the diagnoses correlated with follow-up in most cases. Processing of microbiopsies in smears with an inconclusive cytologic diagnosis or a diagnosis of ASCUS allowed correct diagnosis in 50% of cases in this study.

Review of acute chemical incidents as a first step in evaluating the usefulness of physiologically based pharmacokinetic models in such incidents.

CONTEXT. Acute chemical incidents can have substantial public health consequences in terms of morbidity and mortality. OBJECTIVE. We aimed to characterize acute chemical incidents and near-misses in the Netherlands and compare the results with previous studies. This review is a first step in evaluating whether Physiologically Based Pharmacokinetic (PBPK) models can be of value in acute chemical incidents. MATERIAL AND METHODS. Government, regional, municipal and University Hospital Institutes involved in the management of acute chemical incidents in the Netherlands were contacted, and they provided data between 2008 and 2010 on the characteristics and consequences of the incidents. The study is a retrospective epidemiological study based on data from five institutes. Incidents involving biological agents or radiation were excluded. RESULTS. A total of 764 reports were available which involved 722 incidents after cross-matching the different sources of data. Forty incidents were excluded, leaving 682 incidents for which information was available in accordance with the inclusion criteria. Of the 682 incidents included in this study, most occurred in non-industrial buildings (37%) or industrial sites (34%). The most frequently observed event types were loss of containment (60%) and fire (36%), leading to gas emission (54%), followed by spill of liquid or solid chemicals (36%). The chemicals involved were most often products of combustion (e.g. smoke, soot, particles, 25%) and volatile organic compounds (e.g. solvents, styrene, xylene, 23%), followed by inorganic gases (e.g. carbon monoxide, hydrogen, hydrogen sulphide, 13%). A minimum of 847 people experienced adverse health effects following exposure during a chemical incident, and 10 fatalities were reported. The most frequently reported symptoms were respiratory (27%), due to irritant chemicals. The number of incidents related to fire and the number of injured people were higher in this study than in previous studies; 49% of the injured were transported to hospital. DISCUSSION. This study helps to identify which chemicals are frequently involved in acute chemical incidents in the Netherlands. The results will be used in future to assess whether PBPK models may be useful for risk assessment of chemicals often involved in acute chemical incidents and for which human toxicological and kinetic data are scarce.

Nature of laminar-turbulence intermittency in shear flows.

In pipe, channel, and boundary layer flows turbulence first occurs intermittently in space and time: at moderate Reynolds numbers domains of disordered turbulent motion are separated by quiescent laminar regions. Based on direct numerical simulations of pipe flow we argue here that the spatial intermittency has its origin in a nearest neighbor interaction between turbulent regions. We further show that in this regime turbulent flows are intrinsically intermittent with a well-defined equilibrium turbulent fraction but without ever assuming a steady pattern. This transition scenario is analogous to that found in simple models such as coupled map lattices. The scaling observed implies that laminar intermissions of the turbulent flow will persist to arbitrarily large Reynolds numbers.

Scaling of the turbulence transition threshold in a pipe.

We report the results of an experimental investigation of the transition to turbulence in a pipe over approximately an order of magnitude range in the Reynolds number Re. A novel scaling law is uncovered using a systematic experimental procedure which permits contact to be made with modern theoretical thinking. The principal result we uncover is a scaling law which indicates that the amplitude of perturbation required to cause transition scales as O(Re-1).

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.