Exposure to anaesthetic trace gases during general anaesthesia: CobraPLA vs. LMA classic.

BACKGROUND: To prospectively investigate the performance, sealing capacity and operating room (OR) staff exposure to waste anaesthetic gases during the use of the Cobra perilaryngeal airway (CobraPLA) compared with the laryngeal mask airway classic (LMA). METHODS: Sixty patients were randomly assigned to the CobraPLA or the LMA group. Insertion time, number of insertion attempts and airway leak pressures were assessed after induction of anaesthesia. Occupational exposure to nitrous oxide (N(2)O) and Sevoflurane (SEV) was measured at the anaesthetists' breathing zone and the patients' mouth using a photoacoustic infrared spectrometer. RESULTS: N(2)O waste gas concentrations differed significantly in the anaesthetist's breathing zone (11.7+/-7.2 p.p.m. in CobraPLA vs. 4.1+/-4.3 p.p.m. in LMA, P=0.03), whereas no difference could be shown in SEV concentrations. Correct CobraPLA positioning was possible in 28 out of 30 patients (more than one attempt necessary in five patients). Correct positioning of the LMA classic was possible in all 30 patients (more than one attempt in three patients). Peak airway pressure was higher in the CobraPLA group (16+/-3 vs. 14+/-2 cmH(2)O, P=0.01). The average leak pressure of the CobraPLA was 24+/-4 cmH(2)O, compared with 20+/-4 cmH(2)O of the LMA classic (P<0.001; all values means+/-SD). CONCLUSION: Despite higher airway seal pressures, the CobraPLA caused higher intraoperative N(2)O trace concentrations in the anaesthetists' breathing zone.

[Evidence-based homeopathy and veterinary homeopathy, and its potential to help overcome the anti-microbial resistance problem - an overview]. / Evidenzbasierte Veterinär-/Homöopathie und ihre mögliche Bedeutung für die ­Bekämpfung der Antibiotikaresistenzproblematik ­ ein Überblick.

INTRODUCTION: The basic principles of homeopathy, and its legal and scientific foundations, are discussed in an overview to address the positions of the World Health Organization (WHO) and the commission of the European Union (EU) on complementary medicine. According to the WHO, the antimicrobial resistance problem poses a global threat. The EU Commission's current One Health Action Plan requests research in complementary medicine, the WHO urges member states to include complementary medicine in their national health policies. Regarding external evidence on the general use of human and veterinary homeopathy, evidence level 1a studies are reviewed. Focusing on the external evidence on the use of homeopathy in infections, some evidence level 1a, 1b, 2c studies, and a case report, are described in more detail. In conclusion, evidence for the effectiveness of human and veterinary homeopathy in general, and in particular, of homeopathic treatment for infections, is available. Especially, individualized homeopathy demonstrates effects at all quality levels according to Cochrane criteria, even in the methodologically high-quality studies. As in most areas of veterinary medicine and medicine, further good/excellent studies are necessary. In compliance with the principles of homeopathy, further methodologically high-quality trials focusing on the homeopathic treatment of infections are the next logical step. The selection of the simile (individually fitting homeopathic medicinal product) by appropriately trained homeopathic doctors/veterinarians is essential for the effectiveness of homeopathy. Implementation of studies at university facilities is a prerequisite for quality assurance. Consequently, further integration of homeopathy at universities is a necessary requirement for the patients' best interests.

INTRODUCTION: Les principes de base de l'homéopathie et ses fondements juridiques et scientifiques sont discutés dans cette revue pour répondre aux positions de l'Organisation mondiale de la santé (OMS) et de la commission de l'Union européenne (UE) sur la médecine complémentaire. Selon l'OMS, le problème de la résistance aux antimicrobiens constitue une menace mondiale. Le plan d'action One Health actuel de la Commission européenne demande des recherches en médecine complémentaire et l'OMS exhorte les États membres à inclure la médecine complémentaire dans leurs politiques nationales de santé. En ce qui concerne les preuves externes sur l'utilisation générale de l'homéopathie humaine et vétérinaire, des études de niveau de preuve 1a sont passées en revue. En se concentrant sur les preuves externes de l'utilisation de l'homéopathie dans les infections, certaines études de niveau de preuve 1a, 1b, 2c et un rapport de cas sont décrits plus en détail. En conclusion, des preuves de l'efficacité de l'homéopathie humaine et vétérinaire en général et en particulier du traitement homéopathique des infections, sont disponibles. L'homéopathie individualisée démontre en particulier des effets à tous les niveaux de qualité selon les critères Cochrane, même dans des études de haute qualité méthodologique. Comme dans la plupart des domaines de la médecine et de la médecine vétérinaires, d'autres bonnes voire excellentes études sont nécessaires. Conformément aux principes de l'homéopathie, d'autres essais de haute qualité méthodologique axés sur le traitement homéopathique des infections sont la prochaine étape logique. La sélection des simile (médicament homéopathique adapté individuellement) par des médecins/vétérinaires homéopathes dûment formés est essentielle pour l'efficacité de l'homéopathie. La mise en œuvre d'études dans les établissements universitaires est une condition préalable à l'assurance qualité. Par conséquent, une intégration plus poussée de l'homéopathie dans les universités est une condition nécessaire dans l'intérêt des patients.

Petroleum: a series of 25 cases.

This study is based on 25 well documented reports of cases which responded well to treatment with Petroleum. Materia medica data were compared with results in contemporary clinical practice. Many patients had characteristic skin problems; children often had recurrent or chronic upper respiratory tract problems. The most prominent mental feature is a quiet, withdrawn or stubborn disposition. The mental symptoms may be difficult to recognise. Detailed documentation in daily practice can be helpful for preserving data of the effect of a medicine; confirmation of statements given in materia medica; improving understanding of homeopathic medicines and differentiating the indications for medicines.

The effect of homeopathically prepared thyroxine on highland frogs: influence of electromagnetic fields.

BACKGROUND: Previous experiments show that amphibian larvae are responsive to homeopathically prepared thyroxine. METHODS: We studied the effect of a highly diluted and agitated thyroxine solution exposed to various electromagnetic fields on metamorphosis in highland Rana temporaria. The devices tested were: microwave oven, mobile phone, airport X-ray, and a red light barcode scanner. Animals were treated either with homeopathically prepared thyroxine (10(-30) parts by weight, 10(-35) in the water in which the animals were kept), or analogously prepared blank solution, or analogously prepared thyroxine exposed to the electromagnetic field of one of the devices tested. Solutions were administered at 48h intervals according to a standardized protocol. RESULTS: Animals treated with the standard test solution thyroxine 10(-30) metamorphosed more slowly than the control animals, ie the effect of the homeopathically prepared thyroxine was opposed to the usual physiological effect of molecular thyroxine. The cumulative number of test animals that had reached the four-legged stage at defined points in time was smaller in the group treated with homeopathically prepared thyroxine at most of the points in time. This was found independently by all three research teams involved. In contrast, this effect did not occur when the thyroxine solution had been exposed to the field of the early model microwave oven, or mobile phone. There was no difference between aqueous or alcoholic solutions were used, and there was, if any, only a small protective effect from aluminum foil. Airport X-ray and red light barcode scanning did not diminish the effect of the homeopathic solution.

Comparative study of three different supraglottic airway devices in simulated difficult airway situations.

BACKGROUND: Supraglottic airway devices (SAD) provide an effective way for managing difficult airways. Numerous SADs have been developed in recent years. We compared three SADs utilizing simulated airways. The major aim of this study was to provide evidence for the efficacy of SADs in the management of simulated difficult airway situations. METHODS: The study utilized an airway simulation manikin (Laerdal SimMan® 3G) to assess feasibility and time to final placement of three different airway devices (the classic laryngeal mask airway [LMA], the Laryngeal tube [LT], and the EasyTube® [EzT]). Thirty anesthesiologists inserted each of the SADs under standard physiologic airway conditions (STD) as well as pathological airway conditions, including tongue edema (TE) and trismus combined with limited mobility of the cervical spine (TCS), mimicking a patient with cramps. RESULTS: In STD and TE, all participants were able to successfully place the LMA, LT, and EzT correctly. In TCS, one participant failed to place the LMA correctly, whereas six participants failed to place the LT correctly (P=0.031). Under STD and TE conditions, we found a significantly longer time to final placement with the EzT (P=0.001). Under TCS conditions, there was no significant difference between the tested SADs. Under STD conditions, the participants rated the LMA best (P<0.001). Under TE and TCS condition, the EzT was significantly higher rated (P<0.001). CONCLUSION: The EzT showed benefits in two difficult airway situations (TE and TCS) in a prospective manikin study amongst anesthesiologists.

The immune system and the effects of non-volatile anesthetics on neutrophil transmigration through endothelial cell monolayers.

Inflammation represents the consequence of capillary dilation with accumulation of fluid and transmigration of leukocytes into the surrounding tissue. Leukocytes play a major role in the defense system of the body against invading microorganisms. This defense system has a non-specific branch consisting of granulocytes and macrophages and a specific branch of lymphocytes. Granulocytes release cytotoxic compounds from their intracellular granules into their local environment when encountering microorganisms. This random destruction happens rapidly, but it may also harm healthy tissue of the body. Leukocytes patrol the body by circulating through the blood and lymphatic system ensuring a continuous surveillance which is a prerequisite for an efficient defense. Upon tissue damage and inflammation, leukocytes are recruited from the blood to sites of injury, and this trafficking displays exquisite specificity. In the late 1890 s, Metchnikoff noted the power of certain blood cells to move towards microorganisms and ingest them. In fact, leukocytes adhere to the endothelium of the blood vessels, and subsequently leave the circulation by transmigration through the intercellular junctions of the endothelial cell monolayer. Transmigration is driven by chemoattractants, a process known as diapedesis. Reversible adherence of leukocytes to the endothelium, basement membranes, and other surfaces is an essential event in the establishment of inflammation, whose molecular basis is beginning to be understood. Inflammation may become chronic in many pathophysiologic processes and disease states. In long-term mechanically ventilated critically ill patients, non-volatile anesthetics are needed over a prolonged time period. Perioperative infections are a major cause of morbidity and mortality in critically ill patients. Therefore, the influence of non-volatile anesthetics and opioid agents on the immune system is of high interest. After presentation of the different effectors of the immune system and their fluxes through the body, the aim of this review is to propose a general model of leukocyte transmigration through endothelial cell monolayers. It emphasizes in which way different non-volatile anesthetic drugs may affect the non-specific branch of the immune system, i.e. the leukocyte transmigration through endothelial cell monolayers.

Role of beta-endorphins in silent myocardial ischemia.

The reason for the absence of pain perception in silent myocardial ischemia is unknown. A role of increased endorphinic activity in patients with silent ischemia has been postulated. To further investigate this hypothesis, 10 men with documented coronary artery disease and previous positive electrocardiographic findings during exercise without anginal pain were studied. Six healthy volunteers served as control subjects. The protocol included 2 bicycle exercise tests, the first test serving as baseline and the second performed after administration of naloxone, a specific opiate antagonist. Plasma beta-endorphin levels were measured by radioimmunoassay in both tests at rest, at peak exercise level and after recovery. All patients underwent thallium-201 scintigraphy after coronary vasodilation to provide an additional independent marker of ischemia. All patients showed stress-induced reversible perfusion abnormalities. No patient reported pain after naloxone application. Exercise duration, blood pressure and heart rate were not significantly altered by naloxone. Plasma beta-endorphin levels ranged from 18 +/- 6 pg/100 microliters (mean +/- standard deviation) at rest to 22 +/- 6 pg/100 microliters during exercise in the patient group and from 20 +/- 5 to 27 +/- 9 pg/100 microliters in the control subjects. Thus, there was no significant increase of plasma beta-endorphins during exercise or after naloxone administration, nor was there any difference observed between patients and control group. These data support the view that endorphinic activity does not play an essential role in the pathophysiology of silent myocardial ischemia.

Ventilation with the esophageal tracheal combitube in cardiopulmonary resuscitation. Promptness and effectiveness.

The success of cardiopulmonary resuscitation after cardiac arrest depends not only on the duration of the arrest, but also on the prompt establishment of a patent airway. In this study, we tested the safety and promptness of intubation with esophageal tracheal combitube (ETC) when compared to conventional endotracheal airway. Effectiveness of ventilation via the ETC as shown by blood gas analyses appeared to be comparable to endotracheal airway. Data suggest that the ETC might serve as a useful device during cardiopulmonary resuscitation.

Dexverapamil as resistance modifier in acute myeloid leukaemia.

In order to evaluate dexverapamil as a resistance modifier in acute myeloid leukaemia, we have added dexverapamil (4 x 300 mg/d orally) to DA chemotherapy (daunorubicin, cytosine arabinoside) in six patients with acute myeloid leukaemia. Two patients (1 first and 1 second relapse) achieved complete remission and two patients (1 refractory disease, 1 third relapse) showed some improvement. One patient in first relapse died due to disease progression and one drug-refractory patient remained refractory. The peak plasma levels of dexverapamil and nordexverapamil ranged from about 600 to 4100 ng/ml and from 450 to 1130 ng/ml, respectively. Major sideeffects were hypotension and sinus bradycardia. These results show the need for further evaluation of dexverapamil as a resistance modifier in acute myeloid leukaemia.

Thiopental inhibits migration of human leukocytes through human endothelial cell monolayers in vitro.

OBJECTIVES: The interactions between blood and vascular wall cells are essential for the understanding of pathophysiologic processes, e.g. inflammation. The influence of the anesthetic drug thiopental on leukocyte function is well documented. Recently, an inhibitory effect of thiopental on leukocyte chemotaxis in a Boyden chamber assay (i.e. endothelial cells were not included) was demonstrated. In vivo, leukocytes have to interact with endothelial cell monolayers to invade the tissue. The influence of thiopental on a monolayer of endothelial cells has not yet been investigated. The aim of the current study was to investigate the influence of thiopental on the migration of leukocytes through endothelial cell monolayers (ECM). MATERIAL AND METHODS: Human umbilical vein endothelial cells (HUVEC) were isolated and cultured on microporous membrane filters to achieve a monolayer. Isolated polymorphonuclear leukocytes (PMNL) as well as ECM were preincubated with different concentrations of thiopental. The rate of leukocyte migration against the chemotactic protein formyl-methyl-leucyl-phenylalanine was measured (n = 7). Thiopental was able to reduce the amount of leukocyte migration through ECM significantly. CONCLUSION: In conclusion, we could show that thiopental is able to reduce the migration of PMNL through ECM significantly.

Plasma exchange for treatment of thrombotic thrombocytopenic purpura in critically ill patients.

OBJECTIVE: Description of diagnostic procedures, treatment modalities and intensive care management of patients with thrombotic thrombocytopenic purpura (TTP). DESIGN: Descriptive study. SETTING: Internal medicine Intensive Care Unit (University Hospital of Vienna). PATIENTS: Six patients (two after allogeneic bone marrow transplantation), treated for 12 episodes of TTP. INTERVENTIONS: Treatment with plasma exchange (fresh frozen plasma, 50-80 ml/kg per day), prednisone (0.75 mg/kg b.i.d.) and, in some cases, vincristine. Supportive therapy as needed. MEASUREMENTS AND RESULTS: Patients were admitted to the ICU because of neurological symptoms with acute onset (42% mild, 58% severe), hemolysis and thrombocytopenia. Additional symptoms were fever (50%), bleeding tendency (50%), acute renal failure (42%) and metabolic derangement (8%). Initial laboratory values showed thrombocytopenia (median 17 G/l), hemolysis (median hemoglobin 10.0 g/dl, lactate dehydrogenase 635 U/l, reticulocyte count 175 G/l) with red cell fragmentation. Coagulation tests were normal. Respiratory assist was needed in six episodes (severe seizures, cardiopulmonary resuscitation). In patients without preexisting hematological abnormality the platelet counts exceeded 100 G/l after 3-8 cycles of plasma exchange. In patients after bone marrow transplantation, the platelet counts never exceeded 40 G/l, but the lactate dehydrogenase levels dropped significantly. The neurological symptoms disappeared in all patients and renal function normalized. One patient died before the initiation of therapy. Three patients relapsed 1-3 times between 2 weeks and 5 months after the last episode. The relapses were associated with symptoms similar to the first episode and responded promptly to plasma therapy. CONCLUSIONS: TTP is a rare, but life-threatening disorder. It needs immediate diagnosis and has a good prognosis after adequate treatment with plasma exchange.

Influence of pentoxifylline on cytokine levels and inflammatory parameters in septic shock.

OBJECTIVE: To evaluate the influence of pentoxifylline (PTX), a phosphodiesterase inhibitor, on cytokines and inflammatory proteins in patients suffering from septic shock. DESIGN: Prospective study comparing a therapy group to a matched control group. SETTING: Medical intensive care unit at a university hospital. PATIENTS: Twenty four patients fulfilling the criteria of septic shock were included in this study. Twelve patients received PTX (therapy group) and 12 patients matched for diagnosis, age and gender served as the control group. INTERVENTIONS: Pentoxifylline at 1 mg/kg per hour over 24 h in the therapy group. MEASUREMENTS AND RESULTS: Cytokine levels [tumor necrosis factor-alpha (TNF)], soluble TNF receptor [TNF-R], and interleukin-6 [IL-6] and inflammatory proteins [C-reactive protein, alpha-1-antitrypsin (AAT), fibronectin, and haptoglobin], as well as hemodynamic parameters and the APACHE III score were evaluated before initiation of therapy and 24 h-later. After 24 h, TNF levels were significantly lower in the therapy group (p = 0.013), while IL-6 levels were significantly higher in the therapy group (p = 0.030). Within the 24 h TNF declined significantly in the therapy group (p = 0.006), while IL-6 showed a significant increase (p = 0.043). AAT and the APACHE III score tended to differ significantly after 24 h between the groups [AAT levels higher in the therapy group (p = 0.05), APACHE III score lower (p = 0.05)]. In the therapy group, the systemic vascular resistance index was significantly higher after 24 h (p = 0.0026) whereas the cardiac index declined (p = 0.035). CONCLUSIONS: PTX does influence TNF levels in septic shock patients. Nevertheless, inhibiting a single mediator in severe septic shock cannot stop the inflammatory overreaction.

Influence of prothrombin complex concentrates on plasma coagulation in critically ill patients.

OBJECTIVE: To evaluate thrombogenicity of prothrombin complex concentrates (PCCs) in critically ill patients. DESIGN: Prospective clinical study. SETTING: Medical intensive care unit at a university hospital. PATIENTS: 16 consecutive patients suffering from acquired deficiencies of coagulation factors and with either overt bleeding from any site or a planned invasive procedure. INTERVENTIONS: 2000 factor IX units of PCCs intravenously. MEASUREMENTS AND RESULTS: Prothrombin time (PT), activated partial prothrombin time, fibrinogen, platelet count, plasma levels of coagulation factors II, V, VII, VIII, IX, X, antithrombin, protein C, thrombin-antithrombin complex (TAT), prothrombin fragment F(1+2), and the fibrin degradation product D-dimer were measured prior to and 1, 3, and 24 h after administration of PCCs. PT as well as coagulation factors II, VII, IX, and X, TAT, and F(1+2) showed a significant increase after administration of PCCs. All other parameters remained unchanged. CONCLUSIONS: Administration of PCCs induces thrombin generation. No evidence for induction of disseminated intravascular coagulation in biochemical terms could be found. When rapid correction of acquired coagulation factor disturbances is warranted, the use of PCCs seems reasonable, but the elevated risk of intravascular thrombus formation should be kept in mind.

Purine metabolism of erythrocytes in myotonic dystrophy.

The uptake and subsequent metabolism of adenine and adenosine was studied with the intact erythrocytes of patients with myotonic dystrophy (MD). Washed erythrocytes of both controls and patients were incubated with 14C-labeled adenine or adenosine at 20 degrees C for 5 to 120s to characterize the uptake process. No differences in the uptake process of adenine or adenosine were observed between normal and MD erythrocytes. Formation of adenine nucleotides was determined at 37 degrees C after incubation for 30 min. Compared to controls, the incorporation of adenine into adenine nucleotides was 2.6 times higher in MD erythrocytes. This depends mainly on an increase of ATP formation. The mean ratios of ATP:ADP + AMP for normal red cells and MD erythrocytes were 0.92 and 1.39 respectively. No difference was found in the conversion of adenosine to adenine nucleotides. In spite of a normal amount of intracellular ATP a greater demand for ATP exists. This might be due to leakage of adenine nucleotides out of MD erythrocytes.

Oral contraceptives that contain ethinyl estradiol (0.035 mg) and cyproterone acetate (2 mg) inhibit leukocyte transmigration through endothelial cell monolayers.

OBJECTIVE: To investigate the influence of ethinyl estradiol and cyproterone acetate in oral contraceptives on leukocyte migration through endothelial cell monolayers. DESIGN: Experimental in vitro prospective study. SETTING: An academic research laboratory. INTERVENTION(S): Endothelial cells were cultured on microporous membranes to produce monolayers. Polymorphonuclear leukocytes were used in a previously described migration assay (n = 7). The amount of untreated polymorphonuclear leukocytes that migrated through untreated endothelial cell monolayers was used as a control and set at 100%. In addition, a leukocyte adhesion assay was used. MAIN OUTCOME MEASURE(S): Leukocyte adhesion to and transmigration through endothelial cell monolayers. RESULT(S): Ethinyl estradiol and cyproterone acetate inhibited the migration of polymorphonuclear leukocytes through endothelial cell monolayers significantly (67% +/- 6.4%) when both cell types were treated to simulate in vivo conditions. The adhesion assay produced similar results. CONCLUSION(S): Ethinyl estradiol and cyproterone acetate were identified as potent inhibitors of leukocyte migration through endothelial cell monolayers.

Contamination of central venous catheters in immunocompromised patients: a comparison between two different types of central venous catheters.

Catheters impregnated with silver have been proposed as a means of reducing catheter-related infection. We therefore performed a prospective randomized study to compare a new silver-impregnated central venous catheter (CVC) with a commercially available CVC in a cohort of immunocompromised patients. We studied 157 patients of whom 97 could be analysed. The median indwelling time in the study group (SC) was 10.5 days and 11 days in the control group (CC). The incidence of contamination in the SC group was 15.6 vs 24.6 in the CC group referring to 1000 catheter days. In both groups, we found 6% of catheter-related infections according to the definitions of a published scoring system. The differences between the two groups were not significant. We conclude that the SC decrease the incidence of catheter contamination and may have a positive effect on the reduction of CVC-related infections.

Dexamethasone inhibits leukocyte migration through endothelial cells towards smooth muscle cells.

Leukocyte interactions with endothelial cell monolayers (ECM) and smooth muscle cells (SMC) play an important role during inflammatory processes. Several studies describe an inhibitory effect of dexamethasone on polymorphonuclear leukocytes (PMNL), endothelial cell function, and interleukin-1 (IL-1) release. Aim of the current study was to investigate the influence of dexamethasone on leukocyte migration through an endothelial cell monolayer towards SMC-layers stimulated by tumor necrosis factor-alpha (TNF-alpha). Using a recently developed triple chamber migration system, SMC-layers were cultured on the bottom of a 24-well plate. On the upper surface of the first filter, ECM were cultured, the second filter was a collecting filter. The amount of leukocyte migration through ECM towards TNF-alpha-stimulated smooth muscle cell layers with and without dexamethasone-pretreatment was measured using a fluorescence technique. The pretreatment of SMC-layers with dexamethasone reduced the amount of leukocyte migration down to 92 +/- 8.8% (0.001 mM, p=n.s.), to 67 +/- 5.7% (0.01 mM, p<0.05), to 53 +/- 4.6% (0.1 mM, p<0.05), and to 41 +/- 5.0% (1 mM, p<0.05). In conclusion, dexamethasone treatment of smooth muscle cell layers inhibits leukocyte migration through ECM towards smooth muscle cell layers. The inhibition seems to be due to a decrease in IL-1 release. Treatment of all cell types, PMNL, endothelial cells, as well as smooth muscle cell layers, simulating an in-vivo situation, seems to have an additive effect.

Leukocyte migration: a new triple migration chamber assay allows investigation of various cell interactions simultaneously.

Examination of the interactions between various cells of the vascular wall and blood components are essential for understanding different pathophysiological processes. Such investigations require appropriate techniques. Several groups have attempted to establish different methods. In all blood vessels except capillaries, endothelial cells (EC) and smooth muscle cells (SMC) coexist and interact very closely. The current study describes a new 3-dimensional triple chamber migration assay, studying leukocyte migration through human endothelial cell monolayers (ECM) towards human SMC layers simultaneously. To test the new assay, SMC-layers were prestimulated with different concentrations of tumor necrosis factor alpha (TNF-alpha, 1 ng/ml, 10 ng/ml, 100 ng/ml) over 6 hours. Then, two microporous membranes, a collecting membrane and a third membrane with cultured ECM, were inserted. Freshly isolated peripheral blood mononuclear cells (PBMNC) were seeded on the ECM and transmigrated cells were measured after further 3 hours incubation. The migration against non stimulated SMC-layers was used as control. Prestimulated SMC-layers led to a dose dependent increase of PBMNC migration into the subendothelial cell space. Antibodies against interleukin-1 reduced the PBMNC migration. In conclusion, this assay allows to study cell migration into the subendothelial space and interactions between different vascular cells. Moreover, this assay can also be used for studies on other cell-cell interactions in man.

Rapid, fluorescence-based assay for microtiter plates to test drug influences on neutrophil transmigration through endothelial cell monolayers.

Investigation of drug interactions between blood cells and endothelium is of high interest. The current study describes the development of a rapid fluorescence-based leukocyte transmigration system through endothelial cell monolayers for investigation of drug influences. To test the new assay, endothelial cells were cultured on microporous filters, pore size 3.0 microm, in 96-well-plates. Freshly isolated neutrophils were seeded on endothelial cell monolayers and transmigrated cells were measured after incubation for three hours. Migration of non-stimulated neutrophils through non-stimulated endothelial cell monolayer was used as control and set as 100%. The influence of the non-steroidal anti-inflammatory drug diclofenac was investigated. Assay precision tests were done using intraassay (within-day variability) and interassay (day-to-day variability) controls. Transmigration rate was decreased to 53 +/- 6.8% SD (diclofenac 0.7 microg/mL). Different concentrations showed a dose dependent effect (0.07 microg/mL: 97 +/- 9.5%, 7 microg/mL: 37 +/- 4.7%). Analysis of assay accuracy of the new 96-well-sized transmigration assay showed reliable results (coefficient of variation: intraassay 8.2 %; interassay 11.8%). In conclusion, this new, rapid, and sample saving 96-well-microtiter transmigration assay allows examination of drug influence on neutrophil migration through endothelial cell monolayers. Moreover, this assay can also be used for other cell-cell interactions.