Topical protection of human esophageal mucosal integrity.

Patients with nonerosive reflux disease exhibit impaired esophageal mucosal integrity, which may underlie enhanced reflux perception. In vitro topical application of an alginate solution can protect mucosal biopsies against acid-induced changes in transepithelial electrical resistance (TER). We aimed to confirm this finding in a second model using 3D cell cultures and to assess prolonged protection in a biopsy model. We assessed the protective effect of a topically applied alginate solution 1 h after application. 3D cell cultures were grown by using an air-liquid interface and were studied in Ussing chambers. The apical surface was "protected" with 200 µl of either alginate or viscous control or was unprotected. The tissue was exposed to pH 3 + bile acid solution for 30 min and TER change was calculated. Distal esophageal mucosal biopsies were taken from 12 patients and studied in Ussing chambers. The biopsies were coated with either alginate or viscous control solution. The biopsies were then bathed in pH 7.4 solution for 1 h. The luminal chamber solution was replaced with pH 2 solution for 30 min. Percentage changes in TER were recorded. In five biopsies fluorescein-labeled alginate solution was used to allow immunohistological localization of the alginate after 1 h. In the cell culture model, alginate solution protected tissue against acid-induced change in TER. In biopsies, 60 min after protection with alginate solution, the acidic exposure caused a -8.3 ± 2.2% change in TER compared with -25.1 ± 4.5% change after protection with the viscous control (P < 0.05). Labeled alginate could be seen coating the luminal surface in all cases. In vitro, alginate solutions can adhere to the esophageal mucosa for up to 1 h and exert a topical protectant effect. Durable topical protectants can be further explored as first-line/add-on therapies for gastroesophageal reflux disease.

Bowel habit, diet and body weight in preadolescent children.

BACKGROUND: The possible influence of diet and body weight on bowel habit in children is unknown. The present study aimed to investigate the inter-relationships between bowel function, excess body weight and dietary intake in a group of preadolescent children. METHODS: Eighty-four preadolescent children aged 7-10 years were recruited [mean (SD) age 9.7 (1.0) years]. All children completed a bowel habit diary, examining specific parameters of bowel function and a weighed food inventory concurrently for seven consecutive days. Height and weight measurements were also taken. Children were grouped according to whether they met dietary recommendations and by overweight status; differences in bowel function between the groups were then analysed. RESULTS: Children who exceeded reference values for fat were more likely to report an incidence of straining to start (P = 0.005) and pain during defaecation (P = 0.021). Subjects who met protein recommendations were less likely to report incomplete evacuation (P = 0.000) and those who met zinc recommendations were less likely to report pain during defaecation (P = 0.044). Excess body weight (according to International Obesity Task Force cut-offs) was also associated with poor bowel habit, with overweight and obese children reporting lower defaecation frequency and a higher incidence of straining and feelings of incomplete evacuation, although these findings were not statistically significant. Defaecation frequency in healthy children was 1.4 defaecations per day compared to 1.2 defaecations for overweight and obese children. CONCLUSION: A poor diet that fails to meet dietary recommendations as well as being overweight and obese appears to be associated with increased defaecation problems in preadolescent children.

A Randomized, controlled, crossover trial to investigate times to onset of the perception of soothing and cooling by over-the-counter heartburn treatments.

This was a randomized, controlled, four-way crossover study in 45 subjects with a tendency to suffer from moderate heartburn following some meals. The study was designed to assess the time to onset of the perceived soothing and cooling effects of the alginate raft-forming products, Gaviscon Liquid (peppermint), Gaviscon Double Action Liquid (peppermint) and Gaviscon Powder Formulation (fresh tropical), compared with a non-active sublingual control. All three Gaviscon products provided significantly faster soothing and cooling effects compared with the control. Based on the upper 95% confidence limits for the median, time to onset of soothing was perceived within 3.15 min, 3.08 min and 4.05 min for Gaviscon Liquid, Double Action Liquid and Powder Formulation, respectively. Similarly, time to onset of cooling was perceived within 1.95 min, 1.23 min and 11.22 min for Gaviscon Liquid, Double Action Liquid and Powder Formulation, respectively. The results show that Gaviscon Liquid and Gaviscon Double Action soothe within 3.15 min and cool within 1.95 min.

The online Cough Clinic: developing guideline-based diagnosis and advice.

The aim of the present study was make chronic cough guidelines more practical and user friendly by developing an internet-based interactive diagnostic questionnaire for chronic cough. A prospective cohort study of chronic cough sufferers was conducted in the UK, following European Respiratory Society guidelines for the diagnosis and management of chronic cough. Depending on the response to 16 specific questions, the medical condition responsible for the patient's chronic cough was ascertained according to a predetermined diagnostic algorithm designed to differentiate the three common causes of chronic cough. Appropriate advice and treatment recommendations were then provided. 8,546 adults with chronic cough completed the Cough Clinic diagnostic questionnaire. 46.1% were suggested to have reflux, 38.7% asthma and 15.2% rhinitis. Participants found the website easy to use (94%), the advice helpful (73%) and that it helped them to communicate with their general practitioner better (60%), and 62% reported taking the recommended treatment. The Cough Clinic, an internet-based diagnostic site for chronic cough, had a large uptake by chronic cough sufferers in the UK. Almost half were diagnosed as having reflux as the probable cause of their chronic cough. Internet diagnosis by expert algorithm provides a novel mechanism for patients to access guideline-recommended therapies and enhances dialogue between patients and physicians.

Habitual micronutrient intake during and after pregnancy in Caucasian Londoners.

Micronutrient status is of fundamental importance both upon conception and throughout pregnancy. There is an abundance of literature investigating nutrient intakes during individual trimesters of pregnancy but few studies have investigated baseline intakes of nutrients throughout gestation as a continuum. The current investigation set out to measure habitual micronutrient intakes at weeks 13, 25, 35 of pregnancy and 6 weeks postpartum using a prospective background information questionnaire, 4-7-day weighed food diary and postnatal questionnaire. Seventy-two primiparous, Caucasian Londoners were recruited at the study start with 42 completing the first, second, third trimester and postpartum study stages respectively. Study findings indicated that sodium intakes were significantly higher than UK guidelines throughout and after pregnancy (P < 0.001). Intakes of folate, iron, vitamin D, potassium, iodine and selenium were lower than UK recommendations during and after pregnancy, but to varying levels of statistical significance (P < 0.05). Only 23-38% of women met UK recommendations for folate (300 microg day(-1)) through dietary sources. Similarly, only a small percentage of women met dietary guidelines for iron (19-28%). The findings from the current study indicate that public health interventions may be required to help expectant mothers achieve an optimal diet, particularly after birth when dietary recommendations increase for some micronutrients.

Thickness and continuity of the adherent colonic mucus barrier in active and quiescent ulcerative colitis and Crohn's disease.

BACKGROUND: The colon is covered by a mucus barrier that protects the underlying mucosa and alterations in this mucus barrier have been implicated in the aetiology of inflammatory bowel disease (IBD). This study investigated the thickness and continuity of the mucus barrier in ulcerative colitis (UC) and Crohn's disease (CD) in comparison to normal controls. METHODS: Rectal biopsies were taken from 59 patients and cryostat sections stained with periodic acid-Schiff's/Alcian blue to visualise the mucus layer. Mucus thickness and continuity and goblet cell density were measured using light microscopy. RESULTS: An essentially continuous adherent mucus layer was observed in normal human rectum and there was no change in the mucus barrier in quiescent UC. In active UC there was a trend for the mucus layer to become progressively thinner and significantly more discontinuous as disease severity increased. In severe active UC the mucus layer thickness and goblet cell density were significantly reduced compared with normal controls while the percentage discontinuity significantly increased. CONCLUSION: It is not until severe UC that there is a global change in mucosal protection as a consequence of large regions lacking mucus, a decrease in secretory potential caused by a loss of goblet cells and a thinner, less effective mucus layer even when it is present.

Administration of an alginate based gastric reflux suppressant on the bioavailability of omeprazole.

BACKGROUND & OBJECTIVE: Omeprazole treats gastro-oesophageal reflux disease (GORD) by inhibition of acid secretion whereas alginate based reflux suppressants work by forming a low density raft of near neutral pH which floats on the stomach contents and physically impedes gastro-oesophageal reflux. There is limited pharmacokinetic information regarding possible drug interaction between these two types of products, although these may be frequently co-prescribed to improve symptom control in GORD patients. This study was designed to determine whether the administration of a 10 per cent w/v liquid alginate suspension affected the pharmacokinetic profile of omeprazole. METHODS: This was a randomized, two-treatment, two-sequence, two-period crossover study in 26 volunteers. Each treatment was dosed for 3 consecutive days with a washout period of 7 days between dosing periods. Blood samples for pharmacokinetic analysis were taken over the 24 h period following the final dose of omeprazole. RESULTS: Geometric means and ratios were as follows: C(max) was 555 for omeprazole/alginate and 558 for omeprazole alone (ratio 99.55%, 90% confidence interval 82.75-119.75%; AUC(0-t) was 2050 for omeprazole/alginate and 2094 for omeprazole alone (ratio 97.90%, 90% confidence interval 87.83-109.12%); AUC(0-a) was 2247 for omeprazole/alginate and 2231 for omeprazole alone (ratio 100.74%, 90% confidence interval 90.05-112.70%). Mean values for T(max), K(el) and T(1/2) were also similar for the two treatment regimens. INTERPRETATION & CONCLUSION: As the 90 per cent confidence intervals for the geometric mean ratios for C(max), AUC(0-t), and AUC(0-alpha) are all contained within the bioequivalence interval of 80-125 per cent, it can be concluded that the administration of this liquid alginate suspension does not affect the pharmacokinetic profile of omeprazole.

Primary tumor and metastasis in ovarian cancer differ in their content of urokinase-type plasminogen activator, its receptor, and inhibitors types 1 and 2.

The relevance of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor (PAI) type 1 in predicting the survival probability of patients with advanced ovarian cancer after radical surgery and adjuvant chemotherapy by assessing the patients' primary tumors has recently been shown by us (W. Kuhn et al., Gynecol. Oncol., 55: 401-409, 1994). In the present study, we determined uPA, uPA receptor, PAI-1, and PAI-2 concentrations in primary tumors and tumor-infiltrated omentum and retroperitoneal lymph nodes of ovarian cancer patients. The group consisted of 39 patients with advanced ovarian carcinoma stages Fédération Internationale de Gynécologie et d'Obstétrique (FIGO) IIIc or IV; for comparison 7 patients with early carcinoma stage FIGO I were also included. In metastases of the omentum from ovarian cancer stage FIGO IIIc or IV patients, we noted a 4-fold elevated uPA content, a 2-fold increase in PAI-1, and also a significant increase in uPA receptor and PAI-2 over primary tumors. In metastases of the lymph nodes the levels of the respective antigens were also increased when compared to primary tumors. These data may indicate that elevated levels of components of the fibrinolytic system at sites of metastases may contribute to the aggressive potential of cancer cells by favoring their reimplantation and/or the consolidation of a new tumor stroma.

Characterisation of adherens and tight junctional molecules in normal animal larynx; determining a suitable model for studying molecular abnormalities in human laryngopharyngeal reflux.

BACKGROUND: The disruption of intercellular junctions in the larynx is a pathological feature of laryngopharyngeal reflux (LPR). Good experimental models are necessary to gain greater insight into the molecular mechanisms and alterations that result from abnormal exposure of the laryngeal epithelium to acid refluxate. AIMS: To characterise laryngeal tissues from different species to determine the most suitable for use in experimental studies of LPR. METHODS: Human and non-human laryngeal tissues (mouse, rat, guinea pig, porcine, and rabbit) were studied. Histological characterisation was performed by light microscopy. The expression and subcellular localisation of adherens junctional molecules (E-cadherin and beta catenin) was evaluated by immunohistochemistry, and tight junction molecules (occludin and zonula occludens 1 (ZO-1)) by western blotting. The ultrastructural features of porcine and human tissue were assessed by electron microscopy. RESULTS: Porcine tissue revealed both respiratory-type and stratified squamous epithelium, as seen in the human larynx. The expression and subcellular localisation of the E-cadherin-catenin complex was detected in all species except mouse and rat. The pattern of ZO-1 and occludin expression was preserved in all species. CONCLUSION: The expression of intercellular junctional complexes in porcine epithelium is similar to that seen in humans. These results confirm the suitability of these species to study molecular mechanisms of LPR in an experimental system.

Prognostic value of the cysteine proteases cathepsins B and cathepsin L in human breast cancer.

The lysosomal cysteine proteases cathepsin B and cathepsin L have been implicated in tumor spread and metastasis. To evaluate the prognostic impact of these proteases for disease-free survival and overall survival in breast cancer, the antigen content of cathepsin B and cathepsin L was determined using ELISA in tumor cytosol fractions of 167 breast cancer patients and in cytosols of 29 benign breast tissue specimens. Median values of 856 ng versus 76 ng cathepsin B/mg protein and of 428 ng versus 56 ng cathepsin L/mg protein were found in tumor versus benign cytosol fractions. A positive correlation between cathepsin B and cathepsin L (r = 0.32, P = 0.0000, Spearman test) was found. Cathepsin L was inversely correlated to hormone receptor status (P = 0.0014, Mann-Whitney U test) and to the presence of tumor necrosis (P = 0.009, Mann-Whitney U test). There were no correlations of cathepsin B or cathepsin L to tumor size, axillary lymph node status, age, menopausal status, tumor grading, and vessel invasion. To perform univariate analyses of disease-free survival, optimal cutoff points were determined by isotonic regression and classification and regression trees analysis. Patients with a high content of cathepsin B (>1092 ng/mg protein) or cathepsin L (>376 ng/mg protein) in their primary tumors had a statistically significantly higher risk of recurrence than patients with a low content of cathepsin B or cathepsin L (5-year disease-free survival: cathepsin B, 70% versus 52%, P = 0.04; cathepsin L, 83% versus 52%, P = 0.0002). Median follow-up was 39 (range, 6-73) months. Multivariate analysis for disease-free survival showed that cathepsin L is a strong and independent prognostic factor with a prognostic impact comparable to that of axillary lymph node status and grading. We conclude that both cathepsin B and cathepsin L may serve as prognostic factors for tumor recurrence in human breast cancer. These data underline the significance of tumor-associated proteolysis for invasion and metastasis.

Alginate rafts and their characterisation.

Alginate/antacid anti-reflux preparations are designed to provide symptom relief by forming a physical barrier on top of the stomach contents in the form of a neutral floating gel or raft. This study tested the in vitro effectiveness of a range of liquid products in forming rafts that were cohesive, buoyant, voluminous, resistant to reflux and durable under conditions of movement (resilient). The products tested had a wide range of acid neutralising capacities (ANCs). It was found that products with a high ANC and no calcium ion source formed rafts of low strength, weight and volume, which appeared more as floating precipitates than coherent gels. Products with a high ANC and a calcium ion source formed medium strength, weight and volume rafts. Products with a low ANC formed strong coherent rafts with medium to large weight and volume, and those with low ANC and a calcium ion source formed the strongest rafts. Products with stronger rafts were found to be more resilient and more resistant to reflux in an in vitro reflux model. Significant overall differences in raft buoyancy were found between products forming coherent rafts but these could not be related to the product formulation or amount of available carbon dioxide.

Straining at stool and stool frequency in free-living and institutionalised older adults.

BACKGROUND: The diagnosis of functional constipation according to Rome II criteria includes assessment of straining. However the prevalence in older adults is unknown. AIMS: To assess the prevalence of straining and its association with stool frequency in free-living (FL) and institutionalised (INS) older adults. METHODS: 50 FL subjects (mean age 74 years, range (65-97), 42% male) and 42 INS subjects (mean age 84 years, range (69-101) 36% male) were recruited. Stool frequency and straining to start and to finish were prospectively recorded by subjects for 7 consecutive days in a bowel habit diary. Statistical analyses were performed using the Chi-square or the Pearson correlation coefficient as appropriate. RESULTS: The mean stool frequency (n/week) was significantly higher (p <0.001) in the FL group compared with the INS group (11.7 and 4.9 respectively). The percentage of subjects experiencing straining to start on more than 25% of occasions was significantly lower in the FL compared with the INS group (34% and 64% respectively, chi2 = 8.4, p = 0.004, df = 1). The percentage of subjects experiencing straining to finish on more than 25% of occasions was significantly lower in the FL compared with the INS group (16% and 41% respectively, chi2 = 6.9, p = 0.009, df = 1). CONCLUSIONS: FL subjects had significantly higher stool frequency and had to strain passing a stool (to start and to finish) less often than their INS counterparts. Moreover, straining to start was experienced more often than straining to finish in both groups.

Laxative use and its relationship with straining in a London elderly population: free-living versus institutionalised.

BACKGROUND: The diagnosis of functional constipation according to Rome II criteria includes assessment of straining. However the prevalence in older adults is unknown. Moreover, laxative use increases with age, especially in the elderly. AIMS: to assess the prevalence of straining and its association with laxative use in free-living (FL) and institutionalised (INS) older adults. METHODS: 50 FL (mean age 74 years, 42% male) and 42 INS subjects (mean age 84 years, 36% male) were recruited. Straining to start and to finish defecation were prospectively recorded by subjects for 7 consecutive days in a bowel habit diary. Concurrently, the subjects recorded any laxative use during the 7 days study. Statistical analyses were performed using the Chi-square statistic. RESULTS: 20% of FL and 65% of INS subjects recorded taking laxatives during the study week. Of the 40 FL subjects not taking laxatives, 30 had to strain to start on 25% or less of occasions and 36 had to strain to finish on 25% or less of occasions (chi(2) = 7.2; p = 0.012 and chi(2) = 5.4; p = 0.041, respectively). In the INS group, although 64% of subjects taking laxatives had to strain on more than 25% of occasions, the Chi-square test was not statistically significant. CONCLUSIONS: From our results, it seems that laxatives were used appropriately in the FL, with the majority of those taking laxatives having to strain to start on more than 25% of occasions.

The effect of omeprazole pre-treatment on rafts formed by reflux suppressant tablets containing alginate.

Alginate-based reflux suppressant preparations provide symptom relief by forming a physical barrier on top of the stomach contents in the form of a neutral floating gel or raft. This study investigated whether reduced acidity in the stomach brought about by omeprazole pre-treatment affected the formation and gastric residence time of alginate rafts. It was a balanced, cross-over study in 12 healthy non-patient volunteers following a single dose of two indium-111-labelled alginate tablets in the presence or absence of 3 days' pre-treatment with omeprazole. Raft formation and gastric residence, in the presence of a technetium-99m-labelled meal, were assessed by gamma scintigraphy for 3 h after alginate tablet administration. The relative raft-forming ability of alginate tablets after omeprazole compared with alginate tablets alone was 0.950 with 95% confidence intervals of 0.882 and 1.018. Pre-treatment and co-administration with omeprazole has no significant effect on the raft-forming ability of alginate tablets.

Time-resolved infrared ATR measurements of liposome transport kinetics in human keratinocyte cultures and skin reveals a dependence on liposome size and phase state.

A novel in vitro method for studying the permeation kinetics of superficially applied liposomes or vesicles through layers of human skin or keratinocytes on a solid support is presented, employing attenuated total reflection infrared spectroscopy. The method is applied to investigate transport kinetics of unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) through cultured human keratinocyte layers and through human skin. We find a strong resemblance of the qualitative features of the permeation kinetics of small unilamellar DMPC vesicles for skin and keratinocytes. Detailed studies of the vesicles transport through keratinocyte layers show that DMPC vesicles with an average diameter of 55 nm can readily permeate through the layer at 37 degrees C with a diffusion constant of D = (4.0 +/- 0.8) x 10(-15) m2/second, whereas larger vesicles of twice that diameter do not permeate at all. In contrast, liposomes containing a chemical permeation enhancer permeate through the layer significantly faster [D = (7.0 +/- 0.5) x 10(-15) m2/second] than the small DMPC vesicles despite their five-times-larger diameter. Moreover, the transport of the DMPC vesicles depends drastically on their phase state. No permeation was observed for small DMPC vesicles at a temperature of 10 degrees C when the lipid is in the crystalline phase state. Our results indicate that keratinocyte culture layers can pose a significant permeation barrier for vesicles. The permeation mechanism can be explained by diffusion of the vesicles through small pores and gaps in the layer, presumably driven by transdermal osmotic gradients.

Neuropharmacological studies in rodents on the action of RX 781094, a new selective alpha 2-adrenoceptor antagonist.

Several neuropharmacological effects of RX 781094, a new selective alpha 2-adrenoceptor antagonist, have been investigated in rodents. In rats, RX 781094 (0.1-1.0 mg kg-1, i.v.) produced a rapid dose-related reversal of cortical EEG synchronisation and behavioural sedation, induced by clonidine or the more selective alpha 2-adrenoceptor agonist, guanoxabenz. The alpha 2-adrenoceptor antagonists yohimbine and mianserin were also effective in blocking guanoxabenz-induced EEG synchronisation but had a lower potency than did RX 781094. In specificity experiments, RX 781094 (1.0 mg kg-1, i.v.) failed to antagonise the EEG synchronisation and pronounced behavioural sedation induced by the CNS depressant sodium pentobarbitone (15 mg kg-1, i.v.). In mice, pretreatment (i.v. or p.o.) with RX 781094 inhibited in a dose-dependent way both guanoxabenz-induced behavioural hypoactivity and clonidine-induced hypothermia. By itself, RX 781094 had no effect on the temperature of normal mice. In sleep-waking studies in rats, RX 781094 (0.1 and 1.0 mg kg-1, i.v.) had no measurable stimulant or depressant effect on the CNS, in contrast to (+)-amphetamine (1.0 mg kg-1, i.v.) which elicited marked CNS stimulation. These results support the conclusion that RX 781094 is a potent antagonist at central alpha 2-adrenoceptors.

Analeptic effects of centrally injected TRH and analogues of TRH in the pentobarbitone-anaesthetized rat.

The effect of intracerebral injection of TRH and several biologically stable TRH analogues in the pentobarbitone anaesthetized rat was examined. Bilateral injection of TRH (5.0 micrograms total dose) and the analogues RX 77368 (0.01-1.0 microgram), CG 3509 (0.1-1.0 microgram), DN-1417 (1.0 microgram) and MK-771 (1.0 microgram) into the nucleus accumbens reduced the pentobarbitone-induced sleeping time. The TRH metabolite DKP (5 micrograms) had no effect on the sleeping time following intra-accumbens injection. Intra-septal injection of TRH (1.0-5.0 micrograms), RX 77368 (0.1-1.0 microgram) and CG 3509 (0.1-1.0 microgram) also reversed the pentobarbitone-induced sleeping time. In contrast, TRH (5 micrograms) injected into the striatum had no effect on the pentobarbitone-induced sleeping time, and CG 3509 (0.1 microgram) and RX 77368 (0.1 microgram) had weaker effects following intrastriatal injection compared to injection of these analogues into the nucleus accumbens and septum. Measurements of core temperature and respiration rate in rats following intra-accumbens or septal injection of TRH, CG 3509 and RX 77368 showed these peptides to reverse pentobarbitone-induced hypothermia and stimulate respiration rate. However, while intrastriatal injections of CG 3509 and RX 77368 caused an increase in respiration rate they had no effect on core temperature. These results suggest a close association between peptide-induced respiratory stimulation and reversal of pentobarbitone-induced anaesthesia. Since intra-accumbens and septal injection of dopamine (20-100 micrograms) failed to reverse anaesthesia, it is unlikely that the peptide-induced responses are mediated via dopamine release.

The effect of alpha 2-adrenoceptor agonists on the acid secretory responses of rat isolated gastric mucosa to electrical field stimulation.

The effects of clonidine, UK-14,304, noradrenaline, para-aminoclonidine and phenylephrine were examined on the acid secretory response of the rat isolated gastric mucosa preparation to electrical field stimulation. Clonidine, UK-14,304, noradrenaline and para-aminoclonidine but not phenylephrine (10 microM) reduced the response of the gastric mucosa stimulated at 2.5 Hz; gastric mucosae stimulated at higher frequencies were insensitive to the action of these alpha 2-adrenoceptor agonists. The inhibitory effect of the selective alpha 2-adrenoceptor agonist UK-14,304 was antagonized by idazoxan but not by prazosin. These findings indicate that clonidine and other alpha 2-adrenoceptor agents inhibit the acid secretory response of the rat gastric mucosa to electrical field stimulation by an action at alpha 2-adrenoceptors, which are probably located on cholinergic nerve terminals.

Effects of idazoxan on catecholamine systems in rat brain.

Experiments have been performed to assess the potency of idazoxan (RX 781094) at alpha and beta-adrenoceptors and dopamine receptors and on catecholamine uptake processes in rat brain. The effects of idazoxan on the turnover rates of noradrenaline and dopamine have been determined. Radioligand binding studies with cerebral cortex membranes have demonstrated that idazoxan exhibits 46-fold selectivity for alpha 2-adrenoceptors labelled by (3H)-idazoxan (Mean Ki +/- S.E.M. = 3.1 +/- 0.4 nM) compared with alpha 1-adrenoceptors labelled by (3H)-prazosin (Mean Ki +/- S.E.M. = 142 +/- 27 nM). Under the same conditions, yohimbine showed 6-fold selectivity for alpha 2-adrenoceptors. Idazoxan had low affinity for beta-adrenoceptors labelled by (3H)-dihydroalprenolol (IC50 value greater than 10 microM), for dopamine receptors labelled by (3H)-domperidone (IC50 value greater than 20 microM), for the (3H)-noradrenaline uptake site in rat hypothalamus (IC50 = 31 microM) and for the (3H)-dopamine uptake site in rat striatum (IC50 value approximately 800 microM). In rats treated with alpha-methyl-p-tyrosine, idazoxan (10-80 mg/kg, po) produced a marked increase (63% at 10, 217% at 20 mg/kg, po) in the apparent rate of turnover of noradrenaline in rat cortex/striatum, without affecting the rate of turnover of dopamine. This was in contrast to yohimbine (5-20 mg/kg, po) which increased the turnover rates of both catecholamines. In the absence of alpha-methyl-p-tyrosine, idazoxan (5-40 mg/kg, po) produced a dose related increase in the MHPG concentration and a small (20-30%) reduction in the steady state concentration of NA; the duration of the reduction was dose-related. DA steady state concentrations were unaffected. Idazoxan is a new selective alpha 2-adrenoceptor antagonist which should prove a valuable investigative tool in neurochemical studies and which may be a useful clinical agent in the management of the affective disorders.

Model system for isolation of competent ovarian-carcinoma cells from fresh tumor-tissue by a magnetic separation technique (macs).

Flow cytometric analysis of tumor cells in carcinomas is hampered by the presence of a variety of different cells in the tumor tissue and the surrounding stroma. To obtain single competent tumor cells, we have established a model system which can be applied to separate living cells from fresh ovarian carcinoma tissue. Due to the lack of tumor-cell surface specific antibodies, we isolated tumor cells by a procedure called 'negative tumor cell selection'. For this purpose, fresh ovarian carcinoma tissue, immediately after surgery, was subjected to mechanical disintegration using an automated mincing device to obtain a single-cell suspension (approximately 10(7) cells/g). Collagenase D (0.005%) was added to prevent further aggregation. Cells other than tumor cells were then labeled with a set of monoclonal antibodies directed to cell surface antigens: CD3 (T-cells), CD14 (monocytes), CD15 (granulocytes), CD45R (T-/B-cells) and 5B5 (fibroblasts). Anti-isotype antibodies coupled to ferrit microbeads were then reacted with the cell suspension and those cells reacting with the microbeads retained on a steel wool matrix in a magnetic field (1). Tumor cells not reacting with the microbeads were recovered by a simple wash of the steel wool matrix. All incubation steps were at 4 degrees C. This procedure, which takes about 2 hours, enables fast and simple isolation of single, living competent tumor cells from fresh tumor tissue and also from ascitic or pleuritic effusions. In a model system with cultured ovarian carcinoma cells and human leukocytes, tumor cell purity was about 93% and about 97% when re-subjected to the same procedure (respective recovery rates 75% and 50%). The still unlabeled tumor cells can subsequently be analyzed by flow cytometry or by central laser scanning microscopy for the presence of various surface antigens including receptors for proteases or growth factors. Moreover, after detergent treatment and fixation, flow cytometric multiparameter analysis such as simultaneous labeling of intracellular and surface antigens as well as nuclear DNA staining for ploidy and S-phase determination becomes possible.