IPL576,092, a novel anti-inflammatory compound, inhibits leukocyte infiltration and changes in lung function in response to allergen challenge.

IPL576,092, a lead compound from a novel class of polyhydroxylated sterols, was tested in models of allergen-induced bronchoconstriction and airway inflammation. In a rat ovalbumin lung inflammation model, orally administered IPL576,092 significantly inhibited the challenge-mediated increase in total bronchoalveolar lavage leukocyte numbers, and macrophage and lymphocyte infiltration (1-10 mg/kg/day). There was a similar trend towards inhibition of eosinophil and neutrophil accumulation. Sheep were treated with IPL576,092 by inhalation (400 microg/kg/day), and lung resistance and airway hyper-responsiveness (AHR) were determined after Ascaris suum challenge. IPL576,092 significantly reduced the early and late phase bronchoconstrictor responses by 63+/-4.6 and 84+/-4.6%, respectively. IPL576,092 also blocked AHR (2.2+/-5.7% change from pre-challenge PC400), whereas control animals showed a 62.2+/-2.6% decrease in the PC400 (p<0.05). Oral IPL576,092 (5 mg/kg/day) also significantly decreased hyper-reactivity in mice. In a guinea pig model, IPL576,092 (5 mg/kg/day) significantly protected against allergen-induced increases in lung resistance (11.4+/-2.3 control versus 4.8+/-01.5 IPL576,092, area under the curve) and inhibited the increase in lung elastance (280+/-58 control versus 167+/-52 IPL576,092, p<0.05). IPL576,092, unlike dexamethasone, did not significantly decrease rat serum corticosterone levels or thymus and spleen weights, supporting a mechanism of action different from classic glucocorticoids. IPL576,092 significantly attenuates characteristics of an asthmatic response, indicating therapeutic potential for this drug class.

Heart donation: the New Zealand experience, 1987-1996.

AIMS: To audit all donor hearts offered to our unit, assess the outcome, identify factors which might enhance the acceptance rate and provide data on which to base and modify acceptance criteria. METHODS: Demographic, clinical and outcome data were collected retrospectively and analysed for all patients in whom brain death had been established and permission for organ donation obtained from the family prior to referral to the transplant donor coordinator. RESULTS: Over a nine year period (1987-96) 267 hearts were offered for transplantation. The mean donor age was 31.2 years (2-72), 163 being male and 104 female. The cause of death was secondary to trauma in 52%, neurological in 44% and due to hypoxia in 4%. The mean donor weight was 68 kg and the distribution of ABO blood group similar to the New Zealand population. Seventy-six hearts (28%) were implanted. Of the 191 not implanted no suitable recipient was available for 98 (51%) according to blood group and body weight criteria, 38 donors (20%) were medically unsuitable, 37 (19%) were declined because of donor age, 16 (8%) because of a markedly abnormal echocardiogram and 2 (1%) for logistical reasons. CONCLUSIONS: Enhanced efforts are needed to increase public awareness of the benefits of organ donation. However, a greater number of heart transplants may be achieved by increasing the recipient pool.

Effects of IZP-94005 (contignasterol) on antigen-induced bronchial responsiveness in ovalbumin-sensitized guinea-pigs.

1. We have investigated the novel naturally occurring marine compound, IZP-94005 (contignasterol), as a potential anti-asthma agent, using both in vivo and in vitro models of allergen-induced bronchoconstriction and airway smooth muscle contraction. 2. Tracheal rings from ovalbumin (OA)-sensitized guinea-pigs were treated with various concentrations of IZP-94005 for 20 min prior to challenge with ovalbumin. IZP-94005 (3-30 microM) inhibited responses of sensitized tracheal rings stimulated with OA in a concentration-dependent manner, with an IC50 of 10 microM. 3. IZP-94005 (10 microM) had no effect on carbachol-induced contractions of sensitized guinea-pig tracheal rings, although it did inhibit histamine-induced responses of OA sensitized guinea-pig tracheal rings. 4. The effects of IZP-94005 in vivo were examined using OA-sensitized guinea-pigs which were tracheotomized under anaesthesia and placed in a body plethysmograph. Measurements of lung resistance and compliance were performed by isovolumetric analysis of volume and trans-pulmonary pressure. 5. IZP-94005 (50 and 200 micrograms kg-1), by inhalation 20 min prior to OA challenge caused significant inhibition of the increase in lung resistance induced by OA in sensitized guinea-pigs, compared to vehicle-treated animals. Nedocromil sodium (20 mg kg-1), with a similar protocol, also inhibited OA-induced responses in this model. 6. We therefore suggest that IZP-94005 is a good candidate for further investigation as a possible antiasthma agent.

Donor families experience of organ donation: a New Zealand study.

AIM: To investigate the experiences of New Zealand families who had given approval for organ donation. METHOD: A postal questionnaire was sent to all families in New Zealand over a 5 year period who had agreed to donate organs on the death of their family member. RESULTS: Of the 102 questionnaires sent, 49 were returned completed. No respondents said it was the wrong decision in agreeing to donate organs. 31/49 respondents said that being asked caused no additional stress. 31/49 had previously discussed organ donation within their family. 16/49 knew the information on the donor's driving license. 38/49 said that they understood brain death at the time. 31/49 said the care, understanding and support given by the staff in the intensive care unit helped them most at the time. 23/49 would have liked further support from the transplant coordinators. 32/49 would have liked written information at some stage. Even though they were not asked 25/49 volunteered that they would have liked information about recipient outcome. CONCLUSION: Our results reinforce and support the findings of previous research from New Zealand and other countries. These have been used to confirm and revise procedures in the planning of care for future donor families.

The effect of Ca2+ on the translocation of protein kinase C in bovine tracheal smooth muscle.

The role of calcium in protein kinase C redistribution was studied in bovine tracheal smooth muscle preparations contracted by methacholine. Previous results have shown that, in the presence of normal extracellular Ca2+, 10 microM methacholine produced a sustained contraction and a sustained translocation of protein kinase C from the cytosol to the membrane. In the present study, when tissues were preincubated in Ca(2+)-free buffer containing 1 mM EGTA, methacholine produced a rapid but transient elevation in membrane-associated protein kinase C activity which was detected at 30 s and had returned to basal within 20 min. The redistribution of protein kinase C from the cytosol to the membrane induced by 1 microM methacholine in normal Ca2+ was reversed by removal of the extracellular Ca2+ and addition of 2 mM EGTA during agonist stimulation. Removal of the Ca2+ caused approximately 50% relaxation after 10 min. Verapamil (30 microM) partially reversed the methacholine-induced protein kinase C redistribution and caused approximately 40% relaxation after 15 min. Sodium nitroprusside (10 microM) caused a rapid relaxation and complete reversal of the protein kinase C redistribution induced by methacholine. High K+ (60 mM) also induced a sustained contraction and redistribution of protein kinase C from the cytosol to the membrane. Suitable antagonists were added to the bathing medium to block the effects of endogenous mediators which could be released by KCl-induced depolarization. Thus, translocation of protein kinase C is obtained in the absence of receptor activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of phospholipase C inhibitor U-73122 on antigen-induced airway smooth muscle contraction in guinea pigs.

The importance of phospholipase C (PLC) in airway smooth muscle contraction was studied, using an inhibitor of PLC, 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl] amino]hexyl]-1H-pyrrole-2,5-dione (U-73122). Tracheas from ovalbumin (OA)-sensitized guinea pigs contracted rapidly after exposure to low concentrations of antigen (OA). However, tracheas treated with U-73122 for 10 min prior to the addition of antigen, demonstrated a 3 log rightward shift in the OA dose-response curve with an IC50 of 7 microM. The analogue of U-73122, 1-[6[[17 beta-3-methoxyestra-1,3,5 trien-17-yl]amino]hexyl]-2,5-pyrrolidine-dione (U-73433), was approximately 5-fold less active in inhibiting smooth muscle contraction. In addition to the inhibition of antigen-induced smooth muscle contraction, U-73122 inhibited carbachol- and leukotriene D4-induced smooth muscle contraction. Furthermore, U-73122 inhibited in a dose-dependent manner antigen-induced histamine release from guinea pig tracheal tissue. The inhibition of smooth muscle contraction by U-73122 correlated well with the inhibition of polyphosphoinositide mediates smooth muscle contractile responses to muscarinic agonists and leukotrienes as well as antigenic-induced contraction.

Translocation of protein kinase C in bovine tracheal smooth muscle strips: the effect of methacholine and isoprenaline.

Investigations into the mechanisms involved in the contraction of smooth muscle have suggested that the generation of diacylglycerol and the activation of protein kinase C (PKC) may be important in the generation or maintenance of smooth muscle tone. The present study examined the possible role of PKC in the contraction of bovine tracheal smooth muscle. Methacholine (10 microM) induced a rapid elevation in PKC activity associated with the membrane fraction. PKC levels were significantly elevated in the membrane fraction 30 s after agonist addition, reached a maximum at 1 min and then declined to and remained at a lower level which was still elevated above basal. A concomitant decrease in cytosolic PKC activity of smaller magnitude was observed during this period of stimulation. This methacholine-induced re-distribution of PKC from the cytosol to the membrane was concentration-dependent and was blocked by atropine. Pre-treatment of tissues for 2 min with 100 microM isoprenaline prevented both the re-distribution of PKC and the contraction produced by 1 microM methacholine. Addition of 1 microM isoprenaline to tissues pre-contracted with 1 microM methacholine reversed the re-distribution of PKC produced during contraction and completely relaxed the tissues. Thus, under these conditions, translocation of PKC from the cytosol to the membrane seems to be well correlated with contractions of bovine tracheal smooth muscle. Whether the PKC translocation is responsible for the observed changes in muscle tone or whether the enzyme translocation is a result of drug-induced changes in [Ca2+]i, remains to be determined.

The effect of methacholine and histamine on cyclic AMP-dependent protein kinase activity in the guinea-pig isolated trachea.

The effects of methacholine and histamine were examined on cyclic AMP-dependent protein kinase (A-kinase) activity in guinea-pig isolated trachea, using kemptide as a substrate for phosphorylation during the determination of the enzyme activity. Methacholine (EC90, 10 microM) induced a rapid reduction in the basal A-kinase activity ratio, which was maximal after 30 s. This initial reduction coincided with the early phase of isometric tension development, and returned to control levels 4 min after the addition of methacholine. Pretreatment with atropine inhibited the methacholine response. In contrast, histamine (EC90, 30 microM) was without effect upon A-kinase activity ratio. The results establish the sensitivity of the A-kinase assay using kemptide and demonstrate that not all contractile agonists have the capacity to inhibit basal activity of A-kinase in airway smooth muscle.

The effect of phenylephrine on inositol 1,4,5-trisphosphate levels in vascular smooth muscle measured using a protein binding assay system.

This study utilizes a protein binding assay system to evaluate agonist-induced changes in inositol 1,4,5-trisphosphate (IP3) in rat aorta. Phenylephrine induced a rapid transient increase in IP3 content of rat aorta which was concentration dependent and blocked by prazosin. The concentration response curve to IP3 formation was shifted to the right of the concentration-response curve for contraction in normal calcium-containing buffer but was close to that obtained in calcium-free medium. This suggests that although IP3 may play an important role in mediating release of intracellular Ca2+, other factors (e.g. Ca2(+)-influx) may be involved in determining the magnitude of vascular smooth muscle contraction in Ca2(+)-containing solutions. Both 8-bromo cyclic GMP and sodium nitroprusside significantly attenuated the phenylephrine-induced IP3 formation. Removal of the endothelium did not alter the generation of IP3.

Determination of soluble cAMP-dependent protein kinase activity in guinea-pig tracheal smooth muscle. Preferential use of kemptide as a phosphorylating substrate.

A method is described for the determination of soluble (cytosolic) cyclic AMP (cAMP)-dependent protein kinase (A-kinase) activity in guinea-pig tracheal smooth muscle. The method relies upon the use of either histone or kemptide as a phosphorylating substrate. The relative merits of each of these substances were compared by studying the effects of a protein kinase inhibitor (PKI) and of Na+ on the phosphorylation of each substrate. PKI induced a concentration-dependent inhibition of basal and cAMP-stimulated phosphohistone formation but could not abolish it. Phosphokemptide formation was abolished by equivalent concentrations of PKI. Elevations in Na+ concentration in the reaction buffer inhibited cAMP-stimulated phosphohistone formation in a concentration-dependent manner with concomitant elevations in the enzyme activity ratio. Basal or cAMP-stimulated phosphokemptide formation was not inhibited by elevated Na+ concentrations. When tissues were homogenized in high Na+ concentration buffers, an increase in the basal A-kinase activity was observed using kemptide as the substrate. No apparent change in cAMP-stimulated activity was observed. Concomitant with this was an elevation in the enzyme activity ratio. However, a high Na+ concentration in the homogenizing buffer elevated basal phosphokemptide formation and the activity ratio. Separation of the isoenzymes of the enzyme yielded three peaks of activity upon assay of the fractions, which comprised free catalytic subunits (5% total activity), type I holoenzyme (5% total activity), and type II holoenzyme (90% total activity). Enzyme activity was increased upon pretreatment of tissues with isoprenaline and forskolin using both histone IIa and kemptide as phosphorylating substrates. The data support the preferential use of kemptide over histone IIa as a phosphorylating substrate during the determination of A-kinase activity in guinea-pig trachealis. The potential benefits of the use of kemptide are discussed.

The effect of M&B 22948 on methacholine- and histamine-induced contraction and inositol 1,4,5-trisphosphate levels in guinea-pig tracheal tissue.

The effect of a cyclic GMP phosphodiesterase inhibitor, M&B 22948, on methacholine- and histamine-induced contraction and inositol 1,4,5-trisphosphate (IP3) elevation was studied in guinea-pig tracheal rings. After addition of methacholine or histamine the rise in IP3 content was rapid and transient reaching a maximum after 5-15 s, which coincided with the maximum rate of tension development. Cyclic GMP levels of the tissue were elevated by M&B 22948 before agonist stimulation and further elevated by addition of methacholine or histamine. Cyclic AMP levels were not altered by any of these agents. M&B 22948 abolished IP3 generation induced by methacholine or histamine, but did not alter the rate or magnitude of tension development. Thus, IP3 generation does not appear to be responsible for the contractions induced by methacholine or histamine in this tissue.

Ventilatory impairment from pre-harvest retted flax.

A new method of retting flax recently developed to suit the United Kingdom climate has allowed the reintroduction of flax growing to this country. The weed killer glyphosate is sprayed on the crop which then rets before harvesting six weeks later. The acute bronchoconstrictor responses of 11 normal subjects to dust from dew retted and from pre-harvest retted flaxes were compared in a double blind crossover fashion. There were no significant differences in the dust levels nor in the size of the dust particles in the experimental dust room. The decreases in pulmonary function after six hours of dust inhalation were significantly larger after pre-harvest retted flax dust than after dew retted flax dust (delta FEV1, -0.21 and -0.40 1; delta MEF50, -0.72 and -1.211/s; delta sGaw (specific airway conductance), -0.17 and -0.65 kPa/s for dew retted and pre-harvest retted respectively). The subjects also reported more symptoms after inhaling pre-harvest retted flax dust. It is concluded that the acute bronchoconstrictor response to flax dust is increased by pre-harvest retting, suggesting an increased risk of byssinosis.

Ventilatory responses of normal subjects to flax dust inhalation: the protective effect of autoclaving the flax.

A homogeneous batch of dew retted hackled flax was divided into two portions. One was untreated and the other was steamed for 45 minutes at 125 degrees C in three pressure/vacuum cycles in an autoclave. Dust was collected when the two flaxes were separately processed by industrial doubler and stapler machines. From untreated flax 7.2 g of dust was collected per kilogram of flax after two processing operations. From the steamed flax 4.4 g of flax was obtained per kilogram after four operations. A method was devised to disperse the dust in a room to produce dust levels similar to those encountered in a dusty mill (4.5-5.7 mg/m3). Twelve normal volunteers from the managerial staff of the linen industry of Northern Ireland inhaled the dust over six hour periods. With the untreated flax decreases were obtained in mean forced expiratory measurements of 7.6% in FEV1 and 4.5% in FVC (p less than 0.01). A double blind crossover comparison of similar levels of untreated and steamed flax dusts showed 30% less impairment of the forced expirations with steamed than with untreated flax (p less than 0.05). If these responses reflect the long term airway effects of flax dust then the steaming of flax may help in reducing byssinosis.

Liver biopsy as a technique for assessing copper status of sheep.

A procedure for taking biopsy samples from the ovine liver by a paracostal route is described. Samples were taken from 120 Merino rams on up to 3 occasions over a 3 month period. At the end of the period, the rams were subjected to 2 further biopsies and were then slaughtered, and the liver was removed and homogenised. Copper contents of all samples were determined. Frequency of biopsy did not affect hepatic copper concentration which was significantly overestimated by the biopsy method by approximately 5%. Variability associated with the biopsy procedure was approximately +/- 30 to 40 mg Cu/kg DM (SD) and was small relative to variability between animals. Experimental designs were preferred in which samples are taken before and after treatments are applied; changes in concentration are then analysed. Such analyses eliminate errors associated with variability between animals and the small bias in the sampling procedure. Liver biopsy did not significantly reduce bodyweight or the rate of gain.

Analysis of a follow-up study: an example from asbestos-exposed insulation workers.

An 11-a follow-up study was carried out on a working population of asbestos insulation workers to investigate the possible progression of symptoms and signs. A total of 166 men had repeat radiology, and the 43 men who had progressed in radiography category of profusion of small irregular opacities (as determined by the ILO U/C International Classification of Radiographs of Pneumoconioses, 1980) comprised 37 (32%) of the 117 smokers, 3 (17%) of the 18 exsmokers, and 3 (10%) of the 31 nonsmokers. For 148 men over 20 a of age at the time of original study, and with lung function measured in both surveys, smoking was associated with a significantly reduced transfer factor (TLCO). For smokers in category 0 at first survey, a low TLCO was associated with subsequent progression to higher radiographic categories.