Ethanol vapour-fixation of rat lung for immunocytochemistry investigations.

Ethanol-vapour fixation of rat lung has been successfully employed in the immunocytochemical detection of the gastrin mucin antigen termed mucin 5AC without the need of additional antigen retrieval steps. This procedure gives good morphological preservation and provides all the benefits associated with the microscopic examination of inflated lung tissue. This simple fixation technique provides another option for use in immunocytochemical investigations of rodent lung and could be adapted for other species.

A morphological study of beta-lactoglobulin absorption by cultured explants of the human duodenal mucosa using immunocytochemical and cytochemical techniques.

The absorption of beta-lactoglobulin (BLG) by cultured explants of the normal adult duodenal mucosa has been studied in vitro using immunocytochemical techniques. Membrane filters (1 mm2) were soaked in medium containing BLG and placed on the mucosal surface. Explants were cultured for < or = 60 min, and histological sections were immunostained for BLG. Increasing the BLG concentration in the filters and/or the exposure time increased the number and immunoreactivity of the immunopositive epithelial cells present, suggesting increased BLG absorption. Increased BLG penetration of the duodenal mucosa in untreated coeliac disease was also shown using these techniques. In some studies horseradish peroxidase (HRP) was used as an alternative probe and pin-pointed in epithelial cells by ultracytochemical techniques, confirming vesicular uptake of macromolecules. Colocalisation experiments using BLG and HRP and immunofluorocytochemical techniques suggested linkage between the mechanisms of absorption of both proteins into epithelial cells. Short-term organ culture and protein challenge followed by immunolocalisation has been shown to be of value in studying the absorption of BLG and HRP by the upper small intestine and possibly of other protein macromolecules as well.

Ocular changes induced in the beagle dog by intravenous infusion of a novel dopaminergic compound, FPL 65447.

FPL 65447, a selective D1 receptor agonist with a potential for the acute treatment of renal and cardiac failure and of sepsis and septic shock, was administered to beagle dogs by continuous intravenous infusion for a maximum of 14 days. Ophthalmoscopical examination revealed dose-related changes in the eye and associated structures, consisting of foci of retinal discolouration, corneal changes including oedema, keratitis, opacities and neovascularisation, and inflammation of the iris, periorbital tissues, and adnexa. Microscopical examination confirmed the presence of inflammatory lesions in the eye. These were predominantly histiocytic and were mainly focal circumscribed lesions. More diffuse inflammation with a granulocytic and lymphocytic component was also encountered in the limbus and uveal tract. A predominantly interstitial histiocytic adenitis involving various glandular structures associated with the eye and adnexa was also identified. Possible mechanisms to account for the histiocytic changes are discussed.

Procoagulant activity of platelet arachidonic acid metabolites.

1 Arachidonate metabolites have been extracted from indomethacin-treated human platelets after incubation with arachidonic acid. 2 After separation from platelet phospholipids, the extracts promoted the generation of large amounts of thrombin in normal plasma, but not in plasma devoid of lipoproteins. 3 The procoagulant activity was associated with a minor component of the mixture, which was active at concentrations below 10 micrograms ml-1. 4 The activity was similar to that of autoxidised arachidonic acid previously described. 5 Platelet arachidonic acid metabolites could play a role in the coagulation system.

The pH dependent platelet utilization of arachidonic acid and a thromboxane A2 mimetic, in vitro.

The effects of carbon dioxide (CO2) and pH on platelet aggregation in vitro have been studied to simulate pH changes observed in tissue fluid stasis and inflammation. Platelet aggregation induced by the 7-oxabicycloheptane mimetic of thromboxane A2 (TxA2) showed increased sensitivity in CO2-treated platelet-rich plasma (PRP). This effect is similar to that previously reported for arachidonate-induced aggregation. In contrast, aggregation of washed platelet suspensions (i.e. protein-free), in response to both arachidonate and TxA2 mimetic, was inhibited under acid conditions. These results confirm the importance of plasma proteins in the control of platelet function and support the hypothesis that the pH effects are dependent on these proteins.

Standardization of prekallikrein activator (PKA): the 1st International Standard for PKA.

A international collaborative study was carried out to establish an international standard for prekallikrein activator (PKA). The candidate material, coded 82/530, was assayed against the Office of Biologics (FDA) PKA reference preparation No. 2 (OoB 2) and the 1st British Reference Preparation for PKA. There was good agreement between participant laboratories on the relative PKA activity of the three preparations. Preparation 82/530 has been established by the World Health Organization as the 1st I.S. for PKA, with an assigned potency of 85 International Units per ampoule.

6-Oxo-prostaglandin F1a production by guinea-pig skeletal muscle in vitro: the effects of oestradiol and progesterone.

Fragments of guinea-pig skeletal muscle produce four times as much 6-oxo-PGF1a as PGE and PGF. This production of 6-oxo-PGF1a was unaffected by relatively high concentrations of oestradiol-17B (100 micrograms/ml) but was increased four-fold in the presence of progesterone (500 micrograms/ml). In skeletal muscle homogenates, oestradiol (100 micrograms/ml) inhibited 6-oxo-PGF1a synthesis and this inhibition was enhanced in the presence of progesterone (500 micrograms/ml); at this concentration, progesterone had no effect on its own. Tamoxifen (100 micrograms/ml), a drug with anti-oestrogenic properties in humans, enhanced the inhibition of 6-oxo-PGF1a synthesis by oestradiol and by oestradiol and progesterone together, suggesting that oestradiol and tamoxifen may exert their effect by a similar mechanism in this experimental model. The significance of these observations is discussed.

Standardization of beta-thromboglobulin (beta-TG) and platelet factor 4 (PF4): a collaborative study to establish international standards for beta-TG and PF4.

An international collaborative study was carried out to determine the suitability of freeze-dried preparations of beta-TG and PF4 to serve as international standards, and to compare these materials with other purified preparations and with plasma samples. Although problems remain with the accurate measurement of these proteins, it has been demonstrated that common standards improve the precision of measurement by RIA and provide an essential foundation for future work into the effects of assay system differences. The World Health Organization established in 1984 the purified preparation of beta-TG (83/501) and the purified preparation of PF4 (83/505) as International Standards, with assigned potencies of 500 International Units per ampoule and 400 International Units per ampoule, respectively.

Triglyceride-rich lipoproteins are responsible for thrombin generation induced by lipid peroxides.

Previous studies have shown that lipid peroxides promote thrombin generation in platelet-poor plasma. In the present study, it has been shown that triglyceride-rich lipoproteins, especially chylomicra of dietary origin, are responsible for this procoagulant activity. The generation of thrombin by lipid peroxides is also enhanced by their inhibitory action on antithrombin III. These results suggest a possible new relationship between dietary fat, lipid peroxidation and thrombus formation.

Increased sensitivity of arachidonic acid-induced platelet aggregation in the presence of carbon dioxide.

The effects of carbon dioxide on citrated human platelet-rich plasma (PRP) have been studied as a means of imitating the changes in pH and PCO2 observed in inflammation and tissue fluid stasis. Adenosine diphosphate (ADP)-induced platelet aggregation was inhibited in CO2-treated PRP. In contrast, CO2-treated platelets were rendered up to eight times more sensitive to sodium arachidonate and this effect could be imitated by the addition of exogenous calcium 1 min before the addition of arachidonate. The effects of CO2 on ADP-induced and arachidonate-induced aggregation were abolished if the CO2 was allowed to disperse from treated PRP subsequently exposed to air, suggesting no permanent alteration in platelet metabolism. The increased sensitivity of arachidonate-induced aggregation with lowered pH may be a significant factor in influencing platelet behaviour in haemostasis.

Standardization of beta-thromboglobulin and platelet factor 4: a collaborative study to investigate the sources and extent of variation in the measurement of platelet specific proteins.

A collaborative study involving seven laboratories has been carried out to investigate the sources and extent of variation in the measurement by radioimmunoassay of beta-thromboglobulin (beta-TG) and platelet factor 4 (PF4). Three plasma samples, one purified beta-TG sample and two purified PF4 samples were assayed against house standards for the respective antigens by each laboratory. The main source of variation of both beta-TG and PF4 measurements was inter-assay (particularly for PF4). It was found that a significant component of this variation was due to the participants' use of different preparations as house standards for beta-TG and for PF4. This indicates that the first step in the standardization of beta-TG and PF4 measurement should be the introduction of a reference for each.

Standardization of prekallikrein activator (PKA): the 1st British reference preparation for PKA.

A collaborative study on prekallikrein activator (PKA) has been carried out involving four laboratories for the purpose of establishing a British reference preparation for PKA. Samples of two plasma protein fractions (PPF) were assayed against participants' own supplies of the Bureau of Biologics Reference Preparation No. 2 (BoB 2). There was good agreement on the relative PKA activities of the two preparations. One of the preparations, coded 79/572, was established as the 1st British Reference Preparation for Prekallikrein Activator, with an assigned potency of 78.9 units/ml.

Detection by fluorescence of peroxides and carbonyls in samples of arachidonic acid.

1 Products of lipid peroxidation were compared in two different grades of commercially-obtained arachidonic acid. 2 The less pure 90% sample was yellow in colour, had low reactivity with 2-thiobarbituric acid and diene conjugation but high u.v. fluorescence, whereas the 99% pure sample, which was clear in colour, showed a reverse pattern of oxidation products. 3 The significance of these findings is discussed.