Primary prevention of atherosclerosis by lovastatin in a genetically hyperlipidaemic rabbit strain.

Lovastatin, a lipid-lowering drug which inhibits cholesterol synthesis, was administered to genetically hyperlipidaemic rabbits from the age of 2 months. Twenty rabbits were selected with similar plasma cholesterol levels and divided into matched treatment and control groups. The treated animals showed a 60% decrease in plasma cholesterol due to reduced levels of low density lipoprotein (LDL) and intermediate density lipoprotein (IDL). Levels of other lipoproteins remained unchanged. In untreated animals cholesterol levels in plasma, LDL and IDL increased with age. The area of aortic atherosclerosis-like lesions was quantified after 2-10.5 months of treatment. At each time point the extent of arterial disease was profoundly less in treated than in untreated animals. The findings demonstrate that primary prevention of arterial lesions resembling human atherosclerosis (increased amounts of fibrous tissue, smooth muscle cell proliferation, foam cell formation and necrosis at the base of the plaques) results from early effective reduction of elevated plasma lipids by lovastatin in this rabbit strain.

An improved metaphase index assay for detecting thyroid growth stimulators using FRTL-5 thyroid cells cultured on a microtitre plate.

The metaphase index assay (MIA) for thyroid growth stimulators, as originally described, used FRTL-5 thyroid cells cultured in Bellco culture chambers and glass microscope slides. The metaphases were observed using the nuclear strain aceto-lacto orcein. However the surface properties of the glass proved to be variable and so polystyrene microscope slides were substituted. The aceto-lacto orcein stain was found to be unsuitable for use with polystyrene because of the solvents and mountant used. Therefore combinations of various other nuclear stains and mounting media were tested. The Giemsa stain, which was found to be the most satisfactory, could be applied to FRTL-5 cells maintained on the large variety of plastic supports now available for tissue culture, e.g., 96 well microtitre plates. This permitted the design of an MIA which is much more convenient, robust and economical in its use of clinical samples. The results with seven IgG preparations derived from the sera of patients with a variety of thyroid disorders are presented. In its revised form, the metaphase index assay provides a rapid screening assay for thyroid growth stimulators, such as autoantibodies and TSH.

Effects of cigarette smoking on the ultrastructure of rat thoracic aorta and its ability to produce prostacyclin.

Following exposure of rats to graded doses of fresh cigarette smoke, the aortic endothelium examined by scanning electron microscopy regularly showed alterations. These essentially consisted of areas of blebbing and microvillus-like projections from the luminal surface, and the presence in the majority of cases, of micro-thrombi in the low shear areas just proximal to intercostal branches. Aortas from rats exposed to smoke showed a reduction in prostacyclin production in vitro and platelets from these animals aggregated more readily than did those of controls.

The effects of nicotine on PGI2 production by rat aortic endothelium.

The production of prostacyclin by rat aortic rings was measured following administration of nicotine by a single subcutaneous injection and after continuous subcutaneous infusion over 7 days. Single subcutaneous injections of nicotine at 1, 5, 10 or 20 mg/kg had no effect on prostacyclin production by rat aortic rings in comparison with controls. However, aortic rings obtained after 7 days continuous subcutaneous infusion of 0.54 g/ml of nicotine at the rate of 1 microliter per hour produced significantly less prostacyclin than control animals.

Phagocytosis of Trypanosoma brucei rhodesiense by peritoneal macrophages: a study by scanning electronmicroscopy.

Phagocytosis of Trypanosoma brucei rhodesiense by peritoneal macrophages takes place by seizure of the trypomastigote by either end but usually by the anterior. A lamellar sheath similar to that seen in phagocytosis of the promastigote of Leishmania is observed, but it is smaller and does not proceed to envelop the living parasite. The attached trypomastigote becomes pitted and appears to have been killed and partially destroyed before it is completely engulfed.

The spatial organisation of keratinocytes in acquired middle ear cholesteatoma resembles that of external auditory canal skin and pars flaccida.

A scanning electron microscopic study of acquired middle ear cholesteatoma was undertaken in order to compare morphological findings with those of keratinising squamous epithelium of the tympanic membrane and external auditory meatus. Analysis of results confirms a highly ordered vertical architecture, with cell stacking, in the stratum corneum of cholesteatoma, similar to that found in the skin of the deep external auditory meatus and the pars flaccida of the tympanic membrane. These findings support the notion that acquired cholesteatoma originates by immigration of epithelium from the pars flaccida and external meatus rather than by metaplasia of middle ear mucosa.

Scanning electron-microscopic study of the uptake of Leishmania parasites by macrophages.

The interaction of promastigotes of the protozoan parasite Leishmania tropica with mouse peritoneal macrophages in vitro was studied by scanning electron microscopy. Motile promastigotes attached to host cells by their flagellar tips to which the macrophages responded by producing rather closely fitting lamellar sheaths and progressively enveloping first the flagellum and then the body of the parasite. Lamellar advance during engulfment was rapid in the first 10 min but much slower later on. Fully engulfed parasites could be seen after 1 h but most parasites associated with host cells remained extracellular even after 4 h. On the other hand, parasites immobilized by fixation adhered by either their flagellar or somatic ends. Engulfment proceeded at a steady rate, and by 4 h most of them were completely engulfed. Both the attachment and engulfment stages of parasite uptake were inhibited by low temperature, cytochalasin D and mild fixation of macrophages. The rheological features of the host cells' response to parasite adherence indicate that invasion by parasites is through phagocytosis rather than penetration.

The effect of a calcium antagonist (D600) on isoprenaline-induced myocardial necrosis in the rat.

The effect of inhibiting isoprenaline-induced intracellular calcium accumulation on the degree of damage produced in the rat myocardium by this amine has been investigated by simultaneously dosing rats with the calcium antagonistic drug D600. The degree of myocardial necrosis produced in animals given isoprenaline alone and those given D600 was measured by the use of a standard point counting method to show absence of formazan from dead muscle fibres in sections treated to demonstrate succinic dehydrogenase. The use of the calcium antagonistic drug D600 considerably reduced the degree of myocardial damage produced by a standard dose of isoprenaline bitartrate. This was associated with a decrease in the isoprenaline-induced cellular calcium uptake. The results are discussed in relation to the possible protection conferred by lowering of the calcium influx at the cell membrane and the maintenance of the cells' high-energy phosphates at a level which permits "normal" cell function.

Morphology of the endothelium over atherosclerotic plaques in human coronary arteries.

The right coronary arteries of six hearts removed from patients with atherosclerosis, who were undergoing cardiac transplantation, were perfused with 2% buffered glutaraldehyde for 20 minutes before preparation for scanning electron microscopy. Perfusion was started within five minutes of explanation. In two patients the artery was angiographically normal, in one it was irregular in outline, and three had focal segments with significant stenosis. None of the patients had concentrations of plasma lipids above 5.5 mmol/l. The endothelial surface showed widespread focal abnormalities ranging from adhesion and migration of monocytes to loss of individual endothelial cells. Larger areas of endothelial denudation with exposure of underlying collagen were also seen consistently. Loss of endothelial cells was associated with accumulation of monocytes, on and deep to the surface, as well as adhesion of platelets to the exposed subendothelial tissue. These results accord with the endothelial damage and platelet adhesion seen in hyperlipidaemic animals fed a high lipid diet.

Lipid and cellular constituents of unstable human aortic plaques.

Unstable plaques are undergoing thrombosis which, in most instances, is due to fissuring and rupture of the plaque cap. This process (deep intimal injury) is a complication of plaques with a lipid-rich core. The cap tear allows blood to enter the core from the lumen, leading initially to intraplaque thrombosis and, subsequently, in some cases intraluminal thrombosis. Cap tears reflect the interplay between the force exerted on the tissue and its inherent mechanical strength. Factors which elevate and concentrate circumferential wall stress on the cap during systole include an increasing proportion of the total plaque volume occupied by the lipid core, thinning of the cap and a loss of internal collagen struts within the core. Factors which lead to an inherent reduction in the mechanical strength of cap tissue include a reduction in collagen and glycosaminoglycan concentrations, an increase in the number and density of macrophages, and a concomitant reduction in smooth muscle cells in the cap tissue. It is therefore possible to define a vulnerable plaque as one in which the lipid core is disproportionately large, the cap thin, and in which monocytes preponderate over smooth muscle cells.

Granulocyte-macrophage colony-stimulating factor induces neutrophil adhesion to pulmonary vascular endothelium in vivo: role of beta 2 integrins.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) causes upregulation of neutrophil surface CD11b/CD18 expression, and enhances the adhesion of neutrophils to cultured human endothelial cells in vitro. Systemic administration of GM-CSF results in a rapid, transient decrease in circulating phagocyte numbers. Using a nonhuman primate model (Cynomolgus), we provide histologic evidence that this transient leukopenia is associated with the margination of neutrophils in the pulmonary microcirculation. In four animals receiving 2 to 15 micrograms/kg recombinant human GM-CSF (rhGM-CSF), light microscopic sections of lung contained 36 +/- 8, 17 +/- 7, 21 +/- 6, and 15 +/- 8 (mean +/- SD, n = 20) neutrophils within a graticule grid, as compared with two control animals receiving saline injections whose lung sections contained 2.1 +/- 1.6 and 3.1 +/- 2.1 (mean +/- SD, n = 20) neutrophils within the same grid. Scanning electron microscopy shows activated leukocytes adherent to pulmonary vascular endothelium, but no morphologic evidence of endothelial damage, and no migration of cells into the extravascular space. Margination is associated with an increase in surface expression of CD11b/CD18 on circulating phagocytes, which could contribute to the adhesion to capillary endothelial cells, but CD11b/CD18 levels remain elevated even when demargination is complete. In vitro, monoclonal antibodies (MoAbs) to CD18 and CD11b were able to inhibit neutrophil aggregation and adhesion to endothelium. FMLP-induced neutrophil aggregation was inhibited by 39.8% +/- 11.5% and 44.8% +/- 12.3%, respectively, by MoAbs to CD18 and CD11b (P less than .0005, n = 4 for both); a similar effect was demonstrated on TPA-induced aggregation. MoAb CD18 reduced the adhesion of unstimulated neutrophils to endothelium by 44% (P less than .01, n = 7), and inhibited the amount of GM-CSF-stimulated adhesion by 74% (P less than .001, n = 7), while MoAb to CD11b produced a reduction of unstimulated neutrophil adhesion by 30%, and of GM-CSF-stimulated adhesion by 40% (P less than .01, n = 5, for both). However, when administered in vivo, MoAb CD18 produced only a small, albeit significant, amelioration of GM-CSF-induced margination in vivo, while MoAb CD11b was without effect. These results show that GM-CSF-induced transient leukopenia is associated with enhanced neutrophil adherence to pulmonary vascular endothelium, but suggest that the beta 2 leukocyte integrins CD11/CD18 play only a minor role in this process.

Cellulose granulomatous peritonitis.

The case history of a patient developing granulomatous peritonitis due to cellulose is described. At a second laparotomy following an operation for intestinal obstruction due to adhesions, the peritoneum was found to be studded with multiple, yellow nodules, closely resembling tuberculous lesions, but shown on microscopy to be tuberculoid granulomata containing cellulose fibres. The main features of the condition, which is self-limiting, and probably more common than is generally realized, are discussed.

The effect of anti-lipolytic agents on isoprenaline-induced myocardial necrosis in the rat.

The effect of inhibiting isoprenaline-induced lipolysis on the degree of damage produced in the rat myocardium by this amine has been investigated by pre-dosing rats with the anti-lipolytic agent 5-fluoro-nicotinic acid. The degree of myocardial necrosis produced in animals given isoprenaline alone and those pre-dosed with the anti-lipolytic agent was measured by the use of an automated flying spot microscope to show absence of formazan from dead muscle fibres in sections treated to demonstrate succinic dehydrogenase. The use of the anti-lipolitic considerably reduced the degree of myocardial damage produced by a standard dose of isoprenaline bitartrate. This was associated with an inhibition of the post-isoprenaline rise in plasma free fatty acid levels. The results are discussed in relation to the possible protective roles of the lowering of plasma free fatty acid levels and inhibition of the adenyl cyclase system at the plasma membrane of the myocardial cell produced directly by the anti-lipolytic.

Nicotine and cigarette smoking: effects on the ultrastructure of aortic endothelium.

The aortic endothelium from rats has been examined by scanning (SEM) and transmission electron microscopy (TEM) following nicotine administration or cigarette smoking. Nicotine was administered to rats by subcutaneous injections at concentrations of 1, 5, 10 and 20 mg/kg or by continuous subcutaneous pumps delivering a solution of 0.54 g/ml of nicotine at the rate of 1 microliter/h for 7 days. Animals were exposed to fresh smoke from eight cigarettes, each yielding 19 mg tar and 1.5 mg nicotine. Twenty minutes after subcutaneous injections, no endothelial abnormalities were evident by SEM, apart from at the high dose where minimal endothelial damage was seen. Limited endothelial damage was discerned following nicotine infusion and this consisted of microvillus formation, platelet adhesion and some cell separation. TEM revealed no endothelial changes in any of the animals. Cigarette smoking, however, resulted in considerable endothelial morphological changes consisting of the formation of blebs, microvillus-like projections, increased numbers of plasmalemmal vesicles and an increase in the numbers of Weibel-Palade bodies. The results suggest that components of cigarette smoke other than nicotine are responsible for the endothelial cell changes associated with smoking.

A new treatment for urethral strictures.

A urethral stent, originally developed for endovascular use, was implanted into eight patients with urethral strictures after experimental studies in the canine urethra. The stent is woven in the form of a tubular mesh from surgical grade stainless steel wire and is self-expanding when released from its small-diameter delivery catheter. At follow-up 6 months to 1 year postoperatively (mean 8 months) all had a good calibre urethra. Urethroscopy showed complete epithelial covering of the implant at 4-6 months.

Hereditary hyperlipidemia and atherosclerosis in the rabbit due to overproduction of lipoproteins. II. Preliminary report of arterial pathology.

A genetically determined hyperlipidemic strain of New Zealand White rabbit that has features in common with combined familial hyperlipidemia in humans has been identified. The morphologic findings in a few animals fed a normal chow diet are reported. These consisted of macroscopically visible aortic intimal elevations found in the greatest number in the descending thoracic aorta. The plaques showed the presence of a cell population consisting of modified smooth muscle cells and lipid-laden macrophages. The lesion bases were necrotic and acellular, and some showed the presence of dystrophic calcification. Scanning electron microscopy revealed numerous monocytes attached to the endothelium. Endothelial defects were common, and these were filled with swollen and "ruffled" macrophages. Transmission electron microscopy confirmed the presence of lipid-laden cells penetrating between adjacent endothelial cells. These findings resemble those reported in a number of different animal species after dietary induction of hyperlipidemia. This strain is a useful new model for the study of atherogenesis.

Pathogenesis of Crohn's disease: multifocal gastrointestinal infarction.

In a prospective study, specimens of resected small and large intestine from fifteen patients with Crohn's disease were prepared by heparin-saline vascular perfusion, followed by either resin casting of the mesenteric vascular supply and tissue maceration or glutaraldehyde perfusion-fixation, resin casting, and tissue clearance. The specimens were examined by macrophotography, histopathology, and either scanning or transmission electronmicroscopy. A pathogenetic sequence of events in Crohn's disease was seen--vascular injury, focal arteritis, fibrin deposition, arterial occlusion mainly at the level of the muscularis propria, followed by tissue infarction or neovascularisation. These features were confined to segments of intestine affected by Crohn's disease and did not occur in normal bowel. The findings suggest that Crohn's disease is mediated by multifocal gastrointestinal infarction. This pathogenetic process is compatible with many of the clinical features of Crohn's disease, and its recognition has important implications for the identification of the primary cause of the illness and advances in clinical management.

Cigarette smoking and platelet adhesion.

Non-abraded rabbit endothelium has been exposed to human blood taken from male non-smoking volunteers before and after the smoking of two medium tar cigarettes, in an in vitro system using a Baumgartner chamber. In each case the blood was allowed to circulate for 10 min at a constant flow rate. Blood from 10 volunteers has been tested in this way. Scanning electron microscopy of the endothelial surfaces demonstrates large numbers of adherent platelets when 'post-smoking blood' is used, but very few and in some cases none with the 'pre-smoking' blood. As a further control to ensure that this phenomenon did not occur as a result from changes in the vessel related to the time during which it had been removed from its normal physiological environment, blood from further non-smoking volunteers was passed over seven of the remaining pieces of vessel at the completion of these runs. Platelets were either absent or very few in number, as with the pre-smoking samples.

Hereditary hyperlipidemia in the rabbit due to overproduction of lipoproteins. I. Biochemical studies.

An inherited metabolic disorder in a strain of New Zealand White rabbits, characterized by marked hypercholesterolemia (394 +/- 100 mg/dl), with moderately elevated or normal triglyceride levels is described. Low density lipoprotein (LDL), intermediate density lipoprotein (IDL) and very low density lipoprotein (VLDL) cholesterol levels were increased. VLDL and IDL, and to a lesser extent LDL, had increased free cholesterol and esterified cholesterol content, and triglyceride content was reduced. Kinetic studies with 131I and 125I-labelled rabbit lipoproteins showed a marked increase in production rates of VLDL apo B and LDL apo B. LDL cholesterol levels were directly related to LDL apo B production rate (r = 0.938, p less than 0.001). Both in hypercholesterolemic and normal rabbits injected with labelled VLDL, the specific activity-time curves of VLDL apo B and LDL apo B did not intersect, indicating that LDL apo B was in part derived from sources other than VLDL. No defect was demonstrated in receptor-mediated catabolism of LDL by cultured skin fibroblasts from hyperlipidemic animals. The fractional catabolic rate of LDL apo B was subnormal, but increased when the expanded LDL apo B pool size was reduced by exchange transfusion; the low fractional catabolism may therefore be attributable, at least in part, to saturation of LDL receptors consequent upon the increased pool size of LDL. The hyperlipidemia in this strain of rabbits may be unique in that the underlying mechanism appears to be overproduction of VLDL and LDL.