The inter-sonographer reliability of carotid duplex ultrasound.

Carotid duplex ultrasound (CDUS) is a non-invasive technique used to assess the severity of carotid artery stenosis. It has been shown to have good correlation with digital subtraction angiography (DSA) but has been criticised for its variability. One source of this is the variation in results between studies responsible for re-validating velocity criteria to match the established treatment thresholds of internal carotid artery (ICA) stenosis. The aim of this study was to develop velocity criteria and determine the presence of inter-sonographer variation of CDUS when grading ICA stenosis in our department. Five sonographers measured the degree of ICA stenosis with CDUS in 33 patients who also underwent DSA. Receiver operator characteristic curve analysis was used to develop optimal velocity criteria for the 50%, 70% and 90% ICA stenosis thresholds as a group and for each individual sonographer. A peak systolic velocity ratio of > or = 3.25 was shown to have the highest accuracy (91.5%) for predicting a 70% stenosis. A moderate value of kappa (0.53 +/- 0.027) was calculated if the optimum velocity criterion was employed for each sonographer. There was no significant variation between the ability of sonographers to grade ICA stenosis (P > 0.05) and an excellent ICC of 0.911 was calculated. This study provides evidence to suggest that CDUS in our department is not an operator-dependant test for the investigation of ICA stenosis.

Purification and partial characterization of haloperoxidase from fresh water algae Cladophora glomerata.

Many haloperoxidases have been purified from diverse organisms, including lichen, fungi, bacteria, and marine algae. In this study a haloperoxidase was purified from the fresh water algae, Cladophora glomerata, by homogenization and centrifugation, ammonium sulfate fractionation, ion-exchange and gel filtration chromatography. Molecular weight was determined by SDS-PAGE and by size exclusion HPLC and found to be approximately 43 kDa. The isoelectric point was determined to be approximately 8.1 by isoelectric focusing. The UV spectrum of the peroxidase showed a strong absorbance in the Soret band indicating a heme protein, unlike vanadium-dependent haloperoxidases from marine algae. Fresh water algal haloperoxidase catalyzed the iodination of tyrosine at a pH of 3.1. This haloperoxidase also catalyzes the oxidation of guaiacol and oxidation of iodide as well as catalyzing a peroxide-dependent reaction in both the presence and absence of chloride and bromide ions.

Congenital vascular anomalies and persistent respiratory symptoms in children.

Congenital cardiovascular anomalies are a rare but treatable cause of stridor in the paediatric population. Despite this, we and the authors of other series have found a delay in referral for endoscopic diagnosis. The duration of symptoms prior to endoscopic diagnosis ranged from 6 weeks to 10 years. It has been estimated that up to 3% of the population have congenital anomalies of the aortic arch complex and we feel that many patients currently being diagnosed as having recurrent croup or atypical asthma must be excluded from this group. We had 16 cases of congenital vascular anomalies causing tracheobronchial compression. Fifteen were due to innominate artery compression and one was due to a double aortic arch. Presenting symptoms included stridor (100%), persistent cough (75%), chronic dyspnoea (75%), reflex apnoea (60%), recurring respiratory tract infections (56%) and dysphagia (25%). Direct laryngoscopy and bronchoscopy was diagnostic in all cases and is our investigation of choice. Eleven underwent aortopexy (69%), one had division of the non-dominant aortic arch (6%) and four were managed conservatively (25%). They were followed for up to 9 years post-operatively and 75% had complete resolution of their symptoms. We feel that only a high clinical index of suspicion will enable these potentially fatal anomalies to be diagnosed.

Ultrastructural differentiation of epidermolysis bullosa subtypes and porphyria cutanea tarda.

Identifying subtypes of epidermolysis bullosa clinically, particularly at the time of disease onset, can be extremely difficult. In this investigation samples of intact skin from cases of epidermolysis bullosa and porphyria cutanea tarda were examined ultrastructurally and compared with normal tissue. The histological composition of the blister roof and floor surfaces was also evaluated. Three of the ten epidermolysis bullosa subtypes examined revealed specific features. Distinctive, circumscribed, clumped tonofilament bodies were present in basal keratinocytes from epidermolysis bullosa herpetiformis Dowling-Meara. Blister formation in epidermolysis bullosa simplex generalisata gravis occurred immediately above the dermo-epidermal junction aspect of stratum basale cells and thick (30 nm diameter), cross-striated anchoring fibrils were absent in epidermolysis bullosa dystrophica generalisata gravis. Features relating to the lamina densa of the dermo-epidermal junction, dermal capillaries and blister composition were distinctive, but not confined to a particular epidermolysis bullosa subtype or porphyria cutanea tarda.

Electron microscopy and morphometry enhances differentiation of epidermolysis bullosa subtypes. With normal values for 24 parameters in skin.

Electron microscopy combined with morphometry was used to establish values for 24 parameters in normal skin. These results were compared with those similarly obtained from samples of epidermolysis bullosa with a view to facilitating classification of the disease. Six of the eight subtypes of epidermolysis bullosa investigated could be differentiated. Four subtypes showed values for structural components in intact skin which were outside the normal range: (1) epidermolysis bullosa simplex generalisata gravis (hemidesmosomes); (2) epidermolysis bullosa dystrophica Pasini and (3) Cockayne-Touraine (anchoring fibrils); and (4) epidermolysis bullosa acquisita (anchoring fibrils, hemidesmosomes, and lamina lucida and lamina densa aspects of the dermoepidermal junction). Two subtypes revealed specific features which could be assessed qualitatively: distinctive, circumscribed, clumped tonofilament bodies were present in basal keratinocytes from epidermolysis bullosa herpetiformis Dowling-Meara and thick (30 nm diameter) cross-striated anchoring fibrils were absent in epidermolysis bullosa dystrophica generalisata gravis. Epidermolysis bullosa simplex Köbner and Weber-Cockayne forms could not be distinguished.

In vivo studies of macrophages and intercellular adhesion molecule-1 following lipopolysaccharide treatment in tumour-bearing rats.

Histological, haematological and immunocytochemical analyses were performed on a mammary adenocarcinoma implanted in Dark Agouti rats treated with LPS to further elucidate the role of TNF-alpha in tumour biology. Tumours were examined 4-12 days after implantation with parallel studies on rats treated with LPS (200 micrograms/100 g bodyweight [b.w.]) 24 h before removal. Tumour tissue from rats with 12-day-old lesions was also examined over the 24 h period after LPS administration. Haemorrhagic necrosis was evident 6 h after endotoxin treatment and became profound by 24 hours, at which stage only a thin band of viable cells was seen at the rim of the tumour. Using immunocytochemical techniques and a monoclonal anti-macrophage antibody, macrophage numbers were seen to increase throughout the tumour following LPS administration at each stage of tumour growth. Immunocytochemical staining for intercellular adhesion molecule-1 (ICAM-1), an adhesion molecule normally involved in monocyte recruitment, could not be detected in vascular channels within the tumours investigated. However, subcutaneous vessels at the tumour periphery produced a strong linear reaction. The results show that macrophage numbers in tumours increase after LPS administration, concurrently with the development of haemorrhagic necrosis. This cellular infiltration was associated with peripheral monocytopenia. ICAM-1 expression on the vascular endothelium within tumours was deficient.

Variation in histomorphometric estimates across different sites of the iliac crest.

Bone specimens were obtained from four different sites in the ilium of necropsy cases using a Jamshidi 8G trephine needle. Undecalcified histological sections were prepared and two structural histomorphometric parameters were estimated. Estimates of the percentage of trabecular bone volume (%TBV) deviated systematically, with variation of the biopsy site around the usual location, increasing in magnitude from the anterior to the posterior ilium. Such differences highlight the importance of understanding the degree of variation which exists in histomorphometric data. The histomorphometry of a vertical biopsy specimen of the iliac crest did not differ significantly from that of a transilial biopsy specimen of similar diameter taken from an adjacent site. When taken at the standard site, and when providing a long core of cancellous bone, with minimal discomfort to the patient, the vertical biopsy specimen compares well with the transiliac biopsy specimen for use for investigating metabolic bone disorders.

Comparison of two bone trephine instruments used for quantitative histomorphometry.

Transilial bone samples were obtained at necropsy using trephine instruments with internal diameters of 2 mm and 7 mm. Undecalcified histological sections were prepared and two structural histomorphometric variables (percentage of trabecular bone volume and surface density of bone) were estimated by a Quantimet 720 image analyser. The differences between the variables measured from the two adjacent cores were due to differences in biopsy site rather than the size of the sample. The smaller diameter needle is therefore recommended for use as the biopsy procedure is simpler and tissue sections can be prepared sooner than is the case with the larger needle.

Use of morphometry in cytological preparations for diagnosing follicular non-Hodgkin's lymphomas.

Morphometric techniques were applied to cytocentrifuge smears from 27 patients with histologically confirmed follicular non-Hodgkin's lymphoma to determine the usefulness of this method in diagnosis. Analysis of quantitative data confirmed that most subtypes were classified correctly on the basis of the proportion of large cells, small cleaved, and large non-cleaved cells, and nuclear, nucleolar, and cytoplasmic features. Morphometry is a more objective and reproducible technique than manual cell counting methods, and the use of cytocentrifuge smears has several advantages compared with conventional histological sectioning. Data derived with the aid of morphometry may be of value in follow up clinicopathological studies.

Phosphoenolpyruvate carboxylase of Escherichia coli. Inhibition by various analogs and homologs of phosphoenolpyruvate.

In an attempt to investigate the topography of the catalytic site of phosphoenolpyruvate (PEP) carboxylase [EC 4.1.1.31] of Escherichia coli, the inhibitor constants (Ki) for more than 20 compounds were determined with the reaction system containing dioxane, a non-physiological activator of the enzyme. The Ki values for the compounds lacking methylene-, carboxylate-, or phosphate groups were all more than 10-fold larger than the Km value for PEP, indicating the significant contribution of these groups to the binding of PEP with the enzyme. The Ki value for L-phospholactate (0.30 mM) was almost equal to the Km value for PEP (0.25 mM), whereas that for D-phospholactate (0.89 mM) was about 3-fold larger than the Km value. It was presumed that PEP binds with the enzyme on its si-side. Among 6 PEP homologs, the Ki values for phosphoenol alpha-ketobutyrate (0.024 mM) and phosphoenol alpha-ketovalerate (0.034 mM) were about one-tenth the Km value, indicating the presence of a hydrophobic pocket around the binding site of the methylene group of PEP, where the carboxylation reaction is supposed to occur. DL-Phosphomalate, a presumptive carboxylated substrate, was a weak inhibitor with a Ki value of 2.20 mM.

The kinetics of mononuclear phagocyte infiltration in neoplasms: reactions to non-specific stimuli in a rat fibrosarcoma.

The kinetics of macrophage infiltration into a transplantable rat fibrosarcoma were investigated. Monocytes obtained from normal rats and labelled with 51Cr were injected into normal or fibrosarcoma-bearing rats which had previously been implanted with cotton pellets impregnated with BCG, B. pertussis carrageenan or levan. The subcutaneous tissues of normal and fibrosarcoma-bearing rats as well as the neoplasm itself were the sites for pellet implantation. The various additives induced an enhancement of macrophage infiltration into cotton pellets implanted for 1 week into subcutaneous tissues of normal rats. No significant effect was found in pellets implanted for 3 or 14 days. Macrophage infiltration into pellets in the neoplasm was consistently reduced when compared with controls. This occurred regardless of the length of time of implantation, the type of substance added to the pellet or (in the case of BCG or B. pertussis) whether the rats were pre-sensitised to the pellet additive or not. Pellets removed from the subcutaneous tissues of fibrosarcoma-bearing rats were either similar to or more often higher in radioactivity than those implanted into the neoplasm itself. The results indicate that the capacity of macrophages to respond to inflammatory stimuli is reduced in fibrosarcoma-bearing rats, but especially in the stroma of the neoplasm.

Morphologic and DNA autoradiographic studies of the microcirculation in a transplantable rat fibrosarcoma: growth phase.

The morphology and tritiated thymidine uptake of the vascular channels in a transplantable W rat fibrosarcoma sampled at various times during growth are documented. New vessels originated primarily from normal venules in the subcutaneous tissue surrounding the neoplastic implant and grew at least twofold faster than did wound-induced vessels. During the 4-week observation period, partial maturation of vascular channels newly induced by the neoplasm was seen. This partial maturation was evidenced by an increase in the concentration of micropinocytic vesicles, a reduction in concentration and localization of intraluminal processes at or near interendothelial cell junctions, changes in the endothelial cell nuclei, and partial deposition of basement membrane material. The development of smooth muscle and nerve tissue was not seen. The proportion (13%) of labeled endothelial cells in normal subcutaneous connective tissue surrounding the 3-day-old fibrosarcoma implant was significantly higher than that seen in controls, as was the labeling index (14-25%) for endothelial cells in the fibrosarcoma up to 2 weeks after implantation. Vascular channels in the established neoplasm were examined by scanning and transmission electron microscopy and freeze-fracture techniques, and a resemblance to venular morphology was detected.

The effect of inflammatory stimuli on the stroma of neoplasms: the involvement of mononuclear phagocytes.

The phlogistic response of the stroma supporting a transplantable rat fibrosarcoma and the ability of animals bearing a neoplasm to mount an inflammatory reaction to non-specific stimuli were investigated. In the unstimulated neoplasm the concentration of macrophages in the stroma was similar to that of normal connective tissue. The mononuclear exudate induced by cotton pellets or glass coverslips implanted in the neoplastic tissue, however, was reduced when compared with that in normal connective tissues at both 7 and 14 days after implantation of these foreign bodies. Since the emigration of radioactively labelled monocytes in both inflamed normal connective tissue and inflamed stromal tissue in the neoplasm proved to be similar, the assumption has been made that neoplastic cells inhibit the local accumulation of the newly emigrated monocytes. This macrophage dispersing effect only occurs in the neoplasm and there is no systemic action. The only systemic effect detected is an anti-inflammatory action which was significant only between the 1st and the 2nd wk after transplantation of the neoplasm into neonatal recipients.

Structural and functional characteristics of the microcirculation in neoplasms.

The microcirculation in a transplantable rat fibrosarcoma was investigated. The vasculature of the neoplasm consisted of irregular channels lined by plump endothelium which displayed mainly pentalaminar although a few heptalaminar junctions were also found. Few pericytes surround the endothelial layer while the basement-membrane varied in thickness and was often duplicated and triplicated. Challenge with histamine resulted in increased permeability in comparison with normal connective tissues similarly treated. The increased permeability was accompanied by the formation of interendothelial gaps in these irregular vascular channels. Carrageenan induced a leucocytic exudate in the neoplasm which, apart from an increase in the mononuclear concentration 2 wk after injection, varied little from normal connective tissues. It is concluded that the majority of vascular channels in this neoplasm bear some structural and functional relationship to small venules.