HN-11500--a novel thromboxane A2 receptor antagonist with antithrombotic activity in humans at arterial blood flow conditions.

In the present study we have investigated the effect of a 100 mg single oral dose of a newly developed thromboxane A2 receptor antagonist on collagen-induced thrombogenesis in flowing human non-anticoagulated blood. Blood was drawn directly from an antecubital vein over immobilised collagen type III fibrils on a cover slip placed in a parallel-plate perfusion chamber. Shear rates at the collagen surface were characteristic for medium sized (650 s-1) and moderately stenosed (2,600 s-1) arteries. Blood-collagen interactions were morphologically quantified as platelet-collagen adhesion, fibrin deposition and thrombus volume. Activation peptides of coagulation, fibrinopeptide A (FPA), and of platelets, beta-thromboglobulin (beta-TG), were measured immediately distal to the perfusion chamber. HN-11500 ingestion reduced significantly the thrombus volume by 32% at 2,600 s-1, but not at 650 s-1. However, transmission electron microscopy revealed loosely packed and less degranulated platelets at 650 s-1. The beta-TG plasma levels were also reduced at both shear rates by the HN-11500 ingestion. The platelet-collagen adhesion was significantly enhanced at both shear rates. This was apparently a consequence of higher platelet concentrations at the collagen surface, because fewer platelets were consumed by the thrombi after the drug ingestion. In contrast, the coagulation, as measured by fibrin deposition and FPA plasma levels, was not significantly affected by HN-11500. Thus, it appears that the thromboxane A2 receptor antagonist HN-11500 reduces the thrombotic response by primarily impairing the platelet function at arterial blood flow conditions, and particularly at high wall shear rates.

Clopidogrel--a platelet inhibitor which inhibits thrombogenesis in non-anticoagulated human blood independently of the blood flow conditions.

The goal of the present study was to investigate the effect of 7 and 14 days of daily oral administration of 75 mg clopidogrel on collagen-induced thrombogenesis in flowing non-anticoagulated human blood. Blood was drawn directly from an antecubital vein over immobilised collagen type III fibrils positioned in a parallel-plate perfusion chamber. The wall shear rates at the collagen surface were those characteristic for veins (100 s-1), and for medium sized (650 s-1) and moderately stenosed (2600 s-1) arteries. Clopidogrel ingestion reduced the thrombus volume significantly (p < 0.05) at 100 and 2600 s-1 (39 and 51% respectively). The beta-thromboglobulin plasma levels were reduced concomitantly. However, it was not possible to measure accurately the thrombus volume at 650 s-1, due to loose packing of the platelet thrombi. Transmission electron microscopy substantiated this observation and showed that clopidogrel profoundly reduced the platelet degranulation process (p < 0.005). The inhibitory effect of clopidogrel on platelet consumption by the growing thrombi resulted apparently in higher platelet concentration at the collagen surface, which enhanced the platelet-collagen adhesion at all three shear rates (p < 0.05). Despite the low deposition of fibrin on collagen, clopidogrel reduced significantly the fibrinopeptide A plasma levels and the fibrin deposition at shear rates below 650 s-1. This was apparently a consequence of the reduced platelet recruitment and the lower activation of platelets, since activated platelets in thrombi promote deposition of fibrin.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoelectron microscopy on epoxy sections without deplasticizing to detect glomerular immunoglobulin and complement deposits in renal diseases.

Twenty renal biopsies were studied by immunoelectron microscopy (IEM) after embedding in epoxy resin. Immunogold labeling for immunoglobulins and complement C3 was performed on the epoxy sections, which were not subjected to any kind of etching or deplasticizing prior to the immunolabeling. The concentration of accelerator, DMP-30 (Tri (Dimethyl Amino Methyl) Phenol), was increased in the infiltration and embedding steps far beyond the values normally used to make immunolabeling of these antigens possible on epoxy sections. The sections were stained with tannic acid accompanied by uranyl acetate and lead citrate. Immunofluorescence (IF) for light microscopy was carried out on frozen sections of parallel tissue samples. Some cases with IgA-nephritis demonstrated a higher sensitivity for IEM than IF, in the sense that smaller amounts of antigen were detectable with IEM. Ultrastructural preservation with this method was approximately the same as that usually seen on epoxy-embedded material. By combining excellent immunolabeling with nearly optimal ultrastructural morphology in one procedure, this method is useful particularly in situations where the material available is limited, such as in studies of renal biopsies. As far as we know, this is the first time that immunoglobulins have been satisfactorily immunolabeled on epoxy sections without etching or deplasticizing.

Neuronal uptake of plasma proteins in cryogenic brain lesions. An immunoelectron microscopic study.

A previous light microscopic study on cryogenic brain lesions in rats demonstrated uptake of plasma proteins into damaged neurons within a few minutes after the lesion. The protein concentration was much higher inside the nerve cell bodies than in the surrounding neuropil. This is puzzling since the neuropil to a large extent consists of damaged neuronal processes. The present investigation describes the intracellular localization of albumin in this model using a post-embedding immunoelectron microscopic technique. The distribution of albumin in the lesions was studied after 1, 6 and 12 h survival periods. The intraneuronal albumin was mainly bound to the particulate elements of the cytoplasm and nuclei, while the watery parts of the cells showed no immunoreactivity. The intracellular organelles contained very little albumin, indicating that their membranes may be more resistant to freezing than those of the cells. Most of the neuronal and glial processes in the neuropil were swollen and contained almost no albumin. This explains the contrast between the strong immunoreactivity of the neurons and the vague reactivity of the neuropil in light microscopy.

Reproducibility of tumour typing of lung carcinomas performed according to WHO's recommendation.

Tumour sections from 139 patients with bronchial carcinoma were classified blindly by a panel of three pathologists according to the WHO recommendation of 1981. Only sections from resected specimens were included. There was agreement among all reviewers with regard to the main WHO tumour type of 71% of the specimens. For the remainder, two reviewers agreed upon 19% whereas the tumour classification differed substantially in 10% of all cases. About half of the original diagnoses were maintained when the latter group was re-evaluated blindly. Best agreement in the whole material was noted for squamous cell carcinoma. The main difficulty was differentiation between adenocarcinoma and squamous cell carcinoma with a high malignancy grading score. Despite the improvements of the WHO classification system, our group of diagnostic problem cases was not reduced when compared with earlier classification studies.

The use of post-embedding immunoelectron microscopy in the diagnosis of glomerular diseases. Comparison of immunoelectron microscopic and immunofluorescence studies.

Fifty renal biopsies were studied by immunoelectron microscopy after embedding in a partly hydrophilic polyacrylic resin (LR White). Immunofluorescence studies were carried out on frozen sections of parallel tissue samples. Polyacrylic embedding gave good preservation of the renal ultrastructure and precise localization of immunoglobulin and C3c antibodies within glomerular electron-dense deposits. Non-specific staining of plasma proteins within vascular lumina could easily be detected. There was good correlation between immunoelectron and immunofluorescence microscopy. Immunoelectron microscopy is a very sensitive method, which can detect small amounts of antigen. More cases were, however, positive by immunofluorescence than by immunoelectron microscopy. This discrepancy may be explained by difference in sample size, and by difference in resolution of morphological details (electron microscopy versus fluorescence microscopy).

On the occurrence of focal, occult in situ and invasive carcinoma in 250 mastectomy specimens.

In 1982, a total of 250 breasts were removed for cancer in the surgical departments of the Oslo City Health Department, comprising 81% of all new breast cancers reported in Oslo in 1982. Invasive ductal carcinoma (68%) and invasive lobular carcinoma (12.4%) were the predominant types. Special attention was given to the presence of occult in situ or invasive carcinomas more than 1 cm from the periphery of the main carcinoma. In 24.8% of the specimens, carcinoma in situ was found in such locations, and an additional 6.9% showed a second, occult invasive carcinoma. Carcinoma in situ was equally common in invasive ductal and invasive lobular carcinoma. Occult invasive carcinoma was predominantly found in specimens with invasive lobular carcinoma. There was a significantly increased number of lymph node metastases in patients with carcinoma in situ or second, occult primary carcinoma more than 1 cm from the periphery of the main carcinoma.

Nuclear morphometry of benign and malignant breast lesions.

The mean nuclear area (MNA) of mammary gland epithelium was measured in 403 breast specimens, comprising 239 invasive carcinomas, 49 carcinomas in situ, 45 cases of fibrocystic disease (f.c.d.) with intraductal epithelial hyperplasia, and 60 cases of f.c.d. without intraductal hyperplasia. Normal breast tissue adjacent to other benign or malignant lesions was measured in 170 specimens. Statistical analysis revealed no difference between the MNA of invasive ductal carcinoma and ductal carcinoma in situ. The MNA of lobular and ductal carcinomas were significantly different. Significant differences were also found between ductal carcinoma and the two classes of f.c.d. The MNA of f.c.d. with and without intraductal hyperplasia were also significantly different, the former having the highest MNA. All breast lesions showed MNA significantly higher than that of normal breast epithelium. These findings show that there is a gradual increase in MNA from the baseline value of normal breast epithelium, via fibrocystic disease without and with intraductal proliferation to invasive carcinomas. Measurement of MNA may aid in pinpointing cases of intraductal epithelial hyperplasia with malignant potential.

The histopathology of non-palpable breast lesions detected by clinical mammography.

An histological study of 227 non-palpable breast lesions detected by clinical mammography revealed 64 invasive carcinomas at a mean patient age of 60.5 years. There were 10 carcinomas in situ, mean age 57.2 years. Fibrocystic disease with intraductal epithelial hyperplasia was found in 41 specimens, mean age 54.3 years. Fibrocystic disease without intraductal hyperplasia was found in 57 biopsies, mean age 50.7 years. Histological microcalcifications were found in 113 biopsies, and were considered to be a marker for epithelial proliferation of both benign and malignant kinds. Microcalcifications detectable in histological sections and by mammography differ in size by a factor of 10 or more. This difference has to be considered when comparing histological and mammographic findings.

Polycystic tumor of the atrioventricular nodal region in a man with Emery-Dreifuss muscular dystrophy.

Benign polycystic tumor of the atrioventricular nodal region is a lesion associated with cardiac conduction defects with atrioventricular block and sudden death. We present the clinical and light microscopical, immunohistochemical and ultrastructural findings of such a lesion in a young man with X-linked recessive Emery-Dreifuss muscular dystrophy who died suddenly. The tumor has not previously been described in this group of patients, who frequently suffers from cardiac electric instability and sudden death. Possible mechanisms by which the tumor may cause arrhythmia are discussed and the presence of neuroendocrine cells in the lesion is emphasized.

Solitary fibrous tumor of the pleura. An immunohistochemical, electron microscopic and tissue culture study of a tumor producing insulin-like growth factor I in a patient with hypoglycemia.

We report a patient with recurrent hypoglycemia most likely caused by a solitary fibrous tumor of the pleura. After removal of the tumor, hypoglycemia resolved. The bland histologic picture of this tumor is emphasized. Electron microscopic and immunohistochemical observations support its non-mesothelial derivation. In vitro studies demonstrated that the tumor tissue produced insulin-like growth factor I (IGF-I), and its role as the cause of hypoglycemia is discussed.

The theoretical relationship of immunogold labeling on acrylic sections and epoxy sections.

The purpose of this study was to predict the ratio of immunogold labeling of LR-White sections and epoxy sections using theoretical methods. Tissues used in the experiments were pancreas, pituitary, kidney, thyroid and fibrin. Antigens used as test proteins were glucagon, somatostatin, thyroglobulin, chromogranin A, ACTH (adrenocorticotropt hormone), amyloid A and fibrinogen. These are proteins of different sizes. The quotient labelingLR-White/labelingepoxy was deduced theoretically and compared to calculations based on practical immunogold experiments. The theoretically deduced formula showed acceptable correlation to these calculations. This study gives a theory--expressed mathematically--for what is happening on the molecular level at the surface of resin sections in immunoelectron microscopy. The theory explains why acrylic resins normally are better suited for immunoelectron microscopy than epoxy sections, and indicates increased usefulness of epoxy sections when the diameter of the protein carrying the epitope decreases.

Improved technique for immunoelectron microscopy. How to prepare epoxy resin to obtain approximately the same immunogold labeling for epoxy sections as for acrylic sections without any etching.

The purpose of this study was to improve the immunogold labeling of epoxy sections and to increase our knowledge of the mechanism for how antigens become immunolabeled on resin sections. Tissues from pancreas, thyroid and fibrin clots were embedded in an epoxy resin and LR-White. The epoxy mixture was composed and treated in different ways, especially with respect to altered amounts of accelerator (DMP-30). Immunogold labeling was performed with anti-glucagon, anti-thyroglobulin and anti-fibrinogen respectively. By increasing the amount of DMP-30 in the infiltration steps and/or embedding step, we observed a significant rise in the immunogold labeling. For the largest proteins the labeling was up to 8 times more intense than the labeling achieve with epoxy sections produced by 'normal' amount of accelerator in the embedding mixture and without accelerator in the infiltration mixture. For the smallest protein, glucagon, the differences were almost absent. The labeling of thyroglobulin and fibrinogen on the high accelerator epoxy sections was up to 70% of the labeling of LR-White sections, while conventional epoxy sections showed a labeling of 5-10% of that obtained with acrylic labeling. The cutting qualities of the high-accelerator blocks were similar to that of conventional epoxy embedding. The ultrastructure of the sections from the high-accelerator epoxy blocks were good, and the contrast was improved when tannic acid was used as enhancer. Our theory to explain the improved labeling is that the antigens are less tightly incorporated in the polymer network when the concentration of the accelerator is increased. The method outlined significantly improves the detectability of antigens on epoxy sections, which is the embedding resin routinely used in many laboratories.

The theoretical ratio of immunogold labeling of deplasticized epoxy sections and acrylic sections.

The purpose of this study was to predict the ratio of immunogold labeling of deplasticized epoxy sections and LR-White sections on the basis of theoretical considerations. Tissues used in experiments were pancreas, pituitary, kidney, thyroid, and fibrin. Antigens used as test proteins were glucagon, somatostatin, thyroglobulin, chromogranin A, ACTH (= Adrenocorticotropt hormone), amyloid A, and fibrinogen. These are proteins of different sizes. The quotient labelingdeplasticized/labelingLR-white was deduced theoretically and compared to measurements based on immunogold experiments to obtain a theoretical model with acceptable correlation to the measurements. This study describes a theory--expressed mathematically--for what happens at the molecular level in immunoelectron microscopy at the surface of deplasticized epoxy sections and acrylic sections. The theory explains why we normally get about the same amount of immunogold labeling using LR-White sections (acrylic resin) and deplasticized epoxy sections. Taking the nuances into account, the theory indicates increased usefulness of deplasticized epoxy sections when the diameter of the protein carrying the epitopes decreases.

Mechanism for antigen detection on deplasticized epoxy sections.

The purpose of this investigation was to explain why deplasticizing of epoxy sections gives higher immunogold labeling than non-deplasticizing. The methods used were the following: (1) Comparison of the ratio of immunogold labeling of deplasticized and non-deplasticized sections with gold particles of different sizes and comparison of this ratio with respect to sections of different thickness, (2) the tilt method (Brorson et al., 1994). Human kidney tissue with amyloid A depositions, human fibrin, and human pituitary tissue were embedded, sections were deplasticized on grids, treated with anti-Aa, anti-fibrinogen or anti-ACTH (ACTH = adrenocorticotropic hormone), and reembedded on grids. Indications of significant antibody penetration were found only at the periphery of structures (ACTH-vesicles). This penetration was about 30 nm. The ratios of immunogold labeling of deplasticized and non-deplasticized sections were approximately 2, 5 and 1 for amyloid, fibrin and ACTH, respectively, and were independent of the gold particle size. No significant differences of gold labeling were found between thicker and thinner deplasticized epoxy sections regardless the gold particle size. No significant differences of gold labeling between deplasticized epoxy sections and LR-White sections were found on interior areas of ACTH-vesicles or amyloid A plaques. The increased labeling of deplasticized epoxy sections compared to normal epoxy sections seemed to be mainly a surface phenomenon. The practical significance of this observation is that deplasticizing of epoxy sections may be a better method for localizing antigens at the periphery of structures than the use of other resin embedding media. Deplasticizing of epoxy sections may be a method of choice in a pathological laboratory to detect antigens in routinely embedded tissues.

Antibody penetration into LR-White sections.

The purpose of this investigation is to study the ability of antibodies to penetrate sections of LR-WHITE resin. The methods used in this study were the following: (1) Reembedding of sections labeled with immunogold (1 nm) and peroxidase/DAB/gold chloride, (2) tilting of ultrathin sections treated with immunogold (1 nm), (3) immunolabeling of cylindrical structures embedded in LR-WHITE, (4) application of primary and secondary antibodies on opposite sides of ultrathin sections. Fibrin and human pituitary tissue was embedded in LR-WHITE and treated with anti-fibrinogen or anti-ACTH respectively (ACTH = Adrenocorticotropic hormone). No indication of antibody penetration into the section were found with either of the methods, contrary to findings in earlier publications. The significance of this result is that antigens cannot be demonstrated in the interior of LR-WHITE sections with post-embedding techniques. Furthermore, LR-WHITE resin may be used for quantitative immunoelectron microscopy, and the resin may be used for double immunogold labeling since the application of immunoreagents on opposite sides of the sections is completely safe.

Effect of human fibrin adhesive on the ear. An electrophysiological study of auditory function in the guinea pig correlated to light and electron microscopy of middle ear mucosa and inner ear structures.

The effect of human fibrin adhesive applied to the middle ear has been studied in guinea pig. Auditory function was measured using acoustically evoked brainstem responses. Middle and inner ear structures were studied with light, transmission and scanning electron microscopy. A transitory conductive hearing loss was observed, but after 8 weeks the auditory function appeared normal. Microscopy of the middle and inner ear failed to show any tissue damage.

Bone scintigraphy in the diagnosis of fracture and infection of the temporal bone.

The sensitivity of 99mTc-MDP-bone-scintigraphy in the diagnosis of temporal bone fracture was found equal to that of conventional radiography if the patients were examined 10 days after the trauma. Temporal bone osteomyelitis with concomitant moderate osteosclerosis was demonstrated by bone scintigraphy in 5 cases of mastoiditis with atypical symptoms. A case of apicitis was for the first time demonstrated by scintigraphy. A low sensitivity of 67Ga-scintigraphy was demonstrated by positive 99mTc-bone-scintigraphy and negative 67Ga-scintigraphy in a patient with atypical mastoiditis. 99mTc-scintigraphy was negative in 5 cases of otitis media suppurativa and in 3 cases of otitis media chronica cum cholesteatoma, all with slight degree of osteosclerosis in the mastoid. The sensitivity of 99mTc-bone-scintigraphy in fracture and osteomyelitis of the temporal bone seems to be a function of the amount of reactive new bone formed.

A study of the value of resin-embedded semi-thin sections and electron microscopy in the diagnosis of renal biopsies.

Two hundred consecutive renal biopsies originally studied by light microscopy of paraffin- and semi-thin sections and electron microscopy were reviewed and re-classified according to the proposed WHO classification for glomerular disease. The accuracy of diagnoses based on paraffin sections alone, and of those based on paraffin- and semi-thin sections were compared with the results of the final evaluation, when electron microscopy also was taken into account. Paraffin material showed a diagnostic accuracy of 59 per cent for glomerulonephritis, and 82 per cent for other renal diseases. The diagnostic accuracy of semi-thin sections was 61 per cent for glomerulonephritis and 76 per cent for other renal diseases; i.e. not improved. It is concluded that the study of semi-thin sections cannot replace electron microscopy in the diagnoses of renal biopsies. In the present study, electron microscopy altered the diagnoses in 34 per cent and yielded additional useful information in another 45 per cent of patients with glomerulonephritis. Therefore, the electron microscope should be employed routinely in the study of renal biopsies from this group of patients.

Ultrasonographic evaluation of invasive lobular carcinoma.

PURPOSE: To compare ultrasonographic (US) and mammographic findings and tumor size measurements of invasive lobular carcinoma (ILC). MATERIAL AND METHODS: US diagnoses and mammographic findings were compared in 95 patients with pure ILC, including 46 palpable and 49 nonpalpable tumors. The diameters of tumors measured by mammography, US, and pathology were compared in 70 of the 95 patients using scatter plots and correlation analysis. RESULTS: Eighty-two (86.3%) of the ILCs were correctly diagnosed as malignant tumor, 5 (5.3%) were diagnosed as focal abnormality, and only 2 patients had normal findings on US. The most common mammographic findings were a spiculated mass (57%) and a focal asymmetric density (15%). US correctly diagnosed 8 of 12 patients with normal or equivocal mammographic findings. The correlation of tumor size assessment on imaging and pathology revealed that US measurements including the "halo" (r=0.69) was superior to that of mammography (r=0.59). ILCs larger than 30 mm were heavily underestimated by both methods. CONCLUSION: Malignant tumor was diagnosed on US in most of the patients with ILC. US tumor measurement including the "halo" predicted tumor size most accurately. The correlation between imaging measurements and tumor diameter on histology was lower for ILCs than reported for populations of mixed carcinomas.