Biomedical subjects
Publications and source records attributed to M N Hart.
Adenosine uptake by cultured cerebral endothelium is a carrier-mediated process. The Km value for adenosine uptake is 5.0 microM and the vmax is 1.15 nmol/min/mg cell protein. The uptake system is inhibited by the adenosine analog 2-chloroadenosine at low adenosine concentrations. The results prove the existence of a nucleoside transport system associated with cerebral capillary endothelium.
This study compares freeze substitution with formaldehyde fixation as a means of preserving meningeal vessel morphometry. The diameters of vessels fixed by freeze substitution correlated very well with the in vivo diameters of the same vessels (correlation coefficient 0.9766). On the other hand, diameters of vessels perfusion fixed with 10% buffered formalin displayed poor correlation with in vivo diameters of the same vessels (correlation coefficient 0.1594).
The inflammatory myopathies are a heterogeneous group of diseases currently categorized by clinical findings, laboratory data, and routine histopathologic features. In the ten years since the direct immunofluorescent (DIF) findings in idiopathic inflammatory myopathy were described, there have been a variety of series reporting DIF findings in various rheumatic and non-rheumatic diseases. This paper reviews this experience and attempts to relate the observations to immunologic mechanisms applicable to skeletal muscle. The potential contributions of direct immunohistochemical methodologies have yet to be realized, because 1) there is a paucity of data actually correlating these findings to clinicopathologic data in patients and experimental models and 2) these techniques have been limited to probing for the humoral response. The future expanded use of this technique should include histochemically tagged antisera to T-lymphocyte antigens, macrophages, biologic modifiers, and suspected etiologic agents.
Although macrophages are the earliest cells found in association with vessels in an area of cerebral injury, the role of this cell in the subsequent regeneration of the microvasculature is unknown. DNA synthesis in cerebral endothelial cells at the margin of injury of mouse brain was assayed by quantitation of the labeling indices from 3H-thymidine autoradiographs of normal animals and animals with X-ray-induced leukopenia. A mean endothelial cell labeling index of 10% in the irradiated animals was significantly lower than control animals (26.7%) (p less than 0.01). In vitro tissue culture studies utilizing peritoneal macrophages and cerebral endothelium were then used to isolate the endothelial response to macrophages and their products. Macrophage-conditioned media did not stimulate cerebral endothelial proliferation when evaluated by a growth factor assay, although this macrophage-conditioned media did stimulate DNA synthesis in fibroblasts and bovine aortic endothelium. A migration study of the cerebral endothelial cells utilizing an agarose technique showed enhanced random migration in the presence of macrophage-conditioned media compared to controls (p less than 0.01). The results indicate that macrophages do not directly stimulate proliferation of cerebral endothelial cells, but influence their migration. A loss of contact inhibition and subsequent DNA synthesis and replication may follow.
This is a report of an unusual, densely cellular, midcerebellar neoplasm in a seven-year-old boy. Although clinically consistent with a medulloblastoma, immunohistochemistry and electron microscopy demonstrated glial and rhabdomyoblastic differentiation in the tumor. We discuss the differential diagnosis of this tumor as it relates to glial differentiation in medulloblastomas and the myogenic potential of primitive neuroectoderm.
Adenosine uptake by cerebral smooth muscle cells is a carrier-mediated process. The Km value for adenosine uptake is 10.0 microM and the Vmax is 0.95 nmol/min-mg cell protein. This uptake system is inhibited by the adenosine analog 2-chloroadenosine at low adenosine concentrations. These results prove the existence of a nucleoside transport system associated with cerebral smooth muscle.
BALB/c splenic lymphocytes were activated by co-culturing in vitro and in vivo with an endothelial cell line derived from outbred mouse brain capillaries. The lymphocytes underwent proliferation, and after intravenous injection into syngeneic mice, vasculitis was found in lungs, brain, and other organs between 3 and 8 days postinjection. Controls consisted of normal lymphocytes, lymphocytes activated by phytohemaglutinin in vitro, and lymphocytes activated in vivo by cultured fibroblasts or Freund's complete adjuvant. The control lymphocytes did not cause vasculitis when injected into syngeneic mice. Lymphocytes activated by endothelium displayed nonspecific cytotoxicity by 51Cr release against both endothelial and fibroblast cell lines.
Clinical evidence of skeletal muscle involvement is frequent in systemic lupus erythematosus (SLE). In order to characterize the manifestations of SLE in skeletal muscle, a biopsy series on 19 patients with SLE was studied in terms of the histologic, histochemical, and direct immunofluorescent features of skeletal muscle. The results were correlated with clinical and laboratory data. The histologic spectrum included inflammatory myopathy, vasculitis, perifascicular atrophy, and neurogenic atrophy. Histochemical examination revealed type I fiber predominance in 44 per cent of patients and selective type II fiber atrophy in 33 per cent. Direct immunofluorescent examination of skeletal muscle biopsy specimens revealed immunoglobulin and complement deposition in vessel walls, in sarcolemmal basement-membrane areas, and within non-necrotic muscle fibers. The histologic, histochemical, and immunochemical findings are correlated with the findings in other organ systems.
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An endothelial cell line has been established from a primary culture of cerebral microvessels isolated from Swiss-Webster mice. The microvessels were isolated by a mechanical dispersion and filtration technique. The cells that emerged from these microvessels, maintained in organoid cultures, proliferated and formed plaques of a single or mixed cell type. The endothelial cell line, designated ME-2, was isolated from one such morphologically homogeneous cell plaque, using both cloning ring techniques and C6 glioma-conditioned medium. An endothelial specific antiserum was made in rabbits and was used immunocytochemically to confirm the cell type of origin of the ME-2 cell line. Not only did the cell type specific antiserum react exclusively with endothelial cells in vivo, but in the brain the antiserum localized preferentially to the luminal membrane of the endothelium. The ME-2 endothelial cells have retained several of their unique properties such as cytomorphology, growth characteristics, and cell type specific surface antigens throughout the life of the line (in one case 40 passages before senescence).
The purpose of this study was to assess the morphologic effects of antiendothelial antibodies (EAB) on mouse brain endothelium in vivo. The antigen utilized was the plasma membranes fraction of cultured mouse brain endothelial cells, which was injected into rabbits with complete Freund's adjuvant. The resultant antibody-containing serum was injected back into mice via tail veins in varying time courses and dosages, followed by perfusion of the animals. The antibody was primarily IgG, and was visualized on the brain endothelium by electron microscopy, using the peroxidase antiperoxidase (PAP) labeling technique. Normal rabbit sera and saline were used as controls. Results showed a significantly greater number of micropinocytotic vesicles in the endothelium of the test animals compared to controls. The number of multivesicular bodies and the thickness of the endothelium were also greater in the test animals. At no time was antibody visualized internal to the endothelial luminal membrane, and no lesions such as inflammation or necrosis were observed. This study shows that serum-containing antiendothelial antibodies has a direct, but apparently limited, effect on endothelium.
Orbicularis oculi muscle biopsies were performed in 38 patients (ten with chronic progressive external ophthalmoplegia and 28 controls) to determine whether ragged red fibers were present and, if so, whether they were specific to progressive external ophthalmoplegia. To our knowledge, the orbicularis muscle has not been previously studied in this regard. Ragged red fibers were seen in the orbicularis oculi in patients with and without ophthalmoplegia, although they were more abundant in patients with ophthalmoplegia. The limb muscles of patients with ophthalmoplegia showed ragged red fibers. Electron microscopy demonstrated that these fibers contain either abnormal or increased numbers of normal mitochondria. Thus, the presence of ragged red fibers in the orbicularis oculi muscle is not limited to patients with chronic progressive external ophthalmoplegia, and the diagnosis of this disorder with ragged red fibers should be based on a combination of clinical and laboratory findings, including those from a limb muscle biopsy.
A diagnosis of adult-onset metachromatic leukodystrophy (MLD) was established in a living 63-year-old man with progressive dementia and peripheral neuropathy. Decreased nerve conduction velocities and elevated spinal fluid protein concentration led to more specific diagnostic studies required to confirm the diagnosis. This case expands the spectrum of adult-onset MLD to patients with dementia and polyneuropathy past the sixth decade of life. Measurement of nerve conduction velocities may help to uncover cases of adult-onset MLD and should be included in the evaluation of dementia.
The ultrastructural features of Bunina type inclusions in the anterior horn cells of a patient dying of amyotrophic lateral sclerosis with dementia appear unique. The Bunina-type inclusions are electron dense aggregates containing, and surrounded by organelle-like membranes. These inclusions appear to be a special type of autophagic vacuole, possibly arising from altered mitochondria.
We describe a simplified technique for the histochemical determination of three fiber types from a single section of skeletal muscle. Preincubation in a solution of formaldehyde, glycine, and calcium followed by routine myofibrillar adenosine triphosphatase (ATPase) incubation clearly differentiates type I, type IIA, and IIB fibers in human, rat, rabbit, and porcine muscle. In addition, glycine-formaldehyde-calcium preincubation offers better preservation of cytoarchitecture and standardization of incubation time.
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Whitaker and Engel in 1972 first described immunoglobulin deposition in muscle biopsy specimens in connective tissue disorders. In order to confirm and extend their observations and in the hope of identifying features that may differentiate skeletal muscle involvement in connective tissue diseases from other neuromuscular diseases, a series of 80 muscle biopsy specimens were examined using direct immunofluorescence. Three distinctive direct immunofluorescence patterns consisting of vascular, sarcolemma-basement membrane, and fiber staining were identified in skeletal muscle biopsy specimens from 35 patients, 31 of whom had a connective tissue disease. A vascular staining pattern showing a granular deposition of immunoglobulin or complement was seen in 15 patients, all of whom had a connective tissue disease. Routine stains generally did not reveal vessel abnormalities. A sarcolemma-basement membrane staining pattern was demonstrated around the sarcoplasmic membrane in 29 cases. Twenty-six of these patients had a connective tissue disease. There was no correlation with inflammation, fiber necrosis, or degree of connective tissue replacement. Fibers staining for immunoglobulin or complement, seen in 14 cases, generally occurred in morphologically normal fibers. Thirteen of these patients had a connective tissue disease. Since the pathologic change in muscle in the collagen vascular diseases often consists of the nonspecific findings of focal fiber necrosis frequently without inflammatory infiltrates, direct immunofluorescence may be useful in the diagnosis and classification of muscle diseases in the collagen vascular disorders. Furthermore, the findings of immunoglobulin deposition either within vessels or within individual muscle fibers suggest that immunological mechanisms may be responsible for muscular abnormalities in the connective tissue diseases.
A case of thrombotic thrombocytopenic purpura in a young woman in which platelet microthrombi were distributed in small vessels throughout the body is reported. IgM and complement (C'3) were demonstrated in capillary and small arteriole walls by immunofluorescent microscopy. These findings support the findings of a previous case report, and are further evidence for a possible primary immune etiology for thrombotic thrombocytopenic purpura.