Thymic nurse cells. Lymphoepithelial cell complexes in murine thymuses: morphological and serological characterization.

We describe a new cellular component of normal mouse thymuses, which is isolated by fractionated trypsin dissociation of minced thymus tissue followed by repeated unit gravity sedimentation. These cells are of unusually large size, with diameters of 30 mum and more. They represent cellular complexes of single large cells filled with high numbers of lymphoid cells. The majority of the engulfed lymphoid cells is not only fully intact, as judged by morphological criteria, but, moreover, includes a high proportion of mitotic figures. Electron microscopic investigations reveal the epithelial character of the large thymic nurse cells (TNC). The peripherally situated cytoplasmic tonofilament streams, and characteristic vacuoles filled with coarse, unidentified material, closely resemble cytoplasmic organelles found in the cortical reticuloepithelial cells described in situ. The internalized lymphocytes are located within caveolae lined by plasma membranes. These TNC caveolae are completely sequestered, and have lost any communication with the extracellular space, as demonstrated by the inability of an electrondense marker, cationized ferritin, to diffuse into the perilymphocytic clefts. The structural interactions between the membranes of the engulfed thymocytes with the surrounding TNC caveolar membranes were investigated both in ultrathin sections and in freeze-etch preparates. Two distinct contact types between both membranes were discerned: (a) complete, close contact along the entire lymphocyte circumference, and (b) more frequently, contact restricted to discrete, localized areas. Judging from their size and distribution, the localized contacts could correspond particle aggregates of freeze-etch preparates, which morphologically resemble certain stages of gap junction. Furthermore, we regularly found square arrays of particles of uniform size, which so far have been thought to be typical for cell membranes actively engaged in ion exchange. Tight junction-like particle arrays, which were present on TNC outer membranes, and probably represented disrupted contacts between adjacent TNC in the intact tissue, could not be found on caveolar or lymphocyte membranes. Finally, one of the most conspicuous specializations of the TNC caveolar membrane were membrane invaginations, which were arranged mainly in groups, and which probably reflect endo- or exocytotoxic events. We investigated the surface antigen phenotype of TNC by indirect immunofluorescence, with monoclonal antibodies against determinants of H-2- complex subregions as well as against lymphocyte differentiation markers. Semiquantification was reached with flow cytofluorimetry, followed by morphological control by fluorescence microscopy. The surface antigen formula of TNC is: Ig(-), Thy-l(-), H-2K(++), I-A (++), I-E/C(+), H-D(++), Ly-1(-), Ly-2(-), Qat-4(-), Qat-5(-), and peanut agglutinin (PNA)(-). Thymic macrophages, which were identified by double fluorescence, with rhodamine- coupled zymosan as a phagocytosis marker, were serologically identical with TNC. Free thymocytes, in contrast, had the following antigen formula: Ig(-), Thy-1(++), H-2K(+/-), I-A(-), I-E/C(-), H-2D(+/-), Ly-1(+/-), Ly-2(+), Qat- 4(-), Qat-5(-), and PNA(+). The unprecedented finding of high numbers of dividing thymocytes sojourning within thymic epithelial cells, and the particular specializations of the TNC caveolar membranes surrounding these engulfed thymocytes is the basis of a hypothesis that postulates that an intraepithelial differentiation cycle is one essential step in, intrathymic T lymphocyte generation.

Dexrazoxane prevents doxorubicin-induced long-term cardiotoxicity and protects myocardial mitochondria from genetic and functional lesions in rats.

BACKGROUND AND PURPOSE: Doxorubicin causes a chronic cardiomyopathy in which reactive oxygen species (ROS) accumulate over time and are associated with genetic and functional lesions of mitochondria. Dexrazoxane is a cardioprotective iron chelator that interferes with ROS production. We aim to analyze the effects of dexrazoxane on mitochondria in the prevention of doxorubicin-induced chronic myocardial lesions. EXPERIMENTAL APPROACH: Wistar rats (11 weeks of age) were injected with intravenous doxorubicin (0.8 mg kg(-1) weekly for 7 weeks) with or without simultaneous dexrazoxane (8 mg kg(-1)). Animals were killed at 48 weeks. Cardiomyopathy was scored clinically and histologically and cardiac mitochondria were analyzed. KEY RESULTS: Compared to control rats receiving saline, rats treated with doxorubicin alone developed a clinical, macroscopic, histological and ultrastructural cardiomyopathy with low cytochrome c-oxidase (COX) activity (26% of controls). The expression of the mtDNA-encoded COX II subunit was reduced (64% of controls). Myocardia exhibited a high production of ROS (malondialdehyde 338% and superoxide 787% of controls). Mitochondria were depleted of mitochondrial DNA (mtDNA copy number 46% of controls) and contained elevated levels of mtDNA deletions. Dexrazoxane co-administration prevented all these effects of doxorubicin on mitochondria, except that hearts co-exposed to doxorubicin and dexrazoxane had a slightly lower mtDNA content (81% of controls) and mtDNA deletions at low frequency. CONCLUSIONS AND IMPLICATIONS: Dexrazoxane prevented doxorubicin induced late-onset cardiomyopathy and also protected the cardiac mitochondria from acquired ultrastructural, genetic and functional damage.

Valproic acid triggers acute rhabdomyolysis in a patient with carnitine palmitoyltransferase type II deficiency.

A 47-year-old man suffering from a bipolar disorder and intermittent myoglobinuria presented with acute rhabdomyolysis with renal failure after starting therapy with valproic acid. On morphological examination, skeletal muscle revealed increased lipid storage. Biochemically, decreased enzyme activity of carnitine palmitoyltransferase (CPT) type II with carnitine levels in the lower limit was found. Genetic analysis detected the common Ser113Leu substitution on one allele of the CPT2 gene. We conclude that valproic acid should be avoided in patients with CPT type II deficiency.

Freeze-fracturing studies of mitochondrial myopathy. A correlated clinical, biochemical and morphological investigation.

Freeze-fracture studies of pathologically changed mitochondria in situ from muscle biopsies of a 9.5-year-old girl with a mitochondrial myopathy were correlated with clinical, biochemical and histochemical investigations. In the ultrathin sections giant mitochondria with densely packed cristae membranes - often reoriented to concentric circles - and, in addition, paracrystalline mitochondrial inclusions were found. The freeze fracture faces of such transformed mitochondria and preparations of their inner and outer membranes provided a morphological insight in the macromolecular structure of the mitochondrial membrane under such pathological conditions. The results lead to the hypothesis that part of the transformed mitochondria stay active functionally for an extended period by maintaining the delimitation from the cytoplasm and by preserving the macromolecular membrane architecture. This hypothesis could explain the slow progression of the myogenic symptoms.

Motor unit involvement in spastic paresis. Relationship between leg muscle activation and histochemistry.

In 4 patients with spastic hemiparesis the electromyograms (EMG) of leg muscles were recorded during walking and the gastrocnemius medialis on both sides was investigated by histochemistry and morphometry. During walking a reciprocal mode of muscle activation was preserved on the spastic side, but the EMG amplitude was reduced. In one patient the discharge behaviour of single motor units was investigated during stance. The mean discharge rate on both the spastic and the unaffected side amounted to about 5.5 Hz. Modification of this rate over a wider range by manoeuvres of the trunk was only observed on the unaffected side. Histochemistry and morphometry of the spastic muscle revealed: Increased levels of muscle fibre atrophy (especially type II); A predominance of type I fibres during later stages, when spasticity was established; Structural changes, such as the appearance of target fibres, mainly in type I fibres. These results suggest that the low level of tonic activation in spastic muscle develops tension enough during the stance phase of gait to support the body. The histopathological profile of the spastic gastrocnemius muscle is considered to be indicative of denervation, due to the combined effects of an impaired supraspinal control of the lower motoneurone and a concurrent transsynaptic muscle membrane dysfunction, muscle cell atrophy and fibre type transformation.

T-lymphocytes in experimental autoimmune myasthenia gravis. Isolation of T-helper cell lines.

The role of T-lymphocytes in Experimental Autoimmune Myasthenia Gravis (EAMG) was investigated. We generated highly purified, acetylcholine receptor (AChR)-specific T-cell populations and subsequently characterized these cell lines with respect to their membrane phenotype and their function. Using a series of mouse monoclonal antibodies directed against rat lymphocyte surface differentiation antigens, the vast majority of line cells was shown to express a leucocyte common antigen, a T-common antigen and a T-helper antigen. Small subpopulations were Ia or T suppressor antigen-positive. Adaptive transfer to sublethally irradiated, thymectomized recipients revealed that 1 X 10(6) AChR-specific line cells could cooperate effectively with 10 X 10(6) AChR-primed, complement (C3) receptor-bearing (B-cell enriched) spleen cells in the production of anti-AChR autoantibodies. Recipients of B-cells along with relevant line cells developed an acute myasthenic syndrome 6-7 days after cell transfer. Electron-microscopical examination revealed the typical features of "acute phase" EAMG with heavy mononuclear infiltration. There was, however, no evidence antibody-independent cytotoxic activity exerted by AChR-specific line cells.

Impairment of motor recovery after late nerve suture: experimental study in the rabbit. Part 2: morphological findings.

To study motor recovery after increasingly late nerve suture, we severed 75 rabbit peroneal nerves and reanastomosed them by microsurgical epineural technique after a delay of up to 12 months. Functional, electromyographic, and morphological parameters were used to evaluate the quality of motor function 6 months after suture. This second part of the study deals with the results of the morphological examinations (nerve, motor end-plate, and muscle). In examining the histological picture of the peroneal nerve, individual variations in its neurotization distal to the suture were more obvious than were variations with increasing presuture denervation periods. Histological and histochemical changes within the muscle increased significantly with later nerve suture. Important alterations at the motor end-plate were observed in the very late suture group. From the findings of both parts of the present study, the following conclusions can be drawn. The normal anatomical (histochemical) picture of the muscle is not restored after a nerve suture, not even after primary suture. The important degenerative muscular changes after very late nerve suture are not due to alterations within the nerve alone; structural changes in the motor end-plates and muscle fibers are equally important. These findings seem to be relevant when discussing the indication for nerve suture in patients with a long-standing nerve injury.

Kearns-Sayre syndrome: primarily a mitochondriopathy?

Histopathological changes of the external eye muscles and of the peripheral skeletal muscles of 2 patients with Kearns-Sayre syndrome are demonstrated histochemically and electron microscopically. In one case the progression of the mitochondrial anomalies in this disease was documented through ultrastructural investigations of muscle biopsies over a period of 17 years. By freeze-fracture the membrane fracture faces of the transformed mitochondrial were examined in both patients. Biochemical results of one patient show that energy production by glycolysis is distinctly decreased with respect to oxydation. Clinical, morphological and biochemical results support the hypothesis that the Kearns-Sayre syndrome is caused by a primary mitochondriopathy which is not limited to the musculature.

Axonal neuropathy and predominance of type II myofibers in infantile spinal muscular atrophy.

Two affected siblings with infantile spinal muscular atrophy (SMA I) presented with generalized muscular hypotonia, which progressed to early death. Quadriceps muscle biopsy did not show the typical neurogenic pattern of spinal muscular atrophy. The histochemical fiber type determination revealed a predominance of type II fibers without type I hypertrophy, an unprecedented finding in spinal muscular atrophy. Sural nerve biopsy exhibited findings typical for axonal neuropathy. In one patient, electrical stimulation of peripheral nerves showed an inexcitability of motor and sensory nerves. Genetic studies revealed homozygous deletions of the telomeric survival motor neuron (SMN) gene and the neuronal apoptosis inhibitory protein (NAIP) gene in the affected children. This is the second case report of molecular genetically proven spinal muscular atrophy associated with axonal neuropathy. We conclude atypical findings on muscle biopsy and evidence of axonal neuropathy are compatible with the diagnosis of infantile spinal muscular atrophy.

Rare combination of Becker muscular dystrophy and Klinefelter's syndrome in one patient.

Becker muscular dystrophy (BMD) was diagnosed in a male patient with Klinefelter's syndrome (47, XXY karyotype). The BMD was confirmed by (i) immunohistological methods and Western blotting, showing decreased quantity of dystrophin in muscle biopsy specimen and (ii) molecular genetic analysis which demonstrated a homozygous deletion of exons 45-47 within the dystrophin gene on both X-chromosomes. The same deletion was found on one of the X-chromosomes in the patient's mother. It can be deduced therefore that Klinefelter's syndrome in this patient is most likely due to a non-disjunctional error which occurred either during the second maternal meiotic division or during early postzygotic mitotic divisions.

Primary systemic carnitine deficiency under successful therapy: clinical, biochemical, ultrahistochemical and renal clearance studies.

Systemic carnitine deficiency is an often fatal, but treatable metabolic disorder which should be considered in any child with repeated episodes of a Reye-like syndrome or a cardiomyopathy. A 4-year-old girl with a typical history and clinical findings was successfully treated with oral carnitine. Despite low liver carnitine, ketogenesis upon fasting was normal. Normal muscle function under therapy was associated with unchanged low muscle carnitine levels. Improvement of mitochondrial structure and function was demonstrated by controlled ultrahistochemical studies. A renal carnitine leak, evident from renal clearance studies, may contribute to the pathogenesis of systemic carnitine deficiency.

[Tumor-like transformation of the median nerve in neural muscular atrophy]. / Tumoröse Umwandlung des Nervus medianus bei neuraler Muskelatrophie.

This paper reports a case of local enlargement of the median nerve in the upper arm due to the very rare condition of hypertrophic neuropathy. A characteristic feature of this affection of peripheral nerves, the cause of which is unknown, is the onion-skin-like hypertrophy of the Schwann cells and chronic neurogenic muscular atrophy. The examination of the nerve distension is described. The appropriate treatment is not known; however, the prognosis is favourable in cases showing very slow progression.

Congenital myopathy with arrest of myogenesis prior to formation of myotubes.

We report a novel type of congenital myopathy, which is characterized by an early arrest of muscle formation prior to formation of myotubes. A female infant born prematurely at 32 weeks of gestational age died after six weeks of continuous ventilatory support. Various muscle specimens including quadriceps, deltoid, pectoral, neck, psoas, tongue, and diaphragm musculature were studied. Light and electron microscopy revealed well-demarcated fascicular structures interspersed with undifferentiated, mononuclear myogenic cells. Multinucleated myotubes and muscle fibres were not detectable, pointing towards a defect prior to the generation of myotubes during myogenesis. Immunohistochemistry identified the absence of dystrophin, N-CAM, MyoD and myogenin expression in these myogenic cells, compatible with a block of the complex transcriptional network necessary for correct embryonic muscle formation at an early stage of muscle development. These myopathological findings were absent in cardiac muscle, indicating that the defect exclusively affects skeletal muscle formation.

Thymus-derived striated muscle clones. An ultrastructural analysis of cell differentiation.

Myogenic clones can be induced in thymus reticulum cultures from adult mice. We investigated the differentiation and maturation of myogenic cells by combined ultrathin section and freeze etching analyses. New insights into the development of cell membranes and cytoplasmic organelles will be discussed in the light of previous investigations of embryonic muscle cell cultures.

Intrathymic pathogenesis and dual genetic control of myasthenia gravis.

We propose a two-step model for the pathogenesis of myasthenia gravis. In the first step, primitive intrathymic stem-cells are induced by abnormal stimuli to differentiate to (abnormal?) myogenic cells. In the second step, immunocompetent T lymphocytes start an autoimmune reaction against these newly differentiated myogenic cells. The clinical stage is reached when autosensitised effector T lymphocytes leave the thymus and either infiltrate the synaptic spaces of peripheral muscles or participate in the formation of autoantibodies, causing the neuromuscular symptoms. At two points the pathogenesis is under genetic control--the first at the differentiation of the stem-cells to myogenic cells and the second at the immune responsiveness of the lymphocytes to these atypical intrathymic muscle cells.

[Light-and electron microscopic appearances of the orbicularis oculi muscle in patients with chronic progressive ocular muscular dystrophy and in normal persons (author's transl)]. / Licht-und elektronenmikroskopische Befunde des M. orbicularis oculi bei chronisch progressiver okulärer Muskeldystrophie und bei Normalpersonen.

In 8 patients with chronic progressive ocular muscular dystrophy, of whom some showed a weakness of lid closure, and in 3 normal persons (aged 62 to 69 years) biopsies of the orbicularis oculi muscle were investigated with the light- and electron microscopes. We tried to find out if the histopathological investigation alone of the orbicularis oculi muscle can establish the diagnosis of ocular muscular dystrophy or not. Both groups of investigated persons showed myopathic changes in the orbicularis oculi muscle. These changes were explained as followings of catabiosis (so called "secondary myopathic reaction") in the normal persons. The changes of the patients with myopathies, however, were explained as primarily myopathic, especially with regard to the anomalies of structure, and the pathological inclusions of the mitochondria. The findings showed that there occurred similar histopathological phenomena seen by light microscope in both groups of investigated persons; the muscle cell changes of the myopathy patients, however, were more prominent in the examination of the ultrastructure.