Autosomal dominant distal myopathy: further evidence of a chromosome 14 locus.

In 1995 Laing et al. (Am J Hum Genet 56(1995)422) described a single family with nine members affected by an autosomal dominant infantile onset distal myopathy. This family generated a LOD score of 2.6 for a locus on chromosome 14. We describe two families with an infantile onset distal myopathy: a new family with four affected members and the family previously described by Scoppetta et al. (Acta Neurol Scand 92(1955)122) in both of which haplotype segregation was compatible with linkage to the same chromosome 14 locus, generating LOD scores of 0.9 at a penetrance of 100% for the markers D14S283 and D14S64 (theta=0) in both families. The loci for autosomal recessive hereditary inclusion body myopathy and Nonaka myopathy on chromosome 9 and for autosomal dominant distal myopathy of Markesberry-Griggs and Udd on chromosome 2q31-33 were excluded by linkage analysis. The disease followed a uniform course with selective wasting of the anterior tibial muscles, starting in infancy and recognizable by a characteristic clinical sign of the 'hanging big toe'. This was followed by slow progression, with involvement of the finger and wrist extensor muscles in the third decade and proximal limb muscles in the fourth decade. Interestingly, we also found evidence of an accompanying mild peripheral neuropathy in the oldest individual with hypomyelination of numerous large myelinated fibres. In addition, this patient's muscle biopsy also showed autophagic vacuoles and numerous intranuclear tubulo-filamentous inclusions of 15-20 nm diameter. Given that all three families with infantile onset distal myopathy are compatible with linkage to the same locus on chromosome 14, this study supports evidence for, and enlarges the clinical and neuropathological spectrum of the distal myopathy on chromosome 14.

Occurrence of adrenergic nerve fibers and of noradrenaline in thymus gland of juvenile and aged rats.

Adrenergic nerve fibers (ANF) were studied in juvenile, adult and old rats by biochemical and morphological methods and by quantitative analysis of images (QAI). After chemical sympathectomy with neurotoxin 6-OH dopamine, the greater part of ANF disappeared. The whole thymus was drawn in juvenile normal or sympathectomized rats, in adult normal or sympathectomized rats and in old normal or sympathectomized rats. Thymuses from the above-mentioned animals were weighed, measured and dissected. Thymic slices were stained with eosin orange for the detection of the microanatomical details and with Bodian's method for the recognition of the whole nerve fibers. Histofluorescence microscopy was used for staining of ANF while immunofluorescence microscopy was employed for staining of neuropeptide Y (NPY)-like immunoreactivity. Biochemical dosage of proteins and of noradrenaline amount was performed. Finally, all morphological results were subjected to QAI. Our results suggest that: (1) total innervation of the thymus increases with age; (2) ANF do not change with age; (3) the content of noradrenaline in the thymus increases with age; and (4) NPY-like immunoreactive structures in the thymus decrease with age. Biochemical results are in accordance with the morphological ones and both are confirmed by means of QAI. The probable function of sympathetic innervation of rat thymus is also discussed.

Effect of interferon on human thymus microenvironment.

Interferon, a thymic immunostimulator, was used with the aim of assessing the importance of adrenergic nerve fibers (ANF) and/or AChE-positive nerve fibers (AChENF) in the regulation of some immunological functions in humans. Thymic normal control fragments and/or thymic fragments of immunostimulated patients were removed during surgical biopsies. Thymic slices were stained with eosin-orange (for the recognition of microanatomical details of the microenvironment) and with Bodian's method for staining of nerve fibers. Histofluorescence microscopy was employed for staining ANF. AChENFs were detected by means of the direct-coloring thiocholine method. All images were submitted to quantitative morphometrical analysis and statistical comparisons of data. Moreover, the amount of proteins and noradrenaline was measured on thymic homogenates of the same patients. Treatment with interferon induces substantial changes in the thymic microenvironment, on ANF, on AChENFs and on the total amount of proteins and noradrenaline in thymic tissue homogenates. In conclusion immunostimulation with interferon induces substantial changes in the whole thymus and in its microenvironment, involving both sympathetic and parasympathetic nerve fibers.

gamma-aminobutyric acid-transaminase activity in the human thymus after administration of interferons.

The purpose of this article is to study the amounts of gamma-aminobutyric acid-transaminase (GABA-t) during immune response in the human thymus. GABA-t was studied by biochemical and histochemical methods in 7 immunostimulated (treated) and 7 non-immunostimulated (untreated) patients (4 young adult, age range: 24-36 years; 3 older adult, age range: 56-66 years). Immunostimulation was performed using interferon drugs for 6 months. After the histoenzymatic staining of GABA-t activity, the slides containing the samples of thymus of treated and untreated patients underwent quantitative analysis of images. The present results provide direct evidence that the immune response increases the level of GABA-t contained in vessels, parenchyma and nerve fibers of the thymus. Treatment with interferon is also capable of increasing the protein content of the thymus. The biochemical data together with the histoenzymatic results provide evidence for a localization of GABA-t in the thymic gland. Moreover, gamma-aminobutyric acid can be considered as one of the linking molecules between the immune and nervous functions of the human thymus.

Occurrence of gamma-aminobutyric acid-transaminase activity in nerve fibers of human thymus.

The specific localization of gamma-aminobutyric acid-transaminase (GABA-t) in the thymus of young and elderly men was studied. Our results show a specific vascular localization of GABA-t in the human thymus, and deal with the amount and distribution of GABA-t and its changes with age. Samples of human thymus were harvested throughout of 12 autopsies in infants (n = 3), as well as young (n = 3), adult (n = 3) and elderly (n = 3) men. Histologic staining of the human thymus was performed with eosin-orange, while histologic staining of nerve fibers was performed with the Bodian method. Histochemical and biochemical demonstration of GABA-t, including protein dosage, was performed by the methods of Van Gelder and Jung, respectively. Finally, quantitative analysis of images was performed. Staining with eosin-orange reveals the micro-anatomical details of the thymic micro-environment. The Bodian method shows the nerve fibers and neurofibrils. Histochemical staining for GABA-t shows an increase of this enzyme with age and a marked localization in the nerve fibers of the thymus in infant, young, adult, and elderly men, as well as specific vascular localization of this enzyme. These biochemical data are in accordance with the histoenzymatic results and confirm all of our previous observations. Finally, quantitative analysis of images performed on slices let us confirm all the morphological changes induced by age. We can conclude that GABA is an inhibitory neurotransmitter of the human thymus, while GABA-t plays an important role in GABA metabolism.

Quantification of acetylcholinesterase-positive structures in human thymus during development and aging.

Acetylcholinesterase (AChE) localization in the human thymus has been studied by biochemical and morphological methods during development and aging. The occurrence, the amount and the distribution of acetylcholinesterase and the changes with age were examined in 24 human thymuses. The whole human thymus was removed during autopsies in males of the following age-groups: prenatal of six months, new-born, infant, young, adult and elderly. The thymuses were weighed, measured and dissected: the microanatomical details were stained with Eosin-orange, nervous structures were identified by means of Bodian's method. Protein content was determined with biochemical methods. Histoenzymatical and biochemical demonstration of acetylcholinesterase was performed. The morphological results obtained were submitted to quantitative image analysis. Our results show that the thymic microenvironment changes with age; moreover, an increase of acetylcholinesterase-positive structures can be observed with age. Biochemical results are in agreement with morphological results and both are confirmed by the outcome of quantitative analysis of images. Acetylcholinesterase activity in human thymus may play a key role in thymic functions.

Localisation of dopamine D2-like receptors in pulmonary artery of the human and rabbit but not of the rat.

The present study was designed to investigate the presence of dopamine D2-like receptor sites in the main trunk of the human, rabbit and rat pulmonary artery using combined radioligand binding and light microscope autoradiography techniques. [3H]Spiroperidol was used as a ligand. The presence and the localisation of the sympathetic neuroeffector plexus were also studied using catecholamine histofluorescence techniques. Radioligand binding experiments demonstrated the labelling of a population of dopamine D2-like receptors in sections of human and rabbit pulmonary arteries by [3H]spiroperidol. No specific binding occurred in sections of the rat pulmonary artery. Light microscope autoradiography showed the development of specific silver grains within the tunica adventitia, including the adventitia-media border, of the human and rabbit pulmonary arteries. No specific silver grains were found in sections of the rat pulmonary artery. Studies on the pharmacological characterisation of [3H]spiroperidol binding sites in the human and rabbit pulmonary arteries showed that they are sensitive primarily to domperidone, haloperidol, (-)-sulpiride or bromocriptine, and to a lesser extent to n-propylnorapomorphine, quinpirole or clozapine displacement. This suggests that [3H]spiroperidol binding sites in the pulmonary artery probably belong to the dopamine D2 receptor subtype. Catecholamine histofluorescence techniques revealed a rich plexus of fluorescent adventitial and adventitial-medial nerve fibres in the human and to a lesser extent in the rabbit pulmonary artery. Comparison of the localisation of dopamine D2-like receptor sites and of the sympathetic neuroeffector plexus in the pulmonary artery, suggests a possible prejunctional localisation of these sites.

Age-related changes in brain microanatomy: sensitivity to treatment with the dihydropyridine calcium channel blocker darodipine (PY 108-068).

The influence of aging and of treatment with the dihydropyridine Ca2+ antagonist darodipine (PY 108-068) on the age-related microanatomical changes of rat brain were studied in male Wistar rats treated from the 18th to the 24th month of age with an oral dose of 5 mg/kg/day of darodipine. Twelve-month-old untreated rats were used as an adult reference group. A decreased number of nerve cells and of alkaline phosphatase-positive capillaries and an increased lipofuscin deposition were observed in the frontal and occipital cortex, in the hippocampus, and in the cerebellar cortex of rats of 24 months in comparison with 12-month-old animals. The number of nerve cells was higher in the occipital cortex and in the hippocampus, but not in the frontal cortex and in the cerebellar cortex, of darodipine-treated rats in comparison with age-matched untreated animals. Lipofuscin deposition is reduced in all the brain areas investigated. The density of alkaline phosphatase-reactive capillaries is also increased in the frontal and occipital cortex and in the hippocampus of aged rats treated with darodipine. The above results suggest that treatment with darodipine is able to counter some microanatomical changes occurring in the brain of aged rats and involving not only microvascular parameters. The occipital (visual) cortex and the hippocampus were the cerebral areas more sensitive to treatment with darodipine. The possible relevance of these findings is discussed.

Diabetic rats treated by low molecular weight heparin OP 2123/parnaparin: morphological changes in the kidney and heart.

AIM: Early morphological alterations in the rat kidney and heart due to experimentally induced diabetes are described in order to evaluate the possible therapeutic role of low molecular weight heparin (LMWH; OP 2123/parnaparin). METHODS: Our findings concern the alterations observed in the rat kidney and heart because these are the organs (together with the retina) mainly involved in the early morphological angiopathic modifications associated with diabetic damage of organs and tissues. In diabetic animals treated with LMWH, the Periodic Acid-Schiff (PAS) reaction showed a slight decrease when compared with the diabetic control group. Photographs were submitted to the quantitative analysis of images using a Quantimet 500 Image Analyzer (Leica) equipped with specific software. The following parameters were measured: (1) total area occupied by alkaline phosphatase (AP)-positive capillaries; (2) number and diameter of AP-positive capillaries; (3) distribution and total area occupied by PAS-positive structures (related to the intensity of the reaction resulting from the different amount of mucopolysaccharides). RESULTS: LMWH treatment is efficient in preventing these modifications, above all in the kidney. The histological study of the heart and kidney shows no significant, relevant alterations. However, the histological study of the mucopolysaccharides in diabetic animals highlighted a tendency for the heart to accumulate these substances. LMWH treatment only modestly reduced this accumulation. CONCLUSIONS: Previous evidence demonstrating a beneficial effect of therapy based on heparan sulphate proteoglycans and/or other heparin-like substances in insulin-dependent diabetes mellitus seems to be confirmed by our experimental results in different organs of adult rats. In fact, parnaparin treatment is effective (in our experience) for ameliorating the morphological pattern observed early in some diabetic tissues of rats and, above all, in the kidney.

Effect of interleukin 1beta on rat thymus microenvironment.

The effect of interleukin 1beta on the thymus of control and chemically sympathectomized adult and aged rats was studied with the aim of assessing the importance of adrenergic nerve fibres (ANF) in the regulation of some immunological functions. The whole thymus was removed from normal, sympathectomized (with the neurotoxin 6-OH-dopamine) and treated (interleukin 1beta) rats. Thymic slices were stained with eosin orange (for the recognition of microanatomical details of the thymic microenvironment) and with Bodian's method for staining of nerve fibres. Histofluorescence microscopy was employed for staining ANF and immunofluorescence was used for detecting NPY-like immunoreactivity. All images were submitted to quantitative morphometrical analysis and statistical analysis of data. Moreover, the amount of proteins and noradrenaline was measured on thymic homogenates. The results indicate that in normal conditions the formation of the thymic nerve plexi in the rat is complex: the majority of ANF are destroyed after chemical sympathectomy with 6-OH-dopamine and do not change after treatment with interleukin 1beta; on the contrary, treatment with interleukin 1beta induces substantial changes in the fresh weight of the thymus, the thymic microenvironment, thymic nerve fibers, ANF, NPY-like positive nerve fibres, and on the total amount of proteins and noradrenaline in rat thymic tissue homogenates. Immunostimulation with interleukin 1beta induces substantial changes in the whole thymus, in its microenvironment and in ANF and NPY-like nerve fibres. After chemical sympathectomy, no significant immune response was evoked by interleukin 1beta, since the majority of ANF was destroyed by chemical sympathectomy.

Acetylcholinesterase activity in rat thymus after immunostimulation with interleukin beta.

The occurrence and distribution of Acetylcholinesterase (AChE) activity were examined in the thymus of normal and immuno-stimulated adult and aged rats using biochemical and enzymehistochemical methods. Specific AChE reactivity was found primarily in the arteries and, to a lesser extent, in the veins. Only a small amount of activity could be observed in association with the subcapsular and medullary part of the parenchyma and nerve fibers. Our findings indicate that AChE activity in the rat thymus increases after treatment with interleukin beta. In fact treatment with interleukin beta induces an increase of protein content, of the amounts of AChE biochemically assayed and at the levels of AChE histoenzymatically stained. Furthermore, staining of the different structures of the thymus in treated or untreated rats shows that the significant modifications concern the parenchyma, the structures resembling nerve fibers and the whole thymus, while only small changes are observed in AChE activity located in the walls of arteries, veins and lymphatic vessels.

Microfilaments in regenerating cells of rabbit cornea: immunological and ultrastructural observations.

The role of myosin-like protein in regenerating and proliferating corneal cells following a standard alkali-injury in the rabbit eye has been studied. Microfilaments were observed by conventional transmission electron microscopy (T.E.M.) in injured epithelium and in fibroblasts. Typical microfilament bundles with electron dense structures and with stress fibers were evident. The presence of myosin-like proteins was demonstrated by means of immunochemical and autoradiographical techniques. Both epithelial cells and fibroblasts bind antimyosin-like antibodies (AMA). The same cells studied with the E.M. showed bundles of microfilaments in the cortical areas of their cytoplasm in correspondence with the same side of fluorescent or labelled AMA. The immunochemical and ultrastructural results suggest that both cells are able to produce in vivo movements involved in morphogenetic phenomena. Therefore, these structures play a role in post-traumatic corneal regeneration.

Sphenoidal metastasis from prostate carcinoma. Case report and review of the literature.

A case of an uncommon sphenoidal metastasis from prostate carcinoma with cranial nerve involvement is described. Current concepts of metastatic spread of this tumor to the skull base, clinical signs and therapeutic approaches are reviewed in the light of the available literature.

Evidence of a kallikrein inhibitor in human kidney. A new ring of the kallikrein-renin-angiotensin-aldosterone chain.

By means of immunohistochemical reactions, the authors proved the inhibitor II-related immunoreactivity in distal convoluted tubules of human kidney. A sharp inhibitor II-related immunoreactivity was also present in the blood vessels' wall. On the contrary, in the wall of proximal tubules and glomeruli only low reactivity was found. The demonstration of an inhibitor II-related immunoreactivity in the distal convoluted tubules and vessels of human kidney represents a strong evidence that an inhibitor of kallikrein exists and acts also in humans as an important key in the kallikrein-renin-angiotensin-aldosterone chain and hitherto confirms the experimental data of the literature. The proved inhibitor in the human kidney may intervene in the modulation of the kallikrein-kinin system and thus represents a key role in the intrarenal mechanisms related to the blood flow and arterial pressure regulation.

Thymectomy and multiple sclerosis: ultrastructural study of an experimental model.

Transmission electron microscopy was performed on specimens of the thymus of rats induced for acute experimental allergic encephalomyelitis (EAE). The ultrastructural alterations of the thymus were progressive and correlated with EAE development. The thymic disorganization was due to a progressive degeneration of both epithelial cells and thymocytes. These data suggest a direct involvement of the epithelial thymic cells and thymocytes in EAE pathogenesis and may suggest the intriguing therapeutic concept of thymectomy in the management of multiple sclerosis.

Anatomical variations of the ulnar and median nerves in the upper limb.

The aim of our study was the evaluation of the anatomy of ulnar and median nerves in the upper limb in order to ameliorate knowledge on the clinical anatomy of these nerves. In fact, further information on this topic may be useful owing to its possible clinical relevance when planning surgical anatomy and reconstructive surgery in tumor affected and injured patients. The relationships between ulnar and median nerve and neighbouring anatomical structures have been examined, together with the course and ramification of the ulnar and median nerves in six fresh cadavers. Moreover, we have performed a review of the literature. Four specific aspects were evaluated during dissection: 1) division modality of the ulnar nerve at the wrist; 2) anatomical details of the medial humeral epicondyle; 3) anatomical relationships between median nerve and retinaculum flexorum; 4) median-ulnar nerves anastomosis. Our results show that: the medial humeral epicondyle shows specific anatomical details in relation to the ulnar nerve; the relationships between the median nerve and the transverse carpal ligament may be characterized by one or two nerve trunks (two cases of bifid median nerve in our experience); median-ulnar nerve anastomosis may be also found at various levels. Comparing our results with those of the available literature we can conclude that anatomical variations of ulnar and median nerve in the upper limb are not an infrequent finding and their clinical, diagnostic and surgical relevance should be considered.

Interleukin 1beta as stimulator of the rat thymus.

The effects of interleukin 1beta administration on the thymus of adult and old rats were studied in order to study the interactions between the nervous and immune systems and to confirm the important role played by catecholaminergic nerve fibres (CNF) in the regulation of thymic functions. Moreover, chemical sympathectomy was performed in a group of rats to study the effects on thymus of the destruction of the majority of CNF. Our results indicate that thymic stimulation (performed by means of interleukin 1beta) induces substantial changes in the fresh weight of the whole thymus, as well as in the thymic microenvironment, thymic nerve fibres, CNF, neuropeptide Y (NPY)-like positive nerve fibres and total amount of both proteins and norepinephrine in rat thymic tissue homogenates. The majority of CNF are destroyed after chemical sympathectomy with 6-OH-Dopamine (DA) and remain unchanged after treatment with interleukin 1beta.

Age-related changes in rat optic nerve: morphological studies.

Age-related changes of the optic nerve were studied in 3-month-old (young), 12-month-old (adult) and 24-month-old (aged) male Sprague-Dawley rats. Cross sections of the intracranial portion of the optic nerves of animals of different age groups were stained with haematoxylin-eosin and examined under a light microscope at low and high magnification. Other sections were stained with crystal violet for demonstration of glial cells. A third group of sections were stained immunohistochemically to detect glial fibrillary acidic protein (GFAP) which is a marker for localizing and characterizing astrocytes. All morphological results were subjected to the quantitative analysis of images and to statistical analysis to identify significant morphometrical data. Tissue protein concentrations were determined on homogenized fragments of optic nerve. Our results demonstrate the following age-related changes: (1) increase of the optic nerve sheaths (meningeal membranes); (2) increased number of astrocytes; (3) increase of areal density of GFAP immunoreactivity; (4) increased diameter and area of the optic nerve; (5) decreased number of nerve fibres; (6) decreased-size of nerve fibres and (7) decrease of the nerve fibres/meningeal membrane ratio from 3:1 to 1:1. Moreover, the protein amount does not change with age. The rat optic nerve, therefore, appears sensitive to ageing processes and can be considered as a useful model for the studies on neuronal ageing.

Uveoscleral outflow in dog's eye: role of several enzymes.

The morphological pattern of several enzymes (succinic dehydrogenase--SDH, glucose-6-phosphate dehydrogenase--G6PDH and lactic dehydrogenase--LDH) was evaluated in normal dog eyes. Special attention was paid to the uveo-scleral tissue. Cryostatic sections of dog eye were stained with toluidine blue for the recognition of the microanatomical details or with histoenzymatic methods for SDH, G6PDH and LDH activities using sodium succinate, glucose-6-phosphate and sodium lactate as substrates respectively, nicotinamide adenine dinucleotide (NAD) as a reducing agent and sodium nitro-blue-tetrazolium as a colouring substance. A moderate positive reaction for SDH and a strong positive reaction for LDH were observed in the uveoscleral tissue, while G6PDH gave negative staining. Some considerations regarding a possible active role of these enzymatic activities to the aqueous humor outflow are suggested.