Multilamellar Inclusions Research Articles

Identification of a novel GLA mutation (Y88C) in a Korean family with Fabry nephropathy: a case report

BackgroundFabry disease is a rare X-linked lysosomal storage disorder caused by α-galactosidase A deficiency. With the advancement of molecular diagnostic tools, more disease-causing mutations in α-galactosidase A (GLA) have been identified in Fabry disease. We found a novel mutation in a Korean family with predominant renal manifestations of the disease.Case presentationA 24-year-old man who wanted to donate a kidney to his 28-year-old brother with end-stage renal disease of unknown cause was evaluated. The 24-year-old man underwent percutaneous renal biopsy because of an accidentally found proteinuria. Electron microscopy of his renal biopsy showed numerous electron-dense multi-lamellar inclusions in the epithelial cytoplasm, typical for Fabry disease. Clinical and laboratory evaluation including the assessment of GLA enzyme activity and direct DNA sequencing in four members of the family were performed. Renal biopsy findings in the two affected male patients were described. Re-evaluation of a renal biopsy specimen of his 28-year-old brother obtained when he was diagnosed with renal failure revealed a very focal area of suspicious multilamellated structures in the Bowman’s space. DNA sequencing on the young man, his brother, and his mother revealed a novel GLA gene mutation, c.263A > G (p.Tyr88Cys). The three all showed decreased α-galactosidase A activity.ConclusionA novel GLA mutation, c.263A > G (p.Tyr88Cys), was found in a Korean family with predominant renal manifestations of Fabry disease.

GFP‐LC3 labels organised smooth endoplasmic reticulum membranes independently of autophagy

Disruption of autophagy leads to accumulation of intracellular multilamellar inclusions morphologically similar to organised smooth endoplasmic reticulum (OSER) membranes. However, the relation of these membranous compartments to autophagy is unknown. The purpose of this study was to test whether OSER plays a role in the autophagic protein degradation pathway. Here, GFP-LC3 is shown to localise to the OSER membranes induced by calnexin expression both in transiently transfected HEK293 cells and in mouse embryo fibroblasts. In contrast to GFP-LC3, endogenous LC3 is excluded from these membranes under normal conditions as well as after cell starvation. Furthermore, YFP-Atg5, a protein essential for autophagy and known to reside on autophagic membranes, is excluded from the calnexin-positive inclusion structures. In cells devoid of Atg5, a protein essential for autophagy and known to reside on autophagic membranes, colocalisation of calnexin with GFP-LC3 within the multilamellar bodies is preserved. I show that calnexin, a protein enriched in the OSER, is not subject to autophagic or lysosomal degradation. Finally, GFP-LC3 targeting to these membranes is independent of its processing and insensitive to drugs modulating autophagic and lysosomal protein degradation. These observations are inconsistent with a role of autophagic/lysosomal degradation in clearance of multilamellar bodies comprising OSER. Furthermore, GFP-LC3, a fusion protein widely used as a marker for autophagic vesicles and pre-autophagic compartments, may be trapped in this compartment and this artefact must be taken into account if the construct is used to visualise autophagic membranes. J. Cell. Biochem. 107: 86–95, 2009. © 2009 Wiley-Liss, Inc.

Renal Pathology in Fabry Disease

Fabry disease is an X-linked recessive lysosomal storage disease that is caused by deficient activity of the lysosomal enzyme α-galactosidase A (α-Gal A), an enzyme that cleaves terminal α-galactosyl residues. This deficiency results in progressive lysosomal accumulation of glycosphingolipid with

Ultrastructural identification of specialized endocytic compartments in macrophages of the thymic cortico-medullary zone and germinal centers of peripheral lymphatic organ of the rat

In this study transmission electron microscopy was used to investigate the ultrastructural features of macrophages which are strategically positioned in the thymic cortico-medullary zone and the dark zone of germinal centers of peripheral lymphatic organs in adult Wistar rats. We show that this, morphologically distinct, type of macrophage displays the entire range of cytoplasmic inclusions, which structurally closely correspond to those of endosomal/MHC-II-enriched compartments of antigen presenting cells. The macrophages of the cortico-medullary zone and germinal centers contain numerous multivesicular bodies, as well as various kinds of cytoplasmic inclusions ranging from single to aggregated multivesicular/multilamellar bodies to large vacuoles. These multilamellar inclusions are composed of elongated, irregularly shaped cisternae, with abundance of internal membrane profiles and dense bodies. Often, cortico-medullary zone and germinal center macrophages contain the typical multilamellar bodies. Polysaccharides are detected by the thiocarbohydrazide-silver proteinate method within the dense bodies of these macrophages. The functional significance of cortico-medullary zone and germinal center macrophages is briefly discussed.

Hepatotoxicity of an HIV Protease Inhibitor in Dogs and Rats

Oral administration of the HIV protease inhibitor L-689,502 caused cholestasis and hepatocyte injury in rats and dogs. These changes occurred rapidly, with elevations in serum transaminase observed as early as 6 hr after oral dosing in dogs. The acute phase of this hepatotoxic response was characterized in more detail in rats. Following intravenous administration, bile flow was decreased in a dose-dependent manner with greater than 90% decrease in less than 30 min at a dose of 5 mg/kg. The decrease in bile flow was associated with a decrease in erythritol clearance. The decrease in bile flow was not due to disruption of biliary tight junctions. Sucrose clearance was not increased and biliary bile acid concentrations in treated animals were not different from controls. Unlike control animals, bile flow was not stimulated by infusion of the bile acid tauroursodeoxycholic acid in animals treated with L-689,502. These cholestatic effects may be due, in part, to direct hepatocyte injury. Histological examination of perfusion-fixed livers 30 min after L-689,502 administration revealed periportal changes including hepatocyte vacuolation and occasional single cell necrosis. On a subcellular level, the nucleus and mitochondria were intact in less-severely affected cells. However, extensive vacuolation with multilamellar inclusions was pronounced in these cells. In addition, canalicular ectasia was also observed which was consistent with the cholestatic changes that were seen. In summary, L-689,502 is a potent, rapid acting hepatotoxin in dogs and rats. The mechanism by which this agent induces cholestasis is novel compared to other well-characterized cholestatic agents such as α-naphtylisothiocyanate and ethinyl estradiol.

Bunina body‐like eosinophilic intracytoplasmic inlusions in a gangliocytomatous brain lesion

Small, round eosinophilic intracytoplasmic inclusions jin the constituent neuronal cells of a gangliocytomatous nodule found in a temporal lobe glioneuronal lesion of a 17‐year‐old boy with intractable complex ipartial seizures is described. These inclusions, measuring several micrometers in diameter, were often multiple in the neuronal cells, forming clusters with a bead‐ or ribbon‐like appearance. Characteristically, they often showed clear areas within them. An ultrastuctural study using selected light microscopic sections revealed that they were electron dense, granular structures with or without fingerprints‐like multilamellar profiles, and often contained a number of translucent inner areas. These inclusions with and without fingerprint‐like multilamellar profiles appeared to be similar to the previously described multilamellar inclusions and Bunina bodies, respectively. The present finding is of great interest when considering that Bunina bodies are a pathological hallmark of amlyotrophic lateral sclerosis (ALS) and that in some patients with ALS, the concurrence of multilamellar inclusions and Bunina bodies in the same neurons has been described.

Effect of amiodarone on the phospholipid and lamellar body content of lymphoblasts in vitro and peripheral blood lymphocytes in vivo

Amiodarone is useful for the treatment of ventricular arrhythmias but has been associated with a significant degree of toxicity especially to lung and liver. The drug produces phospholipid accumulation in multilamella lysosomal inclusions in many tissues due to the ability of amiodarone and desethylamiodarone to inhibit phospholipase A. The adverse effects do not correlate with the plasma levels of amiodarone but relate more closely to the cumulative dose. No clear way of predicting amiodarone toxicity has yet emerged. In this report, normal human lymphoblasts in tissue culture were shown by electron microscopy to have dose-dependent increases in multilamellar inclusions when grown with amiodarone at concentrations which are routinely observed in patients receiving the drug. The content of phospholipid also increased but this parameter was not as sensitive as the number of multilamellar inclusions. Finally, lymphocytes from patients treated with amiodarone were examined by electron microscopy and shown to have increased numbers of multilamellar bodies.

Ultrastructural organization of the exocrine glands in ants

Ants, and social insects in general, are characterized by their very well developed exocrine system. According to their cellular arrangement, the numerous glands either belong to the epithelial type, or are composed of bicellular units, each comprising a secretory cell and a duct cell. The ultrastructural organization with regard to the uptake, biosynthesis and discharge of the secretory products is very similar in both types, and generally involves surface increasing foldings of the plasmalemma. The cuticle, that forms part of each glandular cell, is mostly provided with pore canals. Pheromone producing glands are characterized by numerous mitochondria, a well developed Golgi apparatus and an extensive smooth endoplasmic reticulum in their secretory cells. The often abundant multilamellar inclusions may correspond with secretory material. Glands producing venomous substances or performing digestive functions, on the other hand, are characterized by their well developed granular endoplasmic reticulum.

Cytotoxic interactions of cardioactive cationic amphiphilic compounds in primary rat hepatocytes in culture

Hepatocytes from adult male Sprague-Dawley rats were isolated by the two-stage collagenase perfusion technique; 1 x 10(6) cells/plate were incubated in primary cell culture in Leibovitz's L-15 medium for 24 hr with or without various concentrations (12.5 to 400 mumol/L) of cardioactive cationic amphiphilic compounds such as propranolol, verapamil, sotalol, atenolol and procainamide. Propranolol and verapamil caused a significant release of lactate dehydrogenase (used as cytotoxic index in this study) in the culture media in a concentration-dependent manner, with LC50 values of 220 +/- 10 and 224 +/- 7 mumol/L, respectively. Atenolol, sotalol and procainamide had no effect on lactate dehydrogenase release. Electron microscopy of the hepatocytes showed that subtoxic concentrations of propranolol (12.5 to 125 mumol/L) and verapamil (12.5 to 100 mumol/L) induced multilamellar inclusion bodies after 24 hr of incubation. The two higher concentrations of propranolol (50 and 125 mumol/L) and 100 mumol/L of verapamil produced a significant decrease in the percentage of volume density of the mitochondria as quantitated by morphometrical analysis. An unusual feature of the electron microscopical changes with propranolol and verapamil was the presence of mitochondria within the multilamellar inclusion bodies. When these two drugs were used together or with subtoxic concentrations of amiodarone or desethylamiodarone, release of lactate dehydrogenase was significantly enhanced. No correlation was evident between the cytotoxic response and the volume density of cellular inclusions in hepatocytes treated with different concentrations of propranolol, verapamil, amiodarone or desethylamiodarone. Sotalol, atenolol and procainamide in concentrations up to 400 mumol/L did not produce any ultrastructural changes in hepatocytes after 24 hr of incubation. These results show that (a) cationic amphiphilic structure per se is not the only requirement for induction of multilamellar inclusions, (b) propranolol and verapamil can induce the formation of multilamellar inclusion bodies and cause a concentration-dependent release of lactate dehydrogenase from hepatocytes and (c) combination of different cationic amphiphiles in subtoxic concentrations can enhance cytotoxicity and increase the volume density of multilamellar inclusions.

Role of phospholipase A inhibition in amiodarone pulmonary toxicity in rats

Amiodarone is effective in the treatment of ventricular and supraventricular arrhythmias. In man its clinical use is associated with the accumulation of phospholipid-rich multilamellar inclusions in various tissues including lung, liver and others. This report presents evidence showing that amiodarone is a potent inhibitor of lysosomal phospholipase A from rat alveolar macrophages, J-744 macrophages and rat liver. When compared with other cationic amphiphilic agents which are known to produce phospholipidosis, amiodarone is one of the most potent inhibitors yet discovered. The subcellular localization of amiodarone has been determined in lung and its distribution was consistent with a lysosomal localization. It is hypothesized that amiodarone causes cellular phospholipidosis by concentrating in lysosomes and inhibiting phospholipid catabolism.

Morphology and ultrastructure of the dufour's and venom gland in the ant, Myrmica rubra (L.) (Hymenoptera : Formicidae)

The morphology and fine structure of the Dufour's and venom gland, as well as their entrance into the sting, are described in the myrmicine ant, Myrmica rubra (Hymenoptera : Formicidae). The epithelial cells that constitute the Dufour's gland wall, contain a well-developed smooth endoplasmic reticulum. Older workers, compared with younger ones, show an increasing number of multilamellar inclusions. The venom gland secretory cells are arranged in 2 free filaments that carry the secretion to the reservoir. Their cytoplasm shows an intracellular collecting ductule with surrounding microvillar sheath, and an abundance of free ribosomes. However, a well-organized granular endoplasmic reticulum, which is typical in species with a more powerful sting, does not occur. Both the Dufour's and venom gland ducts are characterized by the insertion of extensive muscle fibres, which act as a precise and mutually independent control mechanism for the discharging activities of the 2 glands.

Immunohistochemical Localization of Glycosphingolipid in Urinary Renal Tubular Cells in Fabry’s Disease

Although the accumulation of neutral glycosphingolipid (GSL), principally globotriaosylceramide ( GbOse3Cer ), in the kidney of patients with Fabry's disease is well documented, little is known about the type and quantity of lipid present in the renal tubular cells shed in the urine. Using a variety of cytologic technics, the authors examined exfoliated cells found in the urine specimens of patients hemizygous and heterozygous for alpha-galactosidase A deficiency. Renal tubular cells contained periodic acid- Shiff positive material that could be identified easily by Papanicolaou stain. A fluorescein-labeled antibody specific for GbOse3Cer localized this lipid to the cytoplasm. Electron microscopy showed numerous electron-dense multilamellar membranous inclusions within phagolysosomes and electronlucent material within lysosomes of tubular cells. Based on immunofluorescence, heterozygote individuals had similar distribution but less quantity of cytoplasmic GSL. The authors conclude that in Fabry's disease GSL accumulates probably in lysosomes of renal tubular cells. These cells are exfoliated and can be identified specifically in voided urine specimens. Examination of renal tubular cells in urine using the fluorescein antibody technic described here affords a noninvasive means of diagnosing and following the effect of therapy in patients with Fabry's disease.

Cataracts in the aging rat lens. Morphology and acid phosphatase histochemistry of incipient forms

The present investigation deals with morphological and certain histochemical changes that precede and accompany cataracts associated with aging in the rat. The cataracts were of two types: supranuclear (SNC) and posterior subcapsular (PSC). The initial biomicroscopie lesions, which were associated with the auterior and posterior lens suture systems, respectively, were attributed to cytological alterations of the ends of the superficial fiber cells. Cortical alterations common to both types of cataract included segmental cellular swelling, and the formation of assorted globular and multilamellar inclusions. With SNC, large multilamellar bodies corresponded to anterior sutural vacuolar opacities; while swelling and dissociation of the fiber cell bodies were associated with the formation of lamellar separations, lesions which eventually appeared in the peripheral cortex. Posterior subcapsular intercellular vacuoles, many of which developed a lamellated outer structure, were characteristic of the early form of PSC. An acid phosphataso which served as a marker for lysosomal hydrolases was demonstrated in the intercellular spaces of lenses with early forms of degeneration. The major source of the extracellular enzyme appeared to be the Golgi/lysosomal system within the epithelium and cortex, although the cortical smooth endoplasmic reticulum was not ruled out. Our studies support the concept that the swelling and degeneration of cortical fibers occurring in the initial stages of the cataracts might have been caused by intercellularly located hydrolases. The value of the aged rat lens as a model for human senile cataract was reinforced by the many similarities between the murine and human forms of the disease.

Mental retardation, hypotonia, and generalized seizures associated with astrocytic “residual” bodies: An ultrastructural study

Two siblings suffering since birth from convulsions, hypotonia, and mental retardation are presented. In the older sibling (eight and one-half years of age) frontal lobe biopsy revealed abnormal cytosomes with lamellar profiles in astrocytes, macrophages, and to a lesser degree in neurons. Similar cytosomes have not yet been reported in cases of sphingolipidoses or in late infantile-juvenile amaurotic idiocy. These cytosomes stained intensely with silver proteinate, an ultrastructural cytochemical stain for carbohydrate moieties. In contrast, lipofuscin did not stain with silver proteinate. Multilamellar (crescentic curvilinear) cytosomes from a reported case of late infantile amaurotic idocy (Batten-Vogt-Spielmeyer disease) did not stain with silver proteinate. Abnormal cytosomes were not found in blood cells, liver, and peripheral nerve. In the younger sibling (14 months old) postmortem ultrastructural studies of cerebral tissue showed very few abnormal cytosomes. On the basis of the clinical and ultrastructural findings, we conclude that these two cases can be distinguished from those with multilamellar (crescentic-curvilinear) inclusions and from cases of the so-called “neuronal ceroid-lipofuscinosis” syndrome.

The significance of cytoplasmic inclusions in late infantile and juvenile amaurotic idiocy. An ultrastructural study.

The electron microscopic findings of cerebral biopsies from two siblings with late infantile-juvenile amaurotic idiocy are presented. One patient had a mixture of “fingerprint” and multilamellar inclusions, often in the same cytosome. The second patient had only “fingerprint” inclusions. Electron microscopic findings in autopsied tissues from two other cases of late infantile-juvenile amaurotic idiocy with multilamellar cytosomes are also presented. Evolution of the multilamellar cytosomes into lipofuscin was not observed. We concluded that cases of late infantile-juvenile amaurotic idiocy with multilamellar and “fingerprint” inclusions should be distinguished from cases with lipofuscin.