Lysosomal storage in human skeletal muscle

Lysosomal Storage in Human Skeletal Muscle STIRLING CARPENTER,MD, AND GEORGE KARPATI,MD Skeletal r0uscle is involved symptomatically in two lysosomal storage diseases, acid maltase deficiency and a similar condition in which enzyme levels are normal. Asymptomatic storage in skeletal muscle ceils is found in Batten-Kufs' disease (ceroid lipofuscinosis), Fabry's disease, and mannosidosis, as well as in rare patients with an unidentified storage disease. Other cell types (vascular endothelium, smooth muscle, fibroblasts, satellite cells) within the confines of the biopsy specimen may reveal storage in other diseases. The differential diagnosis involves predominantly both normal and abnormal conditions in which acid p h o s p h a t a s e activity is p r o m i n e n t in cells. HUM PATHOL 17:683--703, 1986.

T h e term storage disease implies excessive accumulation o f some molecule that normally occurs in cells, although if this concept is applied rigorously, at least one disease traditionally so labeled, branching enzyme deficiency or glycogenosis IV, would have to be excluded. Conceptually, the most homogeneous group of storage diseases are the lysosomal disorders. Lysosomes constitute the main cellular machinery for breaking down complex molecules so that their components can be recycled. More than 50 lysosomal enzymes, almost all of them acid hydrolases, 1 have been identified. Most lysosomal storage diseases are the result of the pronounced genetically determined deficiency of one of these enzymes. Cellular damage in lysosomal storage disease appears to result almost exclusively from the occupation of space inside the cell by distended lysosomes. Rupture of such lysosomes may occur. For example, in acid m a h a s e d e f i c i e n c y the m e m b r a n e surrounding a glycogen collection may have large gaps, but there is no evidence that the residual lysosomal enzymes in st, ch a situation damage normal cell components. For cells of most types, such as neurons or hepatocytes, sufficient storage results in expansion of the cell body. Skeletal muscle cells, in contrast, have a tendency to shrink. There are two possible explanations for this phenomenon. First, storage beyond a certain degree is accompanied by loss of myofibrils, the largest protein component o f the cell, the contraction of which triggers many other intracellular metabolic activities. Second, exocytosis of cytoplasmic components engulfed in autophagic vacuoles may reduce cell size. Storage may be morphologically evident in a cell without compronfising function, if the Receivedfrom the Departments of Neurology-Neurosurgery and Pathology, McGill University and the Montreal Neurological Institute, Montreal, Quebec, Canada. Supported by grants from The Medical Research Cot,ncil of Canada, The Muscular Dystrophy Association of Canada, and the Killam Mentorial Fund of the Montreal Neurological Institute. Address correspondence and reprint requests to Dr. Carpenter: Montreal Neurological Institute, 3801 University Street, Montreal. Quebec tt3A-2B4, Canada.

volume of the stored substances remains relatively small. Storage will occur only in cells programmed to make the substance whose catabolism is altered. For example, in GM 2 gangliosidosis, storage is not seen in muscle fibers, since they synthesize almost no GMz ganglioside. Each storage disease shows a particular distribution in cell types and tissues, although within any entity there may be some variation. For instance, in juvenile Batten's disease cutaneous smooth muscle cells may or may not be involved. Each disease also has its characteristic ultrastructural patterns in the various cell types; ahhough some patterns (e.g., relatively lucent vacuoles containing fine granular materiM) are found in several diseases. Molecules other than tile specific undegraded substrate o f the genetically defective enzyme are often present in storage lysosomes. Some substrate molecules will attract other molecules, to form complicated micelles. For example, the ntembranous cytoplasmic bodies in GM 2 gangliosidosis contain phospholipids and cholesterol as well as the GM 2 ganglioside that accumulates because of the specific enzyme defect. 2 Skeletal muscle may be involved in many storage diseases, relatively few o f which are lysosomal; among those that are lysosomal, symptoms related to skeletal muscle involvement are prominent only in acid maltase deficiency and in a disease that closely mimicks it. s Most of the storage diseases that produce symptomatic involvement of muscle show accumulation of glycogen or its metabolites. These substances are normally utilized in filel- and energy-producing reactions. Therefore, the existence of a lysosomal disease (acid mahase deficiency) in which it is glycogen that cannot be disposed of seems paradoxical. The most likely explanation is that glycogen enters lysosomes randomly and, once there, can be disposed of only by the appropriate lysosomal enzyme. Skeletal muscle does not show definite clinical involvement in the other lysosomal storage diseases, although mild hypotonia may be present, as in mannosidosis, probably reflecting central nervous system dysfunction. Slight dysfimction of the muscle cells might be masked by prominent involvement of the nervous system, or s t o r a g e may not be massive enough to interfere with cell function. In these diseases muscle biopsy may, nevertheless, be useful in suggesting or confirming the diagnosis. In most of the lysosomal storage diseases, accurate biochemical tests for urine, serum, leukocytes, or cnhured fibroblasts are available. Biopsy may, nevertheless, have a role. Confirmation of biochemical test results by microscopic study o f tissue may be considered prudent. In addition, the clinical features may not always be 683

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TABLE 1. Features of Well-defined Lysosomal Storage Diseases with Storage in Skeletal Muscle Cells

Disease and Subtypes Acid mahase deficiency Infantile

Childhood

Age at Onset of Symptoms

Deficient Enzymes

3 mo to I yr

ct-i,4-glucosidase

2 - 5 yr

r cosidase

Adult

15-60 yr

ct-l,4-glucosidase

3 - 1 3 yr

Unknown

1- 112 yr

Unknown

Late infantile

2{--3 yr

Unknown

Juvenile

3 - 1 0 yr

Unknowq

7 - 8 yr

Unknown

15-50 yr

Unknown

Lysosomal glycogen storage with normal acid maltase activity Batten's disease Infantile

Juvenile subgroup

Adult (Ku fs' disease)

Salient Clinical Features

Tissues Other than Muscle Involved

Cardiac and hepatic enlargement, hypotonia Proximal weakness, calf hypertrophy

Mnscle cells, satellite cells, endothelium, peric)tes Muscle cells

Glycogen, amorphous dense material

Muscle cells

Glycogen, amorphous dense material

No tissue data; clinically-brain, heart, liver

Muscle cells

Glycogen, amorphous dense material

Mental and motor retardation, seizures, nlyoclonus, visual impairment Mental and motor retardation, seizures, nlyocIonus, visual impairmem, pigmentary degeneration of retina Mental and motor retardation, seizures, myocIonus, visual impairment, pigmentary degeneration of retina

Almost all cell types

Muscle cells, endothelium, fibroblasts, satellite cells

Granular osmiophilic deposits (GROD)

Almost all cell types

Muscle cells satellite cells, endotheliunl, fibroblasts

Curvilinear bodies

Exocrine and endocrine glands, neurons, retina, Schwann cells, endothelium, smooth muscle, histiocytes

Muscle cells, satellite cells, endothelium

Rectilinear bodies

Mental and motor retardation, seizures, myoclonns, visual impairment, pigmentary degeneration of retiqa Ataxia, seizures, nlyocJonns, dementia

Almost all cell types

Muscle cells, endothelium, satellite cells,

GROD

Neurons

Occasional muscle cells

Rectilinear bodies

Proximal weakness, respiratory failure Proximal weakness, dyspnea, cardiomegaly, mental retardation

684

Almost all cell types

Cells Involved in Muscle

Ultrastructural Pattern of Lysosomal Contents in Muscle Cells

Eccrine glands, endothelium, smooth innscle, Scbwann cells, rare hepatocytes and cardiac myocytes None reported

Glycogen, anaorphons dense material

LYSOSOMALSTORAGEIN SKELETALMUSCLE (.Carpenter & Karpati)

TABLE 1, Continued

Disease and Subtypes

Age at Onset of Symptoms

Fabry's disease Type I

5-15 yr

Mannosidosis

1-4

yr

Deficient Enzymes

et-galactosidase

ct-mannosidase

Ultrastructural Pattern of LysosomalContents in Muscle Cells

Salient Clinical Features

Tissues Other than Muscle Involved

Attacksof pain, s k i n lesions, corneal opacities, renal failure, cardiovascular and cerebrovascular manifestations Ps)chomotor retardation, dysmorphic features, hearing l o s s , gum hyperplasia

Kidney, vessels, perineurium, dorsal root ganglia, cerebral autonomic neurons, cornea and conjunctiva

Musclecells, endothelium, smooth muscle

Straight lamellae with periodicity of 5.5 nm

Neurons, smooth muscle, fibroblasts, Schwann cells, eccrine glands, histiocytes, hepatocytes

Muscle cells, fibroblasts, smooth muscle

Reticulofibrillarmaterial or lucent space with sparse granules

Cells htvolved in Muscle

cell type should be identified. I f the history is not fully consistent with the diagnosis, filrther investigations, such as examination of readily accessible (i,e., intestinal autonomic) neurons, should be considered. Lysosomal enzymes are synthesized by ribosomes attached to tile endoplasmic reticulum. ~ T h e y are secreted into the lumen a n d transferred to vesicles of the Golgi apparatus, where they are glycosylated. T h e y then bind specific m e m b r a n e receptors. Coated vesicles containing the receptors with b o u n d enzymes bud from the Golgi apparatus. These vesicles (prim a r y lysosomes) m a y t h e n fuse with o t h e r m e m brane-bound cytosomes to f o r m secondary lysosomes (defined as acid hydrolase-rich vacuoles that have acquired substrates by endocytosis or autophagia), t~ T h e pH level within the lysosome drops, causing the enzymes to dissociate from their receptors, so that the receptors with attached m e m b r a n e can be recycled to the Golgi a p p a r a t u s or endoplasmic reticu l u m . A p r o p o r t i o n o f the e n z y m e in the Golgi lumen does not immediately attach to receptors and is secreted to the extracellular space, where it binds to receptors on the cell surface. These complexes are internalized by pinocytosis, a n d tim resultant vesicle fuses with lysosomes. Binding of the lysosomal enzymes to their receptors is t h o u g h t to d e p e n d on a mannose-6-phosphate group attached to the enzymeg d u r i n g synthesis. 9 In m a n y cells, there is rapid communication o f components between the cell st, rface, secondary lysosomes, pinocytotic and phagocytic vesicles, and the complex that synthesizes lysosomal hydrolases and vacuolar membrane. 1~ In n o r m a l skeletal muscle cells, lysosomes, as such, appear to be almost absent, and acid phosplaatase activity c a n n o t be d e m o n s t r a t e d histochenai-

obvious e n o u g h to suggest the specific biochemical test indicated. This was true in a retarded 3-year-old patient in whom we tentatively diagnosed fi~cosidosis on the basis o f muscle biopsy. In Batten's disease (ceroid lipofuscinosis), in which the biochemical defect is still u n k n o w n , diagnosis is d e p e n d e n t on biopsy. T h e purpose o f this paper is to review the lysosomal storage diseases that can be detected by muscle biopsy, as well as the conditions most frequently confused with them. O u r discussion includes nonmuscle cells present in the biopsy specimen but excludes storage in peripheral nerves, although intramuscular nerves are seen in m a n y muscle biopsy specimens. Biopsy o f muscle is considerably easier and entails less risk than does biopsy o f brain or even of the gastrointestinal tract for autonomic neurons. Other peripheral tissues, such as skin 4-7 or conjunctiva, 6-s can also be u s e d f o r the d i a g n o s i s o f lysosomal storage diseases. T h e repertoire o f variously differentiated cell types is greater in skin than in skeletal muscle. Skin biopsy may thus be more useful in diagnosis than muscle biopsy, as, for instance, in some cases o f juvenile Batten's disease, in which involvem e n t o f skeletal muscle may be minimal in biopsy specimens, whereas storage is easily f o u n d in sweat gland secretory cells. O f great importance is familiarity with the type o f tissue that is being examined, as well as knowledge of the manifestations o f the disease in question. I f the diagnosis is at all dubious, we prefer to examine both muscle and skin. I f diagnosis is based on examination of these peripheral tissues, caution is salutory. T h e tthrastructt~re must be completely typical. T h e cell type involved must be identifiable. Preferably, the involvement o f more than one 685

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FIGURE t. Adult-onset acid maltase deficiency. Top left,, several muscle fibers contain multiple small vacuoles, most of which are optically empty, while some contain amorphous material. {Hematoxylin-eosin stain.] Top right, similar vacuolar spaces display acid phosphatase reactivity. [Acid phosphatase reaction.) Bottom right, a few muscle fibers contain discrete periodic acid-Schiff-positive collections of glycogen. [Periodic acid-Schiff stain.] (A//, x 350.}

686

LYSOSOMAL STORAGE IN SKELETALMUSCLE [Carpenter & Karpati]

FIGURE 2 (top]. Adult-onset acid maltase deficiency. Between the myofibrils are collections of glycogen, some surrounded by a membrane [white arrow] and others free in the intermyofibrillar space [black arrow]. An autophagic vacuole within the muscle fiber is surrounded by T-tubular profiles, and another large one on the lower right appears to have been exocytosed. [ x 16,750.] FIGURE 3 (bo#om]. Adult-onset acid maltase deficiency. The degrees of retention of glycogen vary within sections. One lysosome (arrow] is densely packed with glycogen; another contains small, pale particles. Between the myofibrils is abundant emply space from which glycogen has been leached. [ x 16,750.]

687

HUMAN PATHOLOGY 9

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differential diagnosis of lysosomal storage disease in muscle, therefore, particularly involves other conditions in which acid phosphatase-positive sites are numerous. T h e distinction necessitates electron microscopic examination as well as evaluation of pathologic and clinical features 9 The lysosomal diseases discussed in this paper are divided .into those invoh, ing skeletal muscle cells (table 1) and those involving ceils of other types, such as satellite cells, fibroblasts, and endothelium.

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Acid Maltase Deficiency Acid maltase deficiency has replaced glycogenosis II as the general term for the three age-related syndromes 13 resulting from deficiency of the lysosomal enzyme ~-l,4-glucosidase. Pompe's disease, the stereotyped infantile form, presents in the first months o f life with hypotonia, progressive muscle weakness, a n d e n l a r g e m e n t o f liver and heart. The patients usually die of heart failure in the first or earl)' in the second year of life. The time of onset is more variable in the childhood form, which presents as a myopathy with progressive weakness. T h e calves may a p p e a r h y p e r t r o p h i e d , and the muscles tend to feel firm. Hepatic and cardiac enlargement usually does not occur. T h e patients tend to die at 6 to 10 years of age, of respiratory complications. The adult form becomes symptomatic at any time from the second to the sixth decade of life, usually presenting as insidiously progressive proximal weakness. Respiratory muscles are sometimes disproportionately involved, leading to early respiratory failure. 14 Electromyographic studies usually show bizarre, lfigh-frequency discharges in all forms. Inheritance is autosomal recessive. One family with a mixture of forms has been reported. 15 In the infantile form, large amounts of glycogen are present in virtually all skeletal muscle fibers, to the point that the muscle fibers scarcely resemble muscle fibers. ~6-1s In cryostat or paraffin sections stained with hematoxylin-eosin, marked vacuolization is seen. Staining with periodic acid-Schiff dentonstrates some excessive glycogen accumulation. The acid phosphatase reaction shows muhiple foci of activity in all fibers. A curious finding is the presence of metachromatic Alcian blue-positive material, thought to contain phosphate groups. 19 This material is apparently removable by salivary amylase) s Electron microscopy shows massive anaounts of [3-glycogen particles, both free in the cytoplasm and in spaces surrounded by a single membrane. However, a considerable p r o p o r t i o n o f the glycogen may have leached out during processing, so that electron microscopic examination will reveal many empty vacuoles and electron-lucent spaces between myofibrils. The extent of this leaching varies front cell to cell. At autopsy, storage can be found in many organs, including heart, liver, kidney, brain, and spinal cord

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FIGURE 4. Late infantile Batten's disease, gastrocnemius muscle biopsy specimen. Almost every muscle fiber contains single or multiple acid phosphatase-reactive structures at the periphery and in the interior. (Acid phosphatase reaction, x 350.]

cally, ix This probably reflects the facts that pinocytosis is relatively minor and secretion is limited to components of the basal lamina in these ceils. Muscle cells have two extensive, specialized internal membrane systems, the T tubules and the sarcoplasmic reticulum. Small amounts of acid hydrolases have been postulated to reside in one or the other system. ~ The genesis of lysosomes in muscle cells is presumably similar to that in other cells, as summarized earlier, but the membrane that lines large lysosomes and autophagic vesicles appears to be partially derived from T tubules, since horseradish peroxidase, used as a marker for connection to the extracellular space, has been shown to appear in autophagic vacuoles and T tubules closely surrounding them. 12 A lysosome is a structure that has a surrounding membrane and contains one or more acid hydrolases; the other contents are variable, depending on the substrates that are present. Identification of lysosomal storage by histochemical methods depends largely on the demonstration of abnormal sites of acid phosphatase activity. Methods are not available for the microscopic cytochemical display of tile relevant enzymes affected in lysosomal storage disease. Tile lysosomal contents may or may not be dentonstrable by histochemical techniques, depending on the disease and the solubility of the substances. The 688

LYSOSOMALSTORAGEIN SKELETALMUSCLE(Carpenter & Karpati] |-

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FIGURE5. Juvenile Batten's disease, 14-year-old girl, gastrocnemius muscle biopsy specimen9 Top left, prominent clear spaces are present in several muscle fibers; some spaces contain amorphous eosinophilic or tiny punctate basophilic structures. [Hematoxylin-eosinstain.) Top right, many spaces in muscle fibers contain periodic acid-Schiff-positive [partiaTlydiastase-removable] material9(Periodic acid-Schiff stain9 Bo#om left, acid phosphatase positivity marks the sites not only of the large spaces, but also of many other small cytosomes in the majority of the muscle fibers9[Acid phosphatase reaction.] Bottom right, numerous autofluorescent granules correspond to the specific storage cytosomes. (A/l, x 350.) 689

HUMAN PATHOLOGY

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Volume 17, No. 7 (July 1986]

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FIGURE 6 (top left]. Granular osmiophilic deposit [GROD] of infantile Batten's disease in a muscle fiber from a 3-year-old girl in whom seizures began at the age of 15 months. (x 100,000.] FIGURE 7 (top right]. Cytosome with curvilinear profiles in the interior of a muscle fiber from a 4-year-old boy with late infantile Batten's disease. Fine lamination (arrows] and the apparent discontinuity of the lysosomal membrane are visible. [ x 67,500.] FIGURE 8 [bottom left]. Curvilinear body in satellite cell in late infantile Batten's disease. [ x 50,000.] MGURE 9 [boftom right]. The lamination of cutvilinear profites can be seen more clearly at high magnification. [ x 'i00,000.]

Affected fibers often show severe involvement, as in infantile cases (fig. I). T h e r e is a predilection of storage for the histochemical type I fibers. Involved fibers may be of normal size or atrophic, but tile), are never hypertrophic. Alcian blue-positive material is scanty in adult cases, but basophilic granules may be seen in some deposits. Electron microscopy, in addition to tile glycogen storage (figs. 2 and 3), often reveals p r o m i n e n t a t t t o p h a g i c vacuoles c o n t a i n i n g m e m b r a n o u s whorls and electron-dense nmterial. ~l Exocytosis of the material f r o m these vacuoles may be seen. Storage is not seen in other organs.

(especially anterior h o r n cells) as well as in autonomic neurons. Skin biopsy shows m e m b r a n e - b o u n d glycogen in n u m e r o u s cell types and is said to be diagnostic. 7 In the childhood form, involvement of muscle fibers is more patctty, and some muscles may show only minor involvement. Storage in skin is present, z~ In the adtflt form, some muscles appear either entirely normal or contain only a very few vacuolated fibers, although enzyme activity is apparently almost absent from all cells3 t Within a single muscle there may be marked regional variation in involvement. 2~ 690

LYSOSOMALSTORAGEIN SKELETALMUSCLE [Carpenter & Karpati]

FIGURE 10, Rectilinear profiles tend to be thinner, straighter, and more heterogeneous titan the curvilinear profiles, although they too are laminated (arrows]. Muscle specimen from an 18-year-old girl with juvenile Batten's disease in whom visual failure began at the age of 7 years. ( x 75,000.]

One of the patients had hepatomegaly. Muscle biopsy showed vacuoles in s c a t t e r e d muscle fibers that stained for glycogen and had acid phosphatase activity. Electron microscopy demonstrated free and lysosomal glycogen as well as autophagic vacuoles. The morphologic features appeared to be those of acid maltase deficiency, but acid maltase activity in the muscle was normal, as was activity in the urine of the patient in whom it was tested. There is at present no e x p l a n a t i o n o f this p h e n o m e n o n . P e r h a p s the findings in these cases should be c o m p a r e d with those in another case in which lysosomal glycogen storage was found in muscle, ahhough acid maltase activity was normal in muscle tissues, but deficient in leukocytes and fibroblasts in culture. 27

The occurrence of extralysosomal glycogen accumulation in muscle fibers has been a subject of speculation. The lysosomal membranes often appear thin and partially ruptured. T h e extralysosomal glycogen may result from the breakdown of lysosomal membranes, but it is not clear why it then could not be disposed of by normal glycolytic mechanisms, unless the surface of the glycogen particles has been altered in some way, preventing normal glycogenolysis. Glycol) tic enzymes, by assay, appear normal. 13 Biochemical confirmation o f the diagnosis is ahvays desirable. Acid maltase activity may be determined in muscle, leukocytes, c u h u r e d skin fibroblasts, or urine. 24 In the adult-onset form, some residual acid maltase activity can be detected with an artificial substrate in muscle and viscera, in contrast to the total absence in infantile casesfl 5 Paradoxically, however, the activity in muscle, which is clinically involved, is no lower than that in viscera, which are not. 23 In addition, muscle fibers that do not show morphologic abnormality have no more enzyme than those that do. 26

Batten-Kufs Disease (Neuronal Ceroid Lipofuscinosis] This is probably the most c o m m o n neuronal storage disease. It can be divided into a number of subtypes, according to the ultrastructure of the deposits and the age of the patient at onset. 2s The name Knfs' disease is reserved for the adult-onset form. A biochemical classification is not possible, since the enzymatic defect is not known, and even the nature of the storage deposits has not been fully determined. The material may be called lipopigment, as defined by its insolubility in lipid solvents, stainability with Sudan stains, and autofluorescence. Isolated storage

Lysosomal Glycogen Storage with Normal Acid Maltase Level Two cases have been reported of a syndrome resembling a protracted childhood variety of acid maltase deficiency, except that the patients were mentally retarded and cardiomegaly was present. 3 691

HUMAN PATHOLOGY

Volume 17, No, 7 [July 1986}

FIGURE 11 (top}, Collection of loose membranous cytoplasmic bodies from a 16-year-old boy who is blind and quadriparetic. The findings suggest a lysosomal sforage disease of undetermined type. [ x 25,000.} FIGURE 12 [bottom]. Endothelial cells from the same patient contain lameIlar bodies with a periodicity of 4.5 nm. [ x 1oo,o00.]

reference to age-matched control subjects, since urinary dolichol levels normally increase with age. T h e test is not diagnostic because o f a 5 to 10 per cent rate of false positivity. The main clinical features are seizures, myoclonus, psycllomotor regression, and progressive visual loss with pigmentary degeneration of

granules have been shown to contain large proportions o f polyisoprenols related to retinoic acid. 29 Considerable amounts o f the lipid-soluble compound dolichol are associated with them. s~ The level of this substance in the urine is used as a screening test for the disease, but abnormality must be established in 692

LYSOSOMALSTORAGEIN SKELETALMUSCLE(Carpenter & Karpati]

FIGURE 13 Cleft). Gastrocnemius muscle biopsy specimen from a 2-year-old girl with hypotonia, cardiac failure, and seizures. Three muscle fibers show prominent acid phosphatase positivity. The fiber in the lower right corner has been almost completely replaced by lysosomes. [Acid phosphatase reaction. x 520.] FIGURE 14 [right]. Loose membranous cytoplasmic bodies in the muscle fiber from the same patient, who had storage disease of undetermined type. [ x 100,000.]

the retina. Patients with the adult-onset form almost always lack visual involvement, so that their clinical recognition is difficult. Storage in skeletal muscle occurs in all varieties of Batten-Kufs disease, 4,31 but occasionally in the juvenile-onset form and usually in the adtdt-onset form, the stored substances may be so limited as to be unavailable for electron microscopy. In the infantile, late infantile, and usual juvenile forms, the acid phosphatase reaction is clearly abnormal. Multiple sites appear beneath the sarcolemma and, to a lesser extent, in the interior of almost every muscle fiber (figs. 4 and 5). The deposits themselves can be visualized by their silvery yellow atltofluorescence. Particularly in the infantile and late infantile forms, acid phosphatase positivity may be seen in vascular endothelium and smooth muscle. Electron microscopy is mandatory for diagnosis. T h e ultrastructural appearances o f storage cytosomes fall into certain categories, which correlate roughly with the age of the patient at onset. Thus, in patients with the infantile variety (Santavori's disease), the storage cytosomes contain predominantly uniform, finely granular electron-dense material 32 (fig. 6). T h e granules are finer and more uniform than those of normal lipofuscin, and lipid vacuoles are usually not present. As a brief code word to signal this type of deposit, we have used the abbreviation GROD for grantdar osmiophilic deposits. 33 In rare patients in whom the onset is later (at 6 to 10 )'ears of age), electron microscop)' shows GROD rather than

the other ultrastructural patterns expected for that age group. 33 In late infantile cases, with the onset at the age of 2 to 31/2 ),ears, cytosomes have curvilinear profiles or, more briefly, curvilinear bodies (figs. 7 to 9). These profiles are made up of stacks of lamellae, usually curving, formed of alternating dark (3.5- to 4-nm) and pale (4- to 4.5-nm) lines. 28 The clarity of the lamellation can vary considerably, and, particularly in skeletal muscle cells, it may be difficult to see in many cytosomes. Demonstration of the lamellae is nevertheless important for identification of the storage. Tile stacks of lamellae tend to be thinner in skeletal muscle cells than in cells of other types. T h e trajectory of the lysosomal membrane is irregular, so that it is often cut tangentially in places and therefore not uniformly visible. In patients with later onset of the disease (generally at 3V2 to 10 years of age), most cells show the uhrastructural features tlmt have been called fingerprint profiles. 28 These profiles are formed of arrays of paired parallel lines, with each line separated from its mate by a 1.5-rim lucent space and each pair separated from neighboring pairs by a 2.5-nm space. The lines are about 2.3-nm wide. The arrays appear to form plates, which may be superimposed to yield a lattice. These fingerprint profiles do not, however, occur in the skeletal muscle cells; they are found in neurons, smooth muscle, Schwann cells, endothelitnn, and muscle satellite cells. The skeletal muscle cells contain cytosomes that resemble poorly formed 693

HUMAN PATHOLOGY

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Volume 17, No. 7 (July 1986]

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FIGURE 15 (fop left). Fucosidosis, 4-year-old girl gastrocnemius muscle biopsy specimen. Prominent acid phosphatase-positive sites are present in the endothelium of larger septal blood vessels and in endomysial capillaries. The muscle fibers show no involvement. (Acid phosphatase reaction, x 140.] FIGURE 16 [top right). Fucosidosis.Clear vacuoles are evident in the endothelial cells of several capillaries in one fascicle. The atrophy of muscle fibers probably reflects anterior horn ceil invoIvement. [Semithin resin section, paraphenylenediamine,phase optics, x 550.] FIGURE 17 [bottom right). Infantile Sandhoff's disease. The endothelium of an intramuscular arteriole contains numerous dense granules, and smaller granules are present in some smooth muscle cells. [Semithin resin section, paraphenylenediamine, phase optics, x 550.]

694

LYSOSOMALSTORAGEIN SKELETALMUSCLE(Carpenter & Karpati)

curvilinear bodies (fig. 10). We introduced tile term rectilinear bodies to indicate this minor distinction. 4 The rectilinear bodies contain tlfin stacks of lamellae, most often in pairs, producing sequences o f three d e n s e lines and two pale spaces. T h e y may be straight, or they may curve irregularly. Since the metabolic error in Batten's disease is not known, biopsy is still necessary for definite diagnosis. T h e heavy involvement of skeletal muscle in most. cases makes it suitable for biopsy. T h e acid phosphatase reaction and fluorescence microscopy serve as excellent screening methods, ahhough electron microscopy is needed to confirm the diagnosis. Skeletal muscle cells are involved less consistently than eccrine secretory cells in the juvenile form, so that skin biopsy may be more useful in such cases. The deposits in eccrine secretory cells and in smooth muscle cells generally have enough size and contrast to be visible in senfithin resin sections by phase microscopy, unlike tim deposits in skeletal muscle cells. They can be distinguished from lipofuscin granules by their relative lack of birefringence. On the other hand, fingerprint profiles are occasionally seen in small numbers in eccrine secretory cells without specificity. It is easier to be confident about the diagnosis if cells of more than one type are im'olved. T h e specificity o f curvilinear or rectilinear bodies in skeletal muscle cells for Batten's disease is high, but these bodies may be found in one other setting--chloroquine treatment. Numerous typical curvilinear bodies were found in muscle cells in four successive biopsy specimens front a woman who had received chloroquine nine years earlier, s4

etal muscle cells. Electron microscopy discloses membrane-bound lamellar bodies, somewhat larger than mitochondria. The lamellae generally have a straight course and a periodicity of 5.5 to 6.3 nm. Mannosidosis

This disease is caused by deficiency of cx-mannosidase and the consequent intralysosomal accumulation of mannose-containing oligosaccharides in many tissues. 39 Dysmorphic features, mental retardation, lens opacities, bearing loss, a tendency for recurrent infections, gum hypertrophy, mild hepatosplenomegaly, and muscular hypotonia are the chief clinical signs. 4~ T h e severity of involvement varies widely, and most patients reach a stage at which the disease does not progress. Diagnosis can be based on the demonstration of the enzyme deficiency in leukocytes or cultured fibroblasts. At autopsy, storage is found in fibroblasts, macrophages, neurons, hepatocytes, and smooth muscle. The results of two muscle biopsies in one 32-year old patient were reported. 41 Skeletal muscle cells contained numerous small membrane-bound inclusions, 0.5 to 5 ~m in diameter, with scattered dark granules as well as an abundance of electron-lucent space present within the inclusions. Similar inclusions were observed in interstitial fibroblasts and occasional endothelial cells. T h e biopsy findings did not seem to be sufficient to explain the patient's mild proximal weakness. Mucolipidosis IV

The enzyme defect in this disease has not been determined. Signs begin in infancy, with motor retardation, spasticity, extrapyramidal signs, and corneal clouding. In cryostat sections, inclusions are visible in almost all muscle fibers. 42 They are red with the modified trichrome stain, and they are PAS-positive. By electron microscopy, they are dense lamellar bodies that rather resemble those of Fabry's disease. In skin, dense granules are visible by phase microscopy in eccrine myoepithelial cells and duct cells, endothelium, Schwann cells, and perineurium, while the eccrine secretory cells are vacuolated.

Fabry's Disease

Deficiency of ~-galactosidase and storage of ceramide trihexoside show sex-linked recessive inheritance. 35 Two main clinical types have been identified. Type I is characterized by skin lesions (angiokeratomata), with corneal opacities, renal and cardiovascular involvement, and episodes o f spontaneous pain. Storage is found in skeletal muscle cells, endothelium, pericytes, smooth muscle, glomeruli, dorsal root ganglia, myocardium, perineurium, some fibroblasts, and regions of tile central nervous system associated with autonomic function. In type II skin lesions are absent, and renal failure appearing in the second or third decade o f life is the dominant clinical feature. Skeletal muscle is not involved, and skin biopsy specimens may be normal or show some storage in vessels and arrector pili muscles. Manifest carriers may occur in this sex-linked disease, with varying degrees of renal insufficiency, angiokeratomata, spontaneous pain, and corneal opacities. In type I Fabry's disease the skeletal muscle cells appear normal in cryostat sections, except for scattered acid phosphatase-positive sites. 36-~s Granular material can be seen in sections of the vessel wall, particularly in smooth muscle cells, on staining with PAS and lipid stains. In semithin resin sections, subsarcolemmal dark granules may be visible in tile skel-

Unidentified Lysosomal Disease

We have seen three patients in whom muscle biopsy specimens contained inclusions suggestive of lysosomal storage, but who did not fit into any established category. T w o brothers, in whom biopsies were performed at the ages o f 15 and 16 years, had spastic quadriplegia, severe retardation, and blindness starting in the first few months of life. In the g a s t r o c n e m i u s 5 to 10 p e r cent o f tile fibers in cryostat sections stained with h e m a t o x y l i n - e o s i n contained vacuoles filled with basophilic and eosinophilic granules. Some of the vacuoles had PAS-positire contents. They gave a strong reaction for acid phosphatase. Electron microscopy revealed aggregates o f 695

HUMAN PATHOLOGY

Volume '17,No. 7 (July '1986]

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FIGURE 18. Top left,,specimen from a 42-year-old patient with unidentified progressive dystonia. In the muscle fibers that appear normal, the subsarcolemmal acid phosphatase-positive areas correspond to lipofuscin. Top right adult-onset centronuclear myopathy. Prominent central acid phosphatase-reactive regions in several muscle fibers mark the site of copious lipofuscin associated with the central nuclei. Bottom left, amyloid neuropathy with severe denervation. Both atrophic and nonatrophic muscle fibers contain abundant acid phosphatase-reactive Iipofuscin. Bottom right muscle specimen from a 35-year-old patient with progressive spinocerebellar syndrome and intestinal malabsorption, with ceroid accumulation in skeletal muscle. Numerous small acid phosphatase-positive sites are present in the interior of several muscle fibers as well as in larger subsarcolemmal fibers. [A//,acid phosphatase reactions, x 350.]

m e m b r a n o u s cytoplasmic bodies f o r m e d o f lamellae with a periodicity o f 4.5 n m in a few muscle cells (fig. 11). Inclusions with a similar periodicity were present in e n d 0 t h e l i u m o f venules in the e p i n e u r i u n l o f tlle

sural nerve (fig. 12). Autopsy o f one o f these patients a few m o n t h s later revealed a small brain. Scattered n e u r o n s in the cortex showed lipid storage. T h e r e was unusual massive.thickening o f the basal lamina

696

LYSOSOMALSTORAGEIN SKELETALMUSCLE(Carpenter & Karpati]

of subarachnoid veins with PAS-positive material that was granular on electron microscopic examination. Information on the presence or absence of visceral storage is lacking. In a third unrelated patient mild retardation and hypotonia were seen during the first year of life. In the second year a severe seizure disorder developed, along with cardiac failure. In muscle biopsy specimens obtained at the ages of 1 and 2 years, occasional fibers in cryostat sections were riddled with acid phosphatase-positive sites (fig. 13). Electron microscopy showed membranous cytoplasmic bodies (fig. 14). Skin biopsy specimens were normal. Results of screening tests for a battery of lysosomal enzymes were normal. STORAGE PRIMARILY IN CELLS OTHER THAN MATURE SKELETALMUSCLECELLS For several lysosomal storage diseases no data related to the pathologic changes in muscle are available. These diseases include Niemann-Pick disease types A and B, Gaucher's disease, Farber's disease, Wolman's disease, cerebrotendinous xanthomatosis, aspartyl-glucosaminuria, Salla disease, and mucopolysaccharidoses III to VIII. Those diseases with involvement of fibroblasts and endothelium might be expected to be demonstrable by muscle biopsy. Our experience indicates that muscle biopsy is negative in Niemann-Pick disease type C, and in GM 2 gangliosidosis (outside of Schwann cells). In the following sections we discuss diseases in which storage in cells other than mature muscle cells was found by us or reported by others. I-cell Disease

I-cell disease is unusual in that it involves a defect in the posttranslational processing of lysosomal enzymes, so that they tend to be secreted rather than retained by lysosomes.43 Serum levels of lysosomal enzymes are high, and levels o f several enzymes within cultured fibroblasts are low. T h e mature muscle fibers in a biopsy specimen from one patient were morphologically normal, but laminated inclusions were present in satellite cells. 44 Myoblasts in culture were crowded with inclusions, but myotubes in which cross-striations had developed had become inclusion-free. Levels of lysosomal enzymes in cultured myoblasts were low, whereas in the biopsied muscle, except for that of [3-galactosidase, they were normal or increased. This case raises the question as to w h e t h e r the processing o f lysosomal enzymes might differ in immature and mature muscle cells. Fucosidosis

In this disease the muscle cells themseh'es are not involved, but storage is sufficiently prominent in endothelial cells that the diagnosis may be based on muscle biopsy. Levels of o~-L-filcosidase are abnormally low, so that fucose-containing oligosaccharides, 697

glycopeptides, mucopolysaccharides, and glycolipids accumulate. 45 Oligosaccharide screening of the urine is helpful in making the diagnosis. T h e enzyme defect is best demonstrated biochemically in lymphocytes. Two clinical types have been described. One t.ype is characterized by severe psychomotor regression, growth retardation, and susceptibility to infections, beginning in the first )'ear of life, with death by the age of 6 years. Storage is present in endothelium, neurons, histiocytes, exocrine glands, hepatocytes, fibroblasts, and adipocytes. 46 Patients with the other form have angiokeratomas and milder neurologic signs, and they survive into the third decade of life. In the severe type of fucosidosis, the examination o f cryostat sections o f muscle shows copious acid p.hosphatase-positive sites in the wall of vessels of all slzes (fig. 15) and in the nerve twigs. Rare muscle fibers contain acid phosphatase-positive sites, but their nature has not been verified ultrastructurally. In semithin resin sections prominent clear vacuoles in the endothelial cells in capillaries, arteries, and veins are seen (fig. 16). Occasional septal fibroblasts contain similar vacuoles. By electron microscopy the largely clear vacuoles may contain small lamellar vesicular structures and pale linear densities. Sandhoff's Disease

This disease is also characterized by fairly prominent involvement of intramuscular vessels, but the storage is more abundant in larger vessels, involving both endothelium and smooth muscle. In resin sections dark granules are visible in the walls of veins and arteries (fig. 17). Electron microscopy shows membranous cytoplasmic bodies and zebra bodies with a periodicity of 5 to 6 nm. The light microscopic appearances in cryostat and resin sections are somewhat similar to those seen in Fabry's disease, but in our experience there is no involvement of skeletal muscle cells, and the clinical features are quite different. Sandhoff's disease is caused by lack of both the A and the B varieties of [3-N-acetyl hexosaminidase. 47 Symptoms of mental and motor regression appear in the first year of life. A cherry red spot is present in the fovea, and there is hepatosplenomegaly. GM 2 ganglioside is stored in neurons and globoside in other cells. Diagnosis can be made from serum or leukocyte hexosaminidase determination. In late-onset Sandhoff's disease, in which the principal clinical features are tremor and progressive motor neuropathy, we have not detected abnormalities in skin or muscle biopsy specimens. GM 1 Gangliosidosis

Deficiency of ganglioside-beta-galactosidase resuits in the storage not only of GM l ganglioside in neurons but also of glycopeptides and mucopolysaccharides in viscera. 47 T h e deficiency of [3-galactosidase can be demonstrated in leukocytes. In a patient with GM l gangliosidosis type I (onset before the age of 6 months, skeletal changes, hepatosplenomegaly,

HUMAN PATHOLOGY

Volume 17, No. 7 [July 1986]

FIGURE t9 [top]. Typical lipofuscin body in adult muscle, characterized by coarse granules, amorphous dense material, and a lipid vacuole. [ x'10(3,000.} FIGURE 20 {bo#om]. Ceroid bodies in a patient with malabsorption have a finely granular composition by electron microscopy. [ x 100,000.]

and psychomotor deterioration), clear storage vacuoles were f o u n d in a muscle biopsy specimen in mnscle satellite cells and endotheliuna,as

Mucopolysaccharidoses

T h e genetic m u c o p o l y s a c c h a r i d o s e s involve storage o f molecules related to the glycosaminoglycans of ground substance. The storage thus tends to be manifested in fibroblasts in all these diseases, and in other cells in specific types, such as Hurler's or Sanfilippo's disease. Little has been reported in relation to skeletal muscle. We have studied biopsy specimens from one patient with typical Hurler's disease and f r o m one patient with a late-onset f o r m o f Hunter's disease, which presented as a syndrome of

Sialidosis

In one patient with infantile sialidosis type 2, with deficiencies o f ~x-neuraminidase and [3-galactosidase, 49 we found prominent interstitial vacuolated fibroblasts in skeletal muscle. Similar cells were seen in skin, and vacuolation of the eccrine secretory cells was seen. 698

LYSOSOMALSTORAGEIN SKELETALMUSCLE [Carpenter & Karpati]

I

m

FIGURE2t (left]. Muscle specimen from a l-year-old child with infantile myotonic dystrophy. Many of the muscle fibers contain prominent subsarcolemmal acid phosphatase-positive sites. [ x 350.] FIGURE 22 [right]. Several large macrophages within a necrotic muscle fiber in a biopsy specimen from a patient with polymyositis. (Acid phosphatase reaction, x 520.]

multiple e n t r a p m e n t n e u r o p a t h i e s . 5~ Specimens from both patients contained acid phosphatase-positive fibroblasts, particularly a r o u n d vessels in the septa but also in the endomysium. Staining of the contents was not seen. In semithin and uhrathin sections vacuolated fibroblasts could be seen. In the patient with Hurler's disease, some of the vacuoles contained electron-dense granules as well as lucent space.

scin can probably be caused by a number of factors, one of which is denervation. There is no evidence that lipofuscin in itself is harmfifl to cells. Lipofuscin granules are surrounded by an irregular membrane and show acid phosphatase activity (fig. 18), but their chemical nature is not known. One component seems to be dolichol. 51 Another component may be debris of membranes that have undergone peroxidation. 52 Lipofuscin is autofluorescent, insoluble i n lipid solvents, and stainable with lipid stains. In these respects it resembles the storage of Batten's disease, although the autofhmrescence is m o r e golden yellow. In semithin resin sections lipofuscin is highly refractile. Electron microscopy shows it to be f o r m e d o f coarse, electron-dense, rather heterogeneous granules, associated sometimes with electron-dense amorphous material and very often with globules that have the density of neutral lipid (fig. 19). D i f f e r e n t i a t i o n f r o m t h e deposits seen in Batten's disease is not difficult. T h e only problem that might arise is in the identification of deposits that have the form o f GROD. T h e nniform, fine granules in these deposits are quite different from the coarse heterogeneous granules of lipidfllscin.

DIFFERENTIAL DIAGNOSIS OF LYSOSOMALSTORAGE

T h e differential diagnosis o f lysosomal storage diseases in skeletal muscle biopsy specimens includes particularly the conditions in which acid phosphatase-positive sites are abundant. The most important examples are discussed in the following paragraphs. Lipofuscin

.Some degree of lipofuscin accumulation is norreally present in almost all muscles after early childhood, and there is a gradual increase with age. Lipofuscin is virtually absent from biopsy specimens from patients with Duchenne dystrophy, probably because repeated necrosis and r e g e n e r a t i o n preclude the cells' surviving for long enough to accumulate significant amounts. An increase in the amount of lipofl~-

M a l a b s o r p t i o n States

Accumulation o f an a u t o f l u o r e s c e n t lipopigment occurs in malabsorption states, probably in relation to vitamin E deficiency5a (fig. 18, bottom right). 699

HUMAN PATHOLOGY

Volume 17, No. 7 [July 1986]

number. As the child grows, the muscle strength tends to increase, and the number of acid phosphatase-positive sites diminishes, until, by the age of 6 years, they are no longer apparent. Subsequently, signs and symptoms of adtflt myotonic dystrophy appear. Acid phosphatase-positive sites are manifested in the adtflt disease, but they are seen only in scattered muscle fibers, particularly in association with sarcoplasmic masses. M o t o r E n d - p l a t e s In C h i l d r e n

!

Acid phosphatase actMty is seen in motor endplates in children but not in adults. The end-plates can be recognized in cryostat sections by their elongated configuration at the surface of muscle fibers and by their proximity to small nerve twigs.

9

Chloroquine Myopathy r

Chronic treatment with chloroquine in some patients has led to the development of a gradually progressive proximal painless myopathy. The reported dose has usually been 250 to 500 rag/day, s5 Chloroquine interferes with the degradation of phospholipids. 56 It has been shown in vitro to diffuse readily into lysosomes, where it raises the pH, thereby inhibiting the release of newly synthesized enzymes from their receptors in the membrane. 9 As a result, re-

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FIGURE 23. Top, lepromatous leprosy, gastrocnemius muscle biopsy specimen. Huge vacuolated macrophages surround a septal blood vessel in the vicinity of atrophic and hypertrophic muscle fibers. [Hematoxylin-eosin stain.] Bottom, lepromatous leprosy. A large collection of giant macrophages, visualized by their acid phosphatase reactivity, is present in an interfascicular septum. [Acid phosphatase reaction.] (Bofh, x 350.]

Unlike lipofuscin, the deposits are not refractile in semithin sections. Electron microscopy shows uniform, fine granularity (fig. 20), closely resembling the pattern of G R O D in the infantile variety o f Batten's disease. Increased amounts of lipopigment may be present in neurons as well. Infantile M y o t o n i c D y s t r o p h y

Infantile myotonic dystrophy may occur when an infant carrying the gene for myotonic dystrophy is born to a mother who has this dominantly inherited disease. T h e infants are hypotonic. They do not have evidence of myotonia, even by electromyography, in the first two years o f life. Biopsy in the first few months shows a tendency for delayed growth of the muscle fibers. Mtdtiple acid phosphatase-positive sites are scattered in skeletal muscle cells 54 (fig. 21). The lysosomes are not visible in semithin sections. By electron microscopy they apparently consist of small FIGURE 24. Adult dermatomyositis. Prominent clear spaces [some groups of irregular tubules with dense contents. Atof which contain myonuclei] represent focal loss of myofibrils, not rophy of the histochenfical type I fibers is shown by storage. The clear space marked by the arrow probably has arisen from t-tubular dilation. [Hematoxylin-eosin stain, x 350.] the ATPase reaction. Central nuclei are increased in 700

LYSOSOMALSTORAGEIN SKELETALMUSCLE(Carpenter & Karpati)

ceptors become unavailable for tim further binding of enzymes, which are then diverted to the extracellular medium. Recycling of lysosomal membranes may also be impaired. In chloroquine myopathy, cryostat sections show a combination of vacuoles and basophilic granules in muscle fibers9 Acid phosphatase-positive sites are numerous. 12 Electron microscopy usually shows a variety of inclusions, ranging from membranous whorls to la.mellar bodies, dense material resembling one component of lipofuscin, and, finally, to curvilinear profiles resembling those of late infantile Batten's disease. 57 Endothelial cells may contain membranous bodies and membrane-bound glycogen. The case of one patient in whom four muscle biopsies were performed nine years subsequent to a four-year course of chloroquine treatment was reportedP 4 All biopsy specimens showed nmhiple acid phosphatase-positive sites in muscle fibers and, by electron microscopy, typical curvilinear bodies. No other type of lysosomal inclusion was found. It appeared that the curvilinear bodies had been generated by the patient's exposure to chloroquine, and that in the subsequent years they 9 had proved resistant to degradation, even though the use of chloroquine had been discontinued nine years earlier. They appeared to cause no deleterious effect on muscle fnnction. The development of chloroquine myopathy has been investigated in rats. t2,56 T h e first change appears to be the encirclement of small areas of cytoplasm, generally containing an organelle, by narrow m e m b r a n e - b o u n d spaces in continuity with T tubules. T h e organelles in the encircled cytoplasm break down, and membranous whorls or myeloid bodies appear. T h e inner membrane of the encircling tubular cisterns tends to disappear. Simple vacuoles coalesce to form large vacuoles with scalloped edg.es and masses of membranous debris, and exocytos~s of vacuoles and membranous whorls occurs.

whorls, glycogen, and abnormal 180-nm filaments. Some of the rimmed vacuoles have acid phosphatase activity, but the majority do not. Inflammation is often scanty or absent. In biopsy specimens from patients with denerrating disease, target fibers are often encountered. The target is essentially a zone inside the muscle fiber in which mitochondria are lacking and the myofibrils show loss of thick filaments and streaming of rite Z discs. In some biopsy specimens the center of the target may have been converted to an autophagic vacuole that has acid phosphatase actMty. A type o f distal m y o p a t h y characterized by prominent vacuolar change in muscle fibers has been described. 59 Electron microscopy disclosed memb r a n o u s whorls and m e m b r a n e - b o u n d vacuoles varying in size and contents. Data as to acid phosphatase positivity are not available. Some of the large vacuoles communicated with T tubules and had undergone exocytosis. Some cases were sporadic, and some were familial, probably with autosomal dominant inheritance 9 In an)' condition in which necrosis o f muscle fibers occurs, p h a g o c y t e s with c o n s p i c u o u s acid phosphatase activity may be expected (fig. 22). The phagocytes may be situated entirely within the confines of a necrotic fiber, so that on an acid phosphatase preparation there might be a question as to whether the acid phosphatase activity was generated by the muscle fiber itself. Collections of large vacuolated interstitial histiocytes with prominent acid phosphatase actMty may be seen in lepromatous leprosy (fig. 23). The condition might well be mistaken for a storage disease if a history were not available and if bacilli were not demonstrable within the vacuoles. Other Conditions Characterized by Vacuoles in Muscle Fibers that Might Be Mistaken For Lysosomal Disease As should by now be evident, acid phosphatase histochemical study is o f prime importance as a screening m e t h o d in the diagnosis o f lysosomal storage in skeletal muscle. In several conditions vacuoles with a variety of contents can be seen in skeletal muscle f b e r s with general stains, such as hematoxylin-eosin or the modified trichrome stain (fig. 24). A question of storage might arise in cases in which adequate acid phosphatase preparations are not available. These conditions are listed in table 2. In addition, tile presence of glycogen or glycogen-like particles in a membrane-bound space on electron microscopic examination is not conclusive evidence of lysosomal glycogen. In Lafora's disease, for example, the membrane-bound spaces in which glycogen particles and other forms of polysacctmride accunmlate in skeletal muscle cells do not have acid phosphatase activity, but are positive for peroxisomal enzymes. 6~ Diagnosis of this disease, which can be considered a nonlysosomal storage disease, is made more confidently on tile basis of the demonstration of PAS-posi" 61 tire bodies in the sweat gland duct cells m skin.

Other Conditions Involving Acid Phosphatase-positive Sites in Skeletal Muscle Cells or Interstitial Cells In many cases of adult polymyositis (as distinct from dermatomyositis), autophagic vacuoles develop within skeletal muscle cells. 58 Relatively few fibers are usually involved, but on cross section these fibers may show multiple acid phosphatase-positive sites9 By electron microscopy the contents are somewhat miscellaneous, with some degenerating organelles, such as mitochondria, amorphous dense material, and membrane-bound glycogen with a semicrystalline organization. Exocytosis of this material may be found. T h e correct diagnosis depends on appreciation of the inflammatory infiltrates, the partial invasion o f nonnecrotic muscle fibers by endomysial m o n o n u c l e a r i n f l a m m a t o r y cells, and the basal lamina abnormalities commonly seen in polymyositis. In inclusion body myositis, rinnned vacuoles are characteristically encountered in scattered muscle fibers. 58 Electron microscopy reveals membranous

9

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HUMANPATHOLOGY Volume1L No, 7 (July1986] TABLE2. Typesof Nonlysosomal Cytoplasmic Vacuolar Spaces that May Suggest Storage with General TissueStains Nature of Space Cytoplasnfic space (lined vacuole) Cytoplasmic space Cytoplasmic space Lipid globule Peroxisome Various membrane-bound spaces Sarcoplasmic reticnhnn Cytoplasmic space resnhing from myofibrillar loss T tubules

Disease Association

Contents

Inclusion body myositis Nonlysosomal glycogen storage diseases Adult dominant centronuclear myopathy Incidental lipid excess and specific lipid storage myopathies Lafora's disease Periodic paralysis Sarcotubular myopathy Dermatomyositis

Membranous whorls, abnormal filaments Glycogen

Miscellaneous, including surviving StUlnpsof fibers with recent segmenial necrosis

Lucent space

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Nuclei, lipofuscin Lipid Polyglucosons Usually lucent space Lucent space Glycogen, mitochondria

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