Macrophagic myofasciitis lesions assess long-term persistence of vaccine-derived aluminium hydroxide in muscle.

Macrophagic myofasciitis (MMF) is an emerging condition of unknown cause, detected in patients with diffuse arthromyalgias and fatigue, and characterized by muscle infiltration by granular periodic acid-Schiff's reagent-positive macrophages and lymphocytes. Intracytoplasmic inclusions have been observed in macrophages of some patients. To assess their significance, electron microscopy was performed in 40 consecutive cases and chemical analysis was done by microanalysis and atomic absorption spectrometry. Inclusions were constantly detected and corresponded to aluminium hydroxide, an immunostimulatory compound frequently used as a vaccine adjuvant. A lymphocytic component was constantly observed in MMF lesions. Serological tests were compatible with exposure to aluminium hydroxide-containing vaccines. History analysis revealed that 50 out of 50 patients had received vaccines against hepatitis B virus (86%), hepatitis A virus (19%) or tetanus toxoid (58%), 3-96 months (median 36 months) before biopsy. Diffuse myalgias were more frequent in patients with than without an MMF lesion at deltoid muscle biopsy (P < 0.0001). Myalgia onset was subsequent to the vaccination (median 11 months) in 94% of patients. MMF lesion was experimentally reproduced in rats. We conclude that the MMF lesion is secondary to intramuscular injection of aluminium hydroxide-containing vaccines, shows both long-term persistence of aluminium hydroxide and an ongoing local immune reaction, and is detected in patients with systemic symptoms which appeared subsequently to vaccination.

Differential microglial response to progressive neurodegeneration in the murine mutant Wobbler.

Activated microglia is associated with neurodegenerative processes, but the precise role of this cell population is difficult to identify. Most experimental models employed to examine microglial responses involve acute alterations of neuronal integrity, in contrast to the progressive nature of neurodegenerative diseases. In order to approach the clinical situation better, the microglial response was analyzed in the murine mutant Wobbler, which exhibits a well-characterized neurodegenerative pathology, manifested by motoneuronal death following a period of cellular dysfunction with characteristic morphological features. Microglial cells were identified using anti-Mac1 or anti-IgG antibodies. Examination of the changes in density, localization, and phenotype of microglia differentiated two types of responses in Wobblers. A first type of response was observed as early as in the third week after birth, when the only apparent neuronal defect was the morphological alteration of a subset of motoneurons in the cervical spinal cord, which was maintained later on. The activated microglia extended long processes that selectively ensheathed vacuolated motoneurons. At later stages, when motoneuron death became prominent, an additional type of response was characterized by an increased density of reactive microglia that was seen extending throughout the cervical enlargement. This secondary microglial response occurred in parallel to the infiltration of T-lymphocytes. Thus, these results point to a differential response of microglial cells to a progressive neurodegenerative process.

Peripheral injections of Freund's adjuvant in mice provoke leakage of serum proteins through the blood-brain barrier without inducing reactive gliosis.

Breakdown of the blood-brain barrier (BBB) and ensuing gliosis are common events following physical trauma to the central nervous system (CNS) or during autoimmune diseases such as experimental allergic encephalomyelitis (EAE). Some studies of EAE in rodents report that peripheral injections of complete Freund's adjuvant (CFA), which contains heat-inactivated Mycobacterium to provoke peripheral inflammation without adversely affecting the CNS, can itself lead to increased BBB permeability to small tracer molecules and certain serum proteins. To study the equivocal relationship between serum protein extravasation and reactive gliosis, we injected C57BL/6 mice with CFA and histologically assessed the permeability of various serum proteins and the reactivity of proximal microglia and astrocytes in the uninjured brainstem and spinal cord enlargements after 1-4 weeks. Our results confirm that CFA injections induce progressive increases in the perivascular extravasation of serum IgG, albumin, IgM, and exogenous horseradish peroxidase, all to varying degrees, most prominently in the brainstem and cervical spinal cord after 2-3 weeks. More importantly, neither microglial cells nor astrocytes in regions of focal serum protein leakage appeared morphologically reactive based on immunoreactivity for CR3 receptors (Mac-1) or glial fibrillary acidic protein (GFAP), respectively. Because we found no evidence of T cell infiltration accompanying the exudates, our results indicate that in the absence of physical trauma or inflammatory cells resident CNS neuroglia remain quiescent upon exposure to extravasated serum proteins.

Regulation of growth factor gene expression in degenerating motoneurons of the murine mutant wobbler: a cellular patch-sampling/RT-PCR study.

Motoneuronal degenerative diseases are characterized by their progressivity; once affected, the motoneurons remain in altered states during an intermediate phase of degeneration prior to their final disappearance. Whether this survival period coincides with active metabolic rearrangements in the affected neuron remains unknown. As a first step toward the elucidation of this question, we developed cDNA pooled samples obtained from degenerating and control motoneuron mRNA populations through cellular patch sampling and RT-PCR, using the murine wobbler mutant as a model of spinal atrophy. Hybridization of the cDNA pools to various markers of intact or degenerating motoneurons allowed us to verify the cellular specificity of the patch sampling and indicated conservation of the original mRNA population complexity. Exploration of transcriptional alterations of genes encoding growth factors thought to be involved in motoneuronal development revealed that gene expression of the neurotrophin BDNF was induced in affected motoneurons, while expression of neurotrophin-3 was present in both neuronal types. Likewise, expression of a member of the epidermal growth factor (EGF) family, the neuregulin transcript sensory motor neuron-derived factor, was detected in both control and degenerating motoneurons, while transforming growth factor alpha, the functional homolog of EGF, was present only in the affected motoneurons. Immunohistochemical detection of corresponding proteins corroborated these observations. These results demonstrate that, during the course of their degeneration, motoneurons can initiate expression of novel genes which lead to the production of molecules endowed with trophic and/or differentiative properties for the neurons themselves and their glial environment. They also validate the use of the developed cDNA pooled samples for further exploration of transcriptional alterations taking place in degenerating motoneurons.

Phenotypic alteration of astrocytes induced by ciliary neurotrophic factor in the intact adult brain, As revealed by adenovirus-mediated gene transfer.

Synthesis of the ciliary neurotrophic factor (CNTF) and its specific receptor (CNTFRalpha) is widespread in the intact CNS, but potential biological roles for this system remain elusive. Contradictory results have been obtained concerning a possible effect on the morphological and biochemical phenotype of astrocytes. To reassess this question, we have taken advantage of adenovirus-mediated gene transfer into the rat brain to obtain the local release of CNTF. Stereotaxic administration of CNTF recombinant adenovirus vectors into the striatum led to phenotypic changes in astrocytes located in regions that were related axonally to striatal neurons at the injection site. Astrocytes appeared hypertrophied and displayed an increase in both GFAP and CNTF immunoreactivity. This response was observed up to 5 weeks after injection, the longest time studied. It was not observed after the administration of a control vector. The methodology used in the present study, allowing us to analyze the effect of the factor in areas remote from the injection site, has provided conclusive evidence that CNTF affects the astroglial phenotype in the intact CNS. The characteristics of these effects may explain why contradictory results have been obtained previously, because this signaling system seems to have a low efficiency and therefore requires a high local concentration of the factor close to the target cells. One might speculate as to the involvement of a CNTF astroglio-astroglial signaling system in the organized response of a population of astrocytes to changes in CNS homeostasis detected locally, even by a single cell.

Transforming growth factor alpha expression as a response of murine motor neurons to axonal injury and mutation-induced degeneration.

We previously showed that degenerating adult motor neurons of the murine mutant wobbler, a model of spinal muscular atrophy, express Transforming Growth Factor alpha (TGF alpha), a growth factor endowed with glio- and neurotrophic activities. Here, we evaluated whether TGF alpha expression is a general response of adult motor neurons to injury. Synthesis of its precursor (pro-TGF alpha) was investigated in another model of motoneuronal degeneration, the murine mutant muscle deficient, and in hypoglossal motor neurons following axonal crush and cut. In control conditions, motor neurons were devoid of pro-TGF alpha immunoreactivity. In the mutant lumbar spinal cord, pro-TGF alpha immunoreactive motor neurons appeared as soon as the disease developed and pro-TGF alpha expression persisted until the latest stages of degeneration. Motor neurons and astrocytes of the white matter weakly immunoreactive for the TGF alpha receptor were also present in both control and mutant lumbar spinal cords. Following hypoglossal nerve crush and cut, motoneuronal pro-TGF alpha expression was precocious and transient, visible at one day post-injury and lasting for only 3 days, during which time astrocyte-like cells immunoreactive for both TGF alpha and its receptor appeared within the injured nucleus. Enhanced TGF alpha mRNA levels following nerve crush showed that activation occurred at the transcriptional level. These results show that upregulation of TGF alpha is an early and common response of adult murine motor neurons to injury, regardless of its experimental or genetic origin.

Bcl-2 sensitivity differentiates two pathways for motoneuronal death in the wobbler mutant mouse.

The molecular events leading to motoneuronal death are still poorly understood. In mammals, the bcl-2 proto-oncogene, which encodes a membrane-associated protein, has been shown to suppress both developmental motoneuronal death and experimental axotomy-induced motoneuronal death. We assessed a potential protective effect of Bcl-2 on pathological motoneuronal death processes in adult rodents. We took advantage of the murine mutant wobbler, which undergoes progressive degeneration of the spinal and brainstem motoneurons. A hybrid carrying both the wobbler mutation and the human bcl-2 transgene under the control of the neuron-specific enolase promoter was produced. Although Bcl-2 protected spinal and brainstem motoneurons from developmental death and the postnatal motoneurons of the facial nucleus from axotomy-induced death, the pathological motoneuronal death was not altered in the adult hybrid. These results demonstrate that Bcl-2 sensitivity distinguishes at least two different motoneuronal death pathways in the wobbler mutant. They support the hypothesis that experimental and pathological motoneuronal death are dependent on different cellular mechanisms.

Short increase of BDNF messenger RNA triggers kainic acid-induced neuronal hypertrophy in adult mice.

Neurotrophin gene expression in adult brain varies according to physiological activity and following brain injury, suggesting a role in neuronal maintenance and plasticity. However, the exact roles and mechanisms of action of neurotrophins in the adult brain are still poorly understood. We have recently demonstrated that neurons of the adult mouse dentate gyrus can develop a conspicuous morphogenetic response to intrahippocampal injection of kainic acid. This response is correlated with long-lasting overexpression of the brain-derived neurotrophic factor gene, suggesting a causal relationship between molecular and structural changes. To test this hypothesis, brain-derived neurotrophic factor messenger RNA were sequestered in vivo by administration of antisense oligodeoxynucleotides. When administered before 20 h post-kainate, antisense oligodeoxynucleotides totally prevented the kainate-induced neuronal hypertrophy, while sense or missense sequences had no effect. On the other hand, the hypertrophic response was observed when antisense administration was begun 24 h post-kainate, indicating an involvement of brain-derived neurotrophic factor messenger RNA in the initiation of structural changes, but not in their evolution. The hypertrophy was blocked by inhibition of tyrosine kinase activities by K252a, suggesting an involvement of Trk high affinity receptors. Administration of human recombinant brain-derived neurotrophic factor without previous treatment by kainate failed to induce any morphogenetic response. These results show that a short activation of the brain-derived neurotrophic factor gene can, in association with neuronal activation by kainate, trigger dramatic and long-lasting morphological changes in adult neurons. A physiological role of brain-derived neurotrophic factor in adult brain could therefore be to link, by autocrine/paracrine action, activation of glutamate receptors and neuronal morphological adaptive responses.

Targeting widespread sites of damage in dystrophic muscle: engrafted macrophages as potential shuttles.

Inherited muscle diseases are characterized by widespread muscle damage in the body. This limits the clinical relevance of cell or gene therapy based upon direct injections into muscles. One way to circumvent this obstacle would be to use circulating cells, capable of homing naturally to the sites of lesion, to deliver therapeutic substances. Certain muscular dystrophies present successive cycles of degeneration-regeneration. These sporadic necrotic lesions trigger local inflammations with subsequent infiltration of blood-borne mononuclear cells. We have, therefore, tested the possibility that homing monocytes and macrophages could be appropriate shuttles for delivering a therapeutic agent to disseminated pathogenic sites, their targeting being triggered by the pathogeny itself. First, fluorescently labeled immortalized monocytes were intravenously injected into mice which had previously undergone freeze-damaging of individual muscles. In agreement with our hypothesis, intense labelling was observed in the muscle, specifically in damaged regions. Second, the technique was adapted to meet the needs of chronic diseases with characteristic continuous, widespread degeneration of muscle fibers, by creating a reservoir of genetically engineered monocytes, via bone marrow transplantation. Mdx mice received bone marrow from transgenic mice expressing the lacZ reporter gene, under the control of the vimentin promoter, which is active in monocytes and macrophages. Histological and molecular analyses demonstrated the homing of engineered macrophages at the sites of muscle damage, for periods as long as 2 months. Bone marrow progenitor cells, appropriately engineered to elicit the synthesis, in macrophages, of therapeutically relevant substances, may be of clinical value in various pathologies involving an inflammatory phase.

The mouse mutation muscle deficient (mdf) is characterized by a progressive motoneuron disease.

Muscle deficient (mdf) is an autosomal-recessive mutation mapped to mouse chromosome 19. The clinical phenotype and the muscle histopathology, briefly described in 1980, and the nervous system histopathology are detailed in the present study. Homozygotes develop a posterior waddle at 4 to 8 weeks of age. Soon thereafter, the hindlimbs become paralyzed and weakness appears in forelimbs, leading to a serious disability. The disease progresses slowly and the mean lifespan is reduced to 8 months. Skeletal muscles exhibit a neurogenic atrophy with signs of reinnervation. Peripheral nerves display axonal degeneration. Neurons within the spinal cord ventral horn, and some motor nuclei of the brain stem, are affected by a cytoplasmic vacuolar degeneration. Ascending and descending spinal cord tracts appear normal. An astrogliosis, restricted to the ventral horn of the spinal cord, occurs in mdf/mdf mice of 10 weeks of age. These clinical and histological features are indicative of a progressive motor neuronopathy. Among the murine spinal muscular atrophies, the programmed cell death of the mdf motoneurons is morphologically similar to wobbler. Because of the long time course, the mdf mutation may represent a valuable tool for understanding juvenile motoneuron diseases with chronic evolution, even though the murine locus is not syntenic with the human ones.

Early detection of mouse wobbler mutation: a model of pathological motoneurone death.

The mouse recessive mutation wobbler, carried by the C57BL/6J strain, is a naturally occurring model of motoneurone death. The gene is unknown and in the absence of predictive markers, mutants have to be diagnosed by phenotypic criteria at 4 weeks after birth. We localized the wobbler gene to chromosome 11 at 0.98 +/- 1.1 cM from the glutamine synthetase (Glns) gene. A polymorphic allele of the Glns gene was then introduced into the congenic wobbler strain by intraspecific crossing. One-quarter of the offspring expressed the same phenotypic mutation as true wobbler and were detectable by PCR, as they are homozygous for the wobbler-linked Glns allele. The new mutants exhibit motoneurone degeneration despite the new genetic background.

Transforming growth factor alpha (TGF alpha) expression in degenerating motoneurons of the murine mutant wobbler: a neuronal signal for astrogliosis?

The enhanced expression of the trophic factor transforming growth factor alpha (TGF alpha) in reactive astrocytes following CNS injury suggests that TGF alpha has a role in the development of astrogliosis. We explored this hypothesis in the murine mutant wobbler, which presents a progressive motoneuronal degeneration associated with an astrogliosis. Evolution of astrogliosis, and expression of TGF alpha precursor (pro-TGF alpha) and of its receptor were examined over the course of the disease, using genetically diagnosed animals and immunocytochemical techniques. We report here that degenerating motoneurons of the cervical spinal cord and a subset of astrocytes express pro-TGF alpha, prior to the onset of astrogliosis, when the first clinical manifestations of the disease are observed at 4 weeks of age. TGF alpha expression appeared strongly correlated with motoneuronal degeneration. All pro-TGF alpha-immunoreactive neurons exhibited a degenerative morphology, and the number of pro-TGF alpha-immunoreactive neurons increased with the progression of the disease. At the glial level, we observed that astrogliosis was a transitory phenomenon in the wobbler mice, developing in coordination with the motoneuronal expression of pro-TGF alpha. Astrogliosis became evident in 6-week-old wobbler mice, when the number of pro-TGF alpha-immunoreactive motoneurons was maximal, and regressed in older mutant mice in correlation with the disappearance of pro-TGF alpha-immunoreactive motoneurons. Furthermore, TGF alpha/EGF receptor immunoreactivity was exclusively localized in a subset of reactive astrocytes, its expression following closely the course of the astrogliosis. These data show that TGF alpha synthesis by the affected motoneurons is an early event in the course of the wobbler disease, and suggest a role for TGF alpha as a neuronal inducer of astrocytic reactivity.

Phosphatidylinositol is involved in the attachment of tailed asymmetric acetylcholinesterase to neuronal membranes.

1. We analyzed the mode of attachment of 16 S tailed acetylcholinesterase (AChE; EC 3.1.1.7) to rat superior cervical ganglion (SCG) neuronal membranes. Using extractions by high-salt (HS) and nonionic detergent (Triton X-100), we found two pools of 16 S AChE. 2. The detergent-extracted (DE) 16 S AChE was tightly bound to membranes through detergent-sensitive, high-salt insensitive interactions and was distinct from high-salt-soluble 16 S AChE. The detergent-extracted (DE) 16 S AChE constituted a significant proportion of about one-third of the total 16 S AChE. 3. Treatment of the neuronal membranes by a phosphatidylinositol-specific phospholipase C (PIPLC) resulted in the release of some, but not all DE 16 S AChE, indicating that a significant amount of the neuronal DE 16 S AChE, about one-third, is anchored to membranes through a phosphatidylinositol containing residue. Thus, a covalent association of a glycolipid and catalytic or structural AChE polypeptidic chains occurs not only for dimeric AChE but also for the asymmetric species of AChE. 4. The complex polymorphism of AChE is due not only to different globular or asymmetric associations of catalytic and structural subunits but also to the alternative existence of a transmembrane domain or a glycolipid membrane anchor.

Tissue-specific processing and polarized compartmentalization of clone-produced cholinesterase in microinjected Xenopus oocytes.

1. To approach the involvement of tissue-specific elements in the compartmentalization of ubiquitous polymorphic proteins, immunohistochemical methods were used to analyze the localization of butyrylcholinesterase (BuChE) in Xenopus oocytes microinjected with synthetic BuChEmRNA alone and in combination with tissue-extracted mRNAs. 2. When injected alone BuChEmRNA efficiently directed the synthesis of small membrane-associated accumulations localized principally on the external surface of the oocyte's animal pole. Tunicamycin blocked the appearance of such accumulations, suggesting that glycosylation is involved in the transport of nascent BuChE molecules to the oocyte's surface. Coinjection with brain or muscle mRNA, but not liver mRNA, facilitated the formation of pronounced, tissue-characteristic BuChE aggregates. 3. These findings implicate tissue-specific mRNAs in the assembly of the clone-produced protein and in its nonuniform distribution in the oocyte membrane or extracellular material.

Expression and tissue-specific assembly of human butyrylcholine esterase in microinjected Xenopus laevis oocytes.

Cholinesterases represent a ubiquitous, polymorphic family of acetylcholine hydrolyzing enzymes. The multileveled tissue-specific heterogeneity which characterizes these enzymes makes the cholinesterases an appropriate model for studying the mechanisms involved in regulating divergent pathways in protein biogenesis. For this purpose, a cDNA coding for human butyrylcholine esterase (BuChE) was subcloned into the SP 6 transcription vector. Synthetic mRNA transcribed from this construct was microninjected into Xenopus laevis oocytes alone, and in conjunction with poly(A)+ RNAs extracted from human brain or muscle. Injected BuChE-mRNA induced the biosynthesis of a protein exhibiting the catalytic activity, substrate specificity, and sensitivity to selective inhibitors characteristic of native human serum BuChE, and clearly distinct from the related enzyme acetylcholinesterase (AChE). The nascent BuChE was reproducibly distributed into low salt-soluble and detergent-extractable pools. Sucrose gradient analysis demonstrated that the nascent human enzyme was capable of limited subunit assembly, appearing as functional dimeric molecules in both of these fractions. Co-injection with brain or muscle-derived mRNAs facilitated higher order oligomeric assembly. Co-injected brain mRNA induced the appearance of tetramers while co-injected muscle mRNA induced the appearance of an array of heavy molecular forms, including a heavy 16 S form. These results indicate that the molecular determinants which distinguish BuChE from AChE are inherent to its primary amino acid sequence and that additional, tissue-specific protein(s) are involved in the modulation of subunit assembly within particular biological milieues.

Definition, at the molecular level, of a thyroglobulin-acetylcholinesterase shared epitope: study of its pathophysiological significance in patients with Graves' ophthalmopathy.

The nature of the putative autoantigen in Graves' ophthalmopathy (Go) remains an enigma but the sequence similarity between thyroglobulin (Tg) and acetylcholinesterase (ACHE) provides a rationale for epitopes which are common to the thyroid gland and the eye orbit. In an attempt to define the shared epitope, we have screened a lambda gt 11 human thyroid cDNA library using a polyclonal antibody to Torpedo ACHE and isolated two clones, which upon sequencing, were shown to contain Tg segments, corresponding to portions of the C terminal part of the molecule which has a high similarity with ACHE. Having demonstrated the existence of an epitope common to Tg and ACHE, the clones have been further tested and found to be positive in lysis plaque assays with 1/10 sera from patients with Hashimoto's thyroiditis (HT), 8/8 from patients with Graves' ophthalmopathy and 0/8 normal sera. We have investigated the physiological significance of this common epitope by in situ immunolocalization studies in which the polyclonal antibody to Torpedo ACHE (which was used for screening the library) and immunoglobulins (Igs) from 6 Go patients tested were shown to bind to end plate regions of human foetal muscle fibres which were concurrently shown to be rich in cholinesterase activity: Igs from 3 normal individuals and 2 patients with Hashimoto's thyroiditis did not bind. The results demonstrate and characterize an epitope which is common to Tg and ACHE and show that Go patients Igs contain antibodies which bind to muscle end plates rich in cholinesterase. The significance of these findings to the pathogenesis of Go is discussed.

A dimeric form of acetylcholinesterase anchored through a glycolipid in mouse skeletal muscle.

A glycolipid anchorage for acetylcholinesterase (AChE) has been found in some tissues. In this paper, the possibility of such an anchorage has been explored in mammalian muscle membranes. We report that a phosphatidylinositol-specific phospholipase C (PIPLC) solubilizes AChE from microsomal membranes of mouse intercostal muscle. Among the several molecular forms of AChE, PIPLC specifically releases in a dose dependent manner one molecular form which migrates on linear sucrose gradients as a single peak of sedimentation coefficient 6.3 s. In other subcellular membrane fractions, including motor endplate enriched fraction, PIPLC fails to solubilize AChE. This type of membrane glycolipid mediated anchorage for AChE is then only detectable in a precise region of skeletal muscle.

Association of tailed acetylcholinesterase to lipidic membranes in mammalian skeletal muscle.

Tailed acetylcholinesterase (AChE) was studied in three subcellular membrane fractions of mouse skeletal muscle: a fraction enriched in isolated motor endplates (C), an extrasynaptic membrane fraction (A) and a microsomal fraction (S). In the (C) fraction, tailed asymmetric 16S AChE required high salt conditions to be extracted, while in (A) and (S) microsomal membranes, a collagenase sensitive 16S form, was extracted by detergent alone. This apparent "hydrophobic" property suggests that there is a pool of 16S AChE which is probably bound to lipidic membranes. The detergent extractable (DE) 16S AChE was not concentrated in motor endplate-rich regions and differential inhibition of external and internal AChE demonstrated that it could have both intra- and extracellular locations in the adult differentiated muscle fibres.

Hydrophilic and hydrophobic attachment of both globular and asymmetric acetylcholinesterase to frog muscle basal lamina sheaths.

We prepared myofiber basal lamina sheaths (BLs) using the in vivo experimental procedure of Sanes et al. (J. Cell Biol.78, 176-198, 1978) on frog cutaneus pectoris muscle. On the 15 days post-operatively, acetylcholinesterase (AChE) is still found concentrated in native BLs and purified BLs preparations and both globular and asymmetric molecular forms coexist (Nicolet et al., J. Cell Biol., 107, 762-768, 1986). We describe here at least two distinct AChE pools, according to their differential solubility in non-ionic detergent and high-salt media. One is detergent-extracted (DE) and the other is detergent-insoluble, high-salt extracted (HSS). In the BLs preparation as well as in control motor end-plate rich regions (MEP-r) of muscle, both globular and asymmetric forms of AChE are found as DE and HSS variants. These observations suggest that all AChE forms are present in the extracellular muscle basal lamina and are bound through not only hydrophilic but also hydrophobic bonds, to probably distinct structural domains of the muscle basal lamina.