Distribution Of Laminin Research Articles

Expression and Distribution of Laminin, Fibronectin and Tenascin is Stage Dependently Modulated in the Skin of Chronic Venous Insufficiency

Objective:The regulation of tissue remodelling is a function of extracellular matrix (ECM) deposition. Our aim was to determine the expression and distribution pattern of the ECM proteins laminin, fibronectin and tenascin in all stages of chronic venous insufficiency (CVI) from telangiectases to ulceration.Methods:In punch biopsies of 25 patients with various stages of CVI and five healthy volunteers, ECM proteins were stained using indirect immunofluroescence. The staining pattern in the affected skin was evaluated by two independent, double-blinded investigators by microscopic examination.Results:In specimens of healthy skin or skin with telangiectases or pigmentation, a faint and partly inhomogeneous ECM staining pattern was detected in the upper dermis. In venous eczema, lipodermatosclerosis and venous leg ulcers, an increased expression and a wide-meshed distribution pattern throughout the dermis was observed for laminin, fibronectin and tenascin. Fragmentation of ECM components was first observed in venous eczema, persisting in the more severe stages of CVI. Laminin staining revealed unusual streak-like distributions in the papillary dermis, pronounced in Pigmentation and lipodermatosclerosis and diminished in leg ulcers.Conclusions:Our results indicate an important role of laminin, fibronectin and tenascin, and in particular of their proteolytic fragments, in the early phases of CVI, such as venous eczema, by creating permissive environments for cell migration and differentiation, essential for wound healing.

Reconstitution of basement membrane after 'sandwich-technique' skin grafting for severe burns demonstrated by immunohistochemistry.

Reconstitution of basement membrane structures after "sandwich-technique" grafting of severe deep burns is demonstrated with use of immunohistochemical techniques. Cryosections of human skin after epifascial burn wound excision and sandwich grafting were stained with monoclonal antibodies against type IV and VII collagen, polyvalent antiserum against type VI collagen, and polyvalent antibody against laminin. Standard hematoxylin and eosin histologies were performed for morphologic correlation. Reorganization of the mesenchymal border zone (basement membrane), after transplantation of extremely expanded split-thickness skin autografts overlaid with glycerolized split-thickness skin allografts onto debrided human full-thickness wounds, occurred from day 5 to day 35. The autografts reepithelize the spaces between the mesh structure, which has been covered primarily exclusively with allogenic skin, and form a layered squamous epithelium, with an underlying three-dimensional basket-weave array of collagen in the remodeled neodermis after epifascial excision. Immunochemical techniques detect the reconstitution of a basement membrane zone with a typical architecture and distribution of laminin, type IV, and type VII collagen being built up 1 week to 5 weeks after sandwich grafting. These structures can be seen in the autografts during the first 2 weeks and are consistent in the whole reconstituted skin after day 35. To our knowledge this is the first report of the expression of type VI collagen in these types of wounds. The findings are compared with the expression of type VI collagen in healthy skin. The results indicate that the modified sandwich-grafting technique is an adequate means for early burn wound closure and resurfacing of third-degree burn wounds and leads to the reconstitution of dermal qualities.

Detection of extracellular matrix protein (laminin) in experimental arterial anastomoses

To investigate the distribution of Laminin in experimental microvascular surgery. Autografts (4 mm) of the left common carotid artery in 20 Wistar rats were harvested after 4 weeks. The specimens were investigated immunohistochemically for the demonstration and distribution of Laminin. The demonstration and distribution pattern of Laminin was a function of the accuracy of each single suture. In the intima, the organisation and amount of Laminin was directed to the lumen and dependent from the distance and vitality of the vessel segments' cells. The media had in its axis direction the lowest reparative potency to regenerate the continuity break. The adventitia formed a thick cuff around the anastomosis, which was circularly grown through with Laminin-positive vasa vasorum. The marked myointimal hyperplasia in the area around the anastomosis and in the entire autograft was constituted of Laminin-positive extracellular matrix. Our results support the demand for a careful and atraumatic suture technique in microvascular surgery.

Levels and molecular size distribution of serum laminin in adult type I diabetic patients with and without microangiopathy

the glycoprotein laminin, a cross-shaped complex of three genetically different polypeptide chains, is a structural component of the capillary basement membrane. Serum laminin concentrations of healthy controls (n = 60) and adult type I diabetic patients (n = 170) were not age-dependent. Laminin was correlated with hemoglobin A 1 (HbA 1) values in normooalbuminuric patients ( r S = .33, P < .0005, n = 116). Type I diabetic patients without nephropathy or retinopathy in good metabolic control had normal laminin levels. However, increasing stages of microangiopathy were associated with higher laminin levels. The molecular size distribution of serum laminin of control subjects (n = 4) and type I diabetic patients (n = 15) was analyzed by molecular-sieve chromatography. Laminin was eluted in two peaks with a molecular mass of 900 and 300 kd, most likely representing intact laminin and its P1 fragment, respectively. The areas of the two peaks were determined by two—gaussian function fitting. In patients without microangiopathy in poor metabolic control, an increase in the high—molecular weight (HMW) fraction could be detected as compared with healthy subjects and patients with acceptable metabolic control. Furthermore, the HMW laminin fraction and the ratio between the areas of the first and second peak increased with the stage of nephropathy ( P < .001, Jonckheere-Terpstra test). These results provide evidence that (1) laminin concentration is increased in chronic hyperglycemia, (2) laminin may be a marker of microangiopathic lesions, and (3) elevated laminin levels may reflect an increased synthesis and/or a defective incorporation of laminin into the capillary basement membrane.

Distribution and function of laminins in the neuromuscular system of developing, adult, and mutant mice.

Laminins, heterotrimers of alpha, beta, and gamma chains, are prominent constituents of basal laminae (BLs) throughout the body. Previous studies have shown that laminins affect both myogenesis and synaptogenesis in skeletal muscle. Here we have studied the distribution of the 10 known laminin chains in muscle and peripheral nerve, and assayed the ability of several heterotrimers to affect the outgrowth of motor axons. We show that cultured muscle cells express four different alpha chains (alpha1, alpha2, alpha4, and alpha5), and that developing muscles incorporate all four into BLs. The portion of the muscle's BL that occupies the synaptic cleft contains at least three alpha chains and two beta chains, but each is regulated differently. Initially, the alpha2, alpha4, alpha5, and beta1 chains are present both extrasynaptically and synaptically, whereas beta2 is restricted to synaptic BL from its first appearance. As development proceeds, alpha2 remains broadly distributed, whereas alpha4 and alpha5 are lost from extrasynaptic BL and beta1 from synaptic BL. In adults, alpha4 is restricted to primary synaptic clefts whereas alpha5 is present in both primary and secondary clefts. Thus, adult extrasynaptic BL is rich in laminin 2 (alpha2beta1gamma1), and synaptic BL contains laminins 4 (alpha2beta2gamma1), 9 (alpha4beta2gamma1), and 11 (alpha5beta2gamma1). Likewise, in cultured muscle cells, alpha2 and beta1 are broadly distributed but alpha5 and beta2 are concentrated at acetylcholine receptor-rich "hot spots," even in the absence of nerves. The endoneurial and perineurial BLs of peripheral nerve also contain distinct laminin chains: alpha2, beta1, gamma1, and alpha4, alpha5, beta2, gamma1, respectively. Mutation of the laminin alpha2 or beta2 genes in mice not only leads to loss of the respective chains in both nerve and muscle, but also to coordinate loss and compensatory upregulation of other chains. Notably, loss of beta2 from synaptic BL in beta2(-/-) "knockout" mice is accompanied by loss of alpha5, and decreased levels of alpha2 in dystrophic alpha2(dy/dy) mice are accompanied by compensatory retention of alpha4. Finally, we show that motor axons respond in distinct ways to different laminin heterotrimers: they grow freely between laminin 1 (alpha1beta1gamma1) and laminin 2, fail to cross from laminin 4 to laminin 1, and stop upon contacting laminin 11. The ability of laminin 11 to serve as a stop signal for growing axons explains, in part, axonal behaviors observed at developing and regenerating synapses in vivo.

Phenotypic modulation of smooth muscle cells after arterial injury is associated with changes in the distribution of laminin and fibronectin.

Earlier in vitro studies suggest opposing roles of laminin and fibronectin in regulation of differentiated properties of vascular smooth muscle cells. To find out if this may also be the case in vivo, we used immunoelectron microscopy to study the distribution of these proteins during formation of intimal thickening after arterial injury. In parallel, cell structure and content of smooth muscle alpha-actin was analyzed. The results indicate that the cells in the normal media are in a contractile phenotype with abundant alpha-actin filaments and an incomplete basement membrane. Within 1 week after endothelial denudation, most cells in the innermost layer of the media convert into a synthetic phenotype, as judged by loss of actin filaments, construction of a large secretory apparatus, and destruction of the basement membrane. Some of these cells migrate through fenestrae in the internal elastic lamina and invade a fibronectin-rich network deposited on its luminal surface. Within another few weeks a thick neointima forms, newly produced matrix components replace the stands of fibronectin, and a basement membrane reappears. Simultaneously, the cells resume a contractile phenotype, recognized by disappearance of secretory organelles and restoration of alpha-actin filaments. These findings support the notion that laminin and other basement membrane components promote the expression of a differentiated smooth muscle phenotype, whereas fibronectin stimulates the cells to adopt a proliferative and secretory phenotype.

The distribution of laminins and fibronectins is modulated during extravillous trophoblastic cell differentiation and decidual cell response to invasion in the human placenta.

We studied the distribution of laminin (Ln) alpha1-alpha3, beta1-beta3, and gamma1 chains, and of the extradomain-A (EDA) and EDB and the oncofetal epitope of fibronectin (Onc-Fn) in extravillous trophoblastic cells and decidua in the human placenta by immunohistochemistry. We found that the transition from villous to extravillous trophoblast was accompanied by emergence of immunoreactivity for EDA-, EDB-, and Onc-Fn among the cells. Furthermore, whereas the villous trophoblastic basement membrane (BM) contains Ln alpha1, alpha2, beta1, beta2, and gamma1 chains, immunoreactivity for Ln alpha1, beta1, and gamma1, but not for Ln alpha2 and beta2 chains, was detected in association with extravillous trophoblastic cells. Interestingly, although immunoreactivity for the Ln alpha1, alpha2, beta1, beta2, and gamma1 chains was detected in all decidual cell BMs, EDB-Fn and Onc-Fn were detected only in decidua that had been invaded by the trophoblast. In summary, our results describe distinct changes in the distribution of Ln and Fn isoforms during the differentiation of villous trophoblast into extravillous trophoblastic cells. Furthermore, EDB- and Onc-Fn are preferentially found in decidua that has been invaded by the trophoblast, indicating that the deposition of these Fn isoforms reflects a decidual cell response to invasion.

FUNCTIONALLY SIGNIFICANT SECONDARY INFLAMMATION OCCURS IN A PRIMARY EPITHELIAL ENTEROPATHY.

Background: Primary epithelial causes of intractable diarrhoea of infancy include microvillous atrophy 1, epithelial dysplasia 2 and heparan sulphate deficiency 3. These conditions are unresponsive to standard anti-inflammatory treatment, and management is generally supportive. However recent experimental work, in which epithelial cadherins were mutated 4, shows that that functionally significant mucosal inflammation may occur as a consequence of such a primary epithelial lesion. Methods: Small intestinal biopsies from 3 Middle-Eastern infants with epithelial dysplasia (confirmed by immunohistochemical analysis for laminin, tenascin, fibronectin, syndecan-1 and collagen IV), were also stained for inflammatory and functional markers (CD's 3, 4, 8, 19, 25, 68, Ki67, HLA-DR, sulphated glycosaminoglycans [GAGs]). On the basis of this analysis, two of the infants were then treated with intravenous tacrolimus (FK506). Results: An isolated abnormality of laminin distribution with associated tufting was confirmed in all biopsies, consistent with the initial report of the lesion by Goulet et al2. However two were also found to show crypt hyperplastic villous atrophy with increased density of CD25+ mucosal T cells, and marked disruption of sulphated GAGs. Tacrolimus therapy substantially improved enteral absorption, particularly for sugars, although neither weaned fully from TPN. Crypt cell proliferation rate decreased markedly on tacrolimus, as did markers of mucosal inflammation, while GAG density increased. Conclusions: This supports the recent contention that basement membrane abnormalities may characterise tufting enteropathy 2. However the mucosal findings and the response to tacrolimus suggests that secondary inflammation may worsen the functional defect of primary epithelial enteropathies. Treatment of the inflammatory component with tacrolimus may usefully improve small intestinal function in such infants.

Increased laminin deposition in capillaries of the stria vascularis of quiet-aged gerbils

The distribution of laminin (LA) and type IV collagen (IV-C) in the gerbil inner ear was investigated by light and electron microscopic immunohistochemistry. Changes in protein expression were assessed from birth to old age to determine the relation of these constituents to maturation of the cochlea and development of presbyacusis. The distribution of LA paralleled that of IV-C during postnatal development, and both were visualized in the basement membrane (BM) of endothelial, epithelial and spiral ganglion cells in neonatal and young adult gerbils. Immunopositive BM underlying the stria vascularis disappeared at 8–12 days after birth coincident with the development and maturation of the strial capillaries. Immunoreactivity for LA afforded an index to the thickness of the BM and was found to increase with age only in the BM of strial capillaries. At 6 months of age, occasional strial capillaries in the apex of the cochlea showed thickening of the LA-positive BM. Abnormal deposition of LA in strial capillary BM spread to lower turns and increased in prevalence with advancing age, affecting apical and basal more than middle cochlear turns. Thickening of the capillary BM appeared to precede capillary obstruction which eventuated in complete strial atrophy. Staining for IV-C in the walls of the strial capillaries did not increase with age. The data show that LA and IV-C play important roles in postnatal development of the cochlea and that LA deposition increases with age only in the BM of strial capillaries.

Expression of laminin and its possible role in adrenal cortex homeostasis.

The adult mammalian adrenal cortex undergoes permanent regeneration. This process implies a cellular proliferation step restricted to the external zone of the tissue, and a subsequent centripetal cell migration during which phenotypic transition from glomerulosa into fasciculata and reticularis cells and elimination of senescent cells through apoptosis occur. As the molecular mechanisms implied in adrenocortical cell migration are still generally unknown, we addressed that question in the present study. Of several extracellular matrix proteins tested, laminin was the most potent chemotactic and haptotactic factor for bovine fasciculata adrenocortical cells. The maximal chemotactic effect (3-fold stimulation) was observed with 50-75 micrograms/ml laminin, whereas the haptotactic effect (3.5-fold stimulation) plateaued for laminin concentrations in the coating solution over 25 micrograms/ml. Using an anti-Engelbreth-Holm-Swarm laminin antibody, we could demonstrate that adrenocortical cells actively synthesize and secrete Engelbreth-Holm-Swarm-laminin, with the A chain produced in limiting quantities. ACTH treatment of adrenocortical cells specifically induced a 2.7- to 4.5-fold increase in A chain synthesis, resulting in a corresponding increase in the amount of secreted laminin. The distribution of laminin in the adrenal cortex tissue was then evaluated by standard immunohistochemistry. The protein appeared to be uniformly expressed in the three zones of the cortex. This observation does not favor the hypothesis that laminin acts as an attractant driving centripetal cell migration. Laminin, which is synthesized under the control of the systemic hormone ACTH, appears as a permissive factor that facilitates proper homeostasis of the adrenocortical tissue.

Prostaglandin action on ciliary smooth muscle extracellular matrix metabolism: Implications for uveoscleral outflow

The cellular mechanisms mediating intraocular pressure reduction following topical prostaglandin (PG) treatments are poorly understood. To determine if PG treatments might induce altered metabolism of extracellular matrix surrounding ciliary muscle cells, confluent human ciliary smooth muscle cell cultures were exposed to PGF 2α, 17-phenyltrinor-PGF2 2α, or 11-deoxy-PGE 1 for one to four days and the distributions of collagen types I, III and IV as well as laminin were determined immunocytochemically. In addition, collagen type 1V and promatrix metalloproteinase III (proMMP-3) content within treated cultures was determined using sandwich ELISAs. Compared with vehicle-treated cultures, there were substantial reductions in the density and branching of the collagen type IV-imnmnoreactive lattice accompanied by thickening of remaining strands in all PG-treated cultures. Similar changes were seen in the distribution of laminin within all PG-treated cultures. Reductions in collagen type III immunoreactivity were seen in cultures treated with either PGF 2α or 17-phenyltrinor-PGF 2α . No changes were observed in collagen type I immunoreactivity. Quantitative analyses revealed increased amounts of collagen type IV in both the culture medium and in extracts of the cell layer in all PG-treated cultures. In addition, there were substantial increases in the concentrations of proMMP-3 in all PG-treated cultures. These results indicate that PGs induce increased turnover and remodeling of ECM adjacent to ciliary muscle cells. Such changes may contribute to increased uveoscleral outflow in vivo following topical PG treatment.

Localization of laminin, fibronectin, E-cadherin, and integrins in endometrium and endometriosis

To compare the localization of adhesion proteins (laminin and fibronectin) and their receptors of the integrin family in endometriosis and endometrium. An immunohistochemical study. University Hospital, Department of Gynecology and Department of Cell Biology. Eighteen endometriosis patients undergoing laparoscopy for pain or infertility and nine control patients undergoing laparoscopy for sterilization or hysterectomy. The expression of adhesion glycoproteins (laminin and fibronectin), their receptors alpha 1 beta 1, alpha 2 beta 1, alpha 3 beta 1, alpha 5 beta 1, and alpha 6 beta 1, and E-cadherin was determined by immunohistochemistry on frozen sections. The distribution of both adhesive glycoproteins, laminin and fibronectin, and their receptors was identical in endometriosis and endometrium. Fibronectin receptors (alpha 4 beta 1, alpha 5 beta 1) displayed distinct expression patterns in endometrium and endometriosis. No endometrial glands showed positive staining for the alpha 5 chain, whereas this integrin subunit was detected in almost all endometriotic lesions. The integrin alpha 4 chain was present in all endometriotic glands but was absent from endometrial glands in the proliferative phase of the cycle. No difference in cell adhesion molecule localization nor receptors was observed between endometriotic and endometrial samples, except for fibronectin receptors. Their expression persisted around endometriotic glands but not in endometrium. These results suggest that fibronectin receptors could play a role in the persistence of endometriotic lesions, despite menstruation in corresponding eutopic endometrium.

Galectin-3 and laminin expression in neoplastic and non-neoplastic thyroid tissue.

Galectin-3 is a 31 kD beta-galactoside-binding lectin which is expressed by several types of non-neoplastic and neoplastic cells and which may be involved in cell-extracellular matrix interactions. An immunohistochemical study has been made of the expression of galectin-3, as well as its ligand, laminin, in a spectrum of benign and malignant thyroid neoplasms and in some non-neoplastic conditions. Immunohistochemistry with anti-human recombinant galectin-3 antibody showed consistent, intense positivity in the neoplastic cells of 18 cases of papillary carcinoma and less intense staining in the five anaplastic carcinomas studied. In addition, two out of three poorly differentiated carcinomas, three out of six medullary carcinomas, and four out of eight follicular carcinomas had less intense or focal positivity. One case of Hürthle cell carcinoma showed scattered strongly positive cells. Eight follicular adenomas, three hyperplastic nodules, five nodular goitres, and normal thyroid tissue were negative. Galectin-3 mRNA expression was also evaluated in three of the papillary carcinomas, two follicular adenomas, and one hyperplastic nodule with matched normal tissue. Northern blot analysis demonstrated mRNA overexpression in the three cases of papillary carcinomas, whereas normal and benign tissues were negative. Laminin distribution in neoplastic and non-neoplastic tissue varied with architectural patterns but did not correlate with galectin-3 immunohistochemical expression. We conclude that expression of galectin-3 is limited to inflammatory foci in normal and benign thyroid tissue and is a phenotypic feature of malignant thyroid neoplasms, especially papillary carcinomas.

Immunohistochemical and biochemical analysis of laminin in neonatal rat first molars.

Rabbit polyclonal antibody against mouse EHS laminin was used to investigate the distribution and composition of laminin in the rat first molar tooth germ. Immunohistochemical analysis showed that laminin is expressed in the inner and outer epithelia of the enamel organ and in small blood vessels in the dental papilla and strellate reticulum. Immunoblots revealed that tooth germ laminin differs from EHS laminin. Tooth germ laminin contains beta chains, while the alpha 1 chain is substituted by a 300-kDa chain. Two-dimensional electrophoresis analysis of tooth germ extract showed that beta chains appeared as four spots with approximate pI values of 6.6, 7.5, 7.8 and 8.5. These results indicate that more than-one type of laminin isoform is present in the first molar tooth germ. Additionally, we have shown that despite the early degradation of tooth germ basement membrane, the laminin molecule is still intact at the time of birth.

Three-dimensional Distribution of Extracellular Matrix in the Mouse Small Intestinal Villi. Laminin and Tenascin

To investigate the three-dimensional distribution of laminin and tenascin in the villi of the mouse small intestine, we designed a new tissue preparation procedure for confocal laser scanning microscopy. Fixed tissue was pretreated with sodium deoxycholate and immunostained. The specimens displayed intense immunofluorescence even in the deeper sites of the lamina propria. Laminin was distributed like a sheet in the basement membrane of the epithelium as well as that of the capillaries. Additionally, it was associated with some tubular structures in the lamina propria. Many pores were found in the laminin sheet. By comparison, tenascin was distributed at the epithelial basement membrane and in the underlying connective tissue exhibiting a striped pattern. Tenascin was observed wrapping around the capillary wall in a spiral but was absent at the tip of the villi except in the capillary wall. Pores in the staining pattern of tenascin were also found as was the case for laminin.This three-dimensional reconstruction made it possible to observe more clearly the localization of these two extracellular matrix molecules.

Expression of extracellular matrix molecules in the embryonic rat olfactory pathway

Primary olfactory neurons arise from placodal neuroepithelium that is separate from the neuroepithelial plate that forms the neural tube and crest. The axons of these neurons course along a stereotypical pathway and invade the rostral telencephalic vesicle where they induce the formation of the olfactory bulb. In the present study we examined the expression of several extracellular matrix constituents during formation of the olfactory nerve pathway in order to identify putative developmentally significant molecules. Double-label immunofluorescence was used to simultaneously map the trajectory of growing primary olfactory axons by expression of growth associated protein 43 (GAP-43) and the distribution of either laminin, heparan sulfate proteoglycans (HSPG), or chondroitin sulfate proteoglycans (CSPG). At embryonic day 12.5 (E12.5) primary olfactory axons have exited the olfactory neuroepithelium of the nasal pit and formed a rudimentary olfactory nerve. These axons together with migrating neural cells form a large mass outside the rostral surface of the telencephalon. This nerve pathway is clearly defined by a punctate distribution of laminin and HSPG. CSPG is selectively present in the mesenchyme between the olfactory nerve pathway and the nasal pit and in the marginal zone of the telencephalon. At E14.5 primary olfactory axons pierce the telencephalon through gaps that have emerged in the basement membrane. At this age both laminin and HSPG are colocalized with the primary olfactory axons that have entered the marginal zone of the telencephalon. CSPG expression becomes downregulated in this same region while it remains highly expressed in the marginal zone adjacent to the presumptive olfactory bulb. By E16.5 most of the basement membrane separating the olfactory nerve from the telencephalon has degraded, and there is direct continuity between the olfactory nerve pathway and the central nervous system. This strict spatiotemporal regulation of extracellular matrix constituents in the olfactory nerve pathway supports an important role of these molecules in axon guidance. We propose that laminin and HSPG are expressed by migrating olfactory Schwann cells in the developing olfactory nerve pathway and that these molecules provide a conducive substrate for axon growth between the olfactory neuroepithelium and the brain. CSPG in the surrounding mesenchyme may act to restrict axon growth to within this pathway. The regional degradation of the basement membrane of the telencephalon and the downregulation of CSPG within the marginal zone probably facilitates the passage of primary olfactory axons into the brain to form the presumptive nerve fiber layer of the olfactory bulb.

The distribution of fibronectin and laminin in the murine periodontal membrane, indicating possible functional roles in the apical migration of the junctional epithelium

Periodontal tissue shows various morphological changes with ageing. A typical example of these changes is the apical migration of the junctional epithelium. The distribution of fibronectin and laminin was investigated by immunofluorescent and immunoelectron-microscope methods in mice to clarify any possible functional roles of these proteins in the apical migration of junctional epithelium. Apical migration begins in 20-week-old mice, and then progresses with increasing age until the mice reach 80 weeks. In the apical tip of the junctional epithelium, fibronectin was demonstrated in the subepithelial fibrillar matrix, preceding the progression of apical migration. Fibronectin was also demonstrated in association with the stromal side of focal contants between epithelial cells and basement membrane. Therefore, these focal contacts are assumed to be fibronectin receptors. There was no apparent relation between the localization of laminin and the migration of the junctional epithelium. These results suggest that the fibronectin provides a provisional matrix for the apical migration of junctional epithelium, but laminin does not appear to play a major part in that migration.

Temporal and spatial patterns of tenascin and laminin immunoreactivity suggest roles for extracellular matrix in development of gustatory papillae and taste buds

Gustatory papillae are complex organs that are composed of 1) an epithelium, 2) specialized sensory cells within the epithelium (the taste buds), 3) a broad connective core, and 4) sensory innervation. During papilla development, cells in the various tissue compartments must divide, aggregate, detach, migrate, and reaggregate in relation to each other, but factors that regulate such steps are poorly understood and have not been extensively studied. All of these processes potentially require participation of the extracellular matrix. Therefore, we have studied temporal and spatial patterns of immunoreactivity for two extracellular matrix molecules, tenascin and laminin, in the developing fungiform and circumvallate papillae of fetal, perinatal, and adult sheep tongue. To determine relations of tenascin and laminin to sensory innervation, we used an antibody to growth-associated protein (GAP-43) to label growing nerves. Immunocytochemical distributions of tenascin and laminin alter during development in a manner that reflects morphogenesis rather than histologic boundaries of the taste papillae. In early fungiform papillae, tenascin immunoreactivity is very weak within the mesenchyme of the papilla core. However, there is a subsequent shift to an intense, restricted localization in the apical papilla core only--directly under taste bud-bearing regions of the papilla epithelium. In early circumvallate papillae, tenascin immunoreactivity is patchy within the papilla core and within the flanking, nongustatory papillae. Later, immunoreactivity is restricted to the perimeter of the central papilla core, under epithelium that contains developing taste buds. In fungiform and circumvallate papillae, the shift in tenascin immunolocalization is associated with periods of taste bud formation and multiplication within the papilla epithelium and with extensive branching of the sensory innervation in the papilla apex. Laminin immunoreactivity, although it is continuous throughout the basement membrane of general lingual epithelium, is interrupted in the epithelial basement membrane of early fungiform and circumvallate papillae in regions where taste buds are forming. The breaks are large in young fetuses, when taste buds first develop, and are evidenced later as punctate disruptions. Heparan sulfate proteoglycan immunoreactivity confirms that these are basement membrane discontinuities. GAP-43 label coincides with innervation of the papilla core and is most extensive in regions where tenascin immunoreactivity is weak or absent. GAP-43 immunoreactivity is also found in early taste buds: Later, it is extensive within more mature multiple taste buds, presumably in relation to synaptogenesis. We propose that tenascin has a role in promoting deadhesion of cells in the papilla epithelium during periods of taste bud formation and multiplication. Discontinuities in the epithelial basement membrane under developing taste buds, indicated with laminin and heparan sulfate proteoglycan immunoreactivity, may interact to facilitate taste bud morphogenesis and multiplication, to permit access of papilla innervation to the forming taste buds, and/or to allow epithelial/mesenchymal interactions during papilla and taste bud development.

An immuno-electronmicroscopical study of the distribution of laminin within autografts of denatured muscle.

We have used an immunogold post-embedding technique to examine, qualitatively and quantitatively, the distribution of laminin along sarcolemmal basal laminae of variously treated muscle autografts placed in transected rat sciatic nerves. We found that freeze-thawing or heating to 60 degrees C prior to grafting did not affect laminin labelling density along the sarcolemmal basal laminae, either at the time of preparation or 7 days after grafting. In sharp contrast, heating to 80 degrees C significantly reduced laminin labelling density. These findings are consistent with our earlier work showing that frozen-thawed or 60 degrees C muscle autografts both support axonal regeneration, whereas 80 degrees C grafts do not, and add further support to the view that laminin is a functionally important molecule in nerve regeneration. We have compared immunostaining using 10 nm gold particles with silver enhancement of 5 nm gold particles: although labelling density was higher in the silver-enhanced preparations, there was no increase in background labelling. Although empty sarcolemmal basal lamina tubes were frequently highly infolded, there was no evidence of preferential labelling of either 'peaks' or 'troughs' of the infolded basal laminae.

Presence of atypical laminin on the surface of mouse Lewis lung carcinoma cells.

We investigated the expression and distribution of laminin in Lewis lung carcinoma LL2-Lu3 cells. The microscopic immunofluorescence study of the non-permeabilized cells and blotting assay after immunoprecipitation with anti-laminin antibodies of biotinylated cell surface proteins demonstrated that LL2-Lu3 cells retained laminin on their cell surfaces. This laminin was atypical in that it lacked A chain as revealed by the immunoblot analysis. The results of the reverse transcription polymerase chain reaction method indicated that LL2-Lu3 cells contained mRNA for B1 and B2 chains, but not A chain corresponding to those of typical laminin derived from murine Engelbreth-Holm-Swarm sarcoma. A precursor form of 67 kDa laminin receptor protein was also shown to exist on the surfaces of LL2-Lu3 cells. These findings suggest that the interaction between atypical laminin and the precursor form of the 67 kDa laminin receptor protein on the cell surfaces may function in regulating cell activities such as metastasis of LL2-Lu3 cells.