Faculty Bibliography

To study the mechanism of basement membrane formation, we determined by immunochemistry temporal and spatial expression of laminin-5 (Ln-5), laminin-1 (Ln-1) and their integrin receptors during early skin morphogenesis. A 3-dimensional skin culture was used that allows the study of the sequential molecular events of basement membrane formation at the epidermodermal interface. During early anchorage of keratinocytes to the extracellular matrix there is expression of Ln-5, BP-230 antigen and alpha3, beta1 integrin subunits. During epidermal stratification and prior to the formation of the lamina densa there is assembly of Ln-5, Ln-1, collagen IV and nidogen accompanied by keratinocyte basal clustering of alpha2, alpha3, alpha6, beta1, and beta4+ integrin subunits. The assembly pattern of Ln-1 and Ln-5 can be disturbed with functional antibodies against the beta1 (AIIB2) and alpha6 (GoH3) integrin subunits. Ln-1 assembly can also be disturbed with antibodies against its E8 domain and by competitive inhibition with a synthetic peptide (AG-73) derived from its G-4 domain. Quantitative RT-PCR showed that the dermis contributes about 80% of the laminin gamma)1 chain mRNA while 20% is produced by the epidermis which emphasizes its dual tissue origin and the major contribution of the mesenchyma in laminin production. The laminin gamma2 chain mRNA, present in Ln-5, was mostly of epidermal origin. This study presents evidence that during the initiation of basement membrane formation, laminins bind to keratinocyte plasma membrane receptors and thus may serve as nucleation sites for further polymerization of these compounds by a self-assembly process.

A 16-year-old Hispanic boy born of consanguineous parents is described as having a history of cataracts, progressive lower-extremity spasticity and atrophy starting at 4 years of age, atretic ear canals with hearing dysfunction and diffuse patchy cutaneous hypopigmented areas. Clinical examination showed the typical signs of spastic paraplegia with increased tone, hyperreflexia, muscle atrophy and contractures. Sensation, autonomic and cerebellar functions were not disturbed. Neuroimaging studies were normal. Laboratory findings did not support a diagnosis of metabolic disturbance or infectious disease. This is considered a new form of complicated hereditary spastic paraplegia (HSP), transmitted presumably in an autosomal recessive pattern

Temporal and spatial expression of alpha1 (IV), alpha2 (IV), alpha3 (IV), alpha4 (IV), alpha5 (IV), and alpha6 (IV) collagen chains was studied during the formation of the basal lamina in an "in vitro" skin model. A sequential study was performed at 7-d and 14-d cultures (lamina densa absent) and at 28-, 36-, and 56-d cultures (lamina densa present). Expression of beta1, beta4, alpha1, alpha2, alpha3, alpha5, alpha6 integrin subunits and co-localization with collagen IV was studied by regular and laser confocal indirect immunofluorescence microscopy. mRNA expression of alpha2 (IV) and alpha6 (IV) chains was estimated by northern blots. The earliest expression of alpha1 (IV) and alpha2 (IV) collagen chains was noted in 7-d cultures restricted to basal keratinocytes. At 14-d cultures, alpha1 (IV) and alpha2 (IV) chains were noted in basal keratinocytes and as a broad band (10 microm) in the adjacent dermis. At this stage 80% of the alpha2 (IV) mRNA was expressed in the dermis and 20% in the epidermis. At 28-, 36-, and 56-d cultures the alpha1 (IV) and alpha2 (IV) chains were present in a linear distribution at the epidermo-dermal junction and in the upper dermis. The alpha6 (IV) collagen chains were expressed much later at 36-d cultures and the alpha5 (IV) at 56 d, both mostly in a linear distribution but also in the adjacent dermis. Alpha6 (IV) mRNA was demonstrated in the dermis of 36-d cultures. There was co-localization of collagen IV and beta1 integrin subunits in 14-d cultures at the matrix site of keratinocytes. Functional perturbation studies with AIIB2 monoclonal antibody (anti-beta1 subunits) and competitive inhibition with a collagen cyanogen bromide digestion derived fragment (CB3[IV]) that contains the collagen IV ligand for alpha1beta1, alpha2beta1 integrins, altered the pattern of collagen IV deposition.

We describe two Hispanic adolescents with Allgrove syndrome (alacrima, achalasia, and sensorimotor polyneuropathy) in whom we documented cholinergic dysfunction by cardiovascular autonomic tests. Both patients had orthostatic hypotension and decreased heart rate variability

The purpose of this study was to determine whether nidogen, the linkage protein of the basal lamina, is of epidermal or dermal origin. The development of the basal lamina was studied in an in vitro skin model. Preputial fibroblasts seeded onto a nylon mesh attached, proliferated, and developed a rich extracellular matrix (dermal model). Preputial keratinocytes were added to the dermal model to form a keratinocyte dermal model that ultrastructurally resembled in many respects human skin. Ultrastructural analysis revealed early stages of dermal development, including an incomplete basal lamina, aggregates of dermal filamentous material connecting to the lamina densa, bundles of 10-nm microfibrils, formation of premature hemidesmosomes, anchoring filaments, and anchoring fibrils. The cell origin of nidogen was determined in the dermal model and in the epidermal and dermal components of the keratinocyte dermal model. Specific antibodies and a cDNA probe for nidogen were used for immunofluorescence microscopy, Western and Northern blots, and for in situ hybridization studies. Our data show that fibroblasts are the only source of nidogen during early basal lamina formation. Although fibroblasts can synthesize nidogen and deposit it in the dermal matrix, no basal lamina will form unless they are recombined with keratinocytes. This suggests that the epidermis plays a major regulatory role in the production and assembly of nidogen into the basal lamina.