Distribution of Fodrin in the Keratinocyte In Vivo and In Vitro

Distribution of fodrin in the keratinocyte, both in vivo and in vitro, was examined by immunofluorescence microscopy. In the rat epidermis in vivo, fodrin was localized in the cell periphery of the spinous layer of all the skins studied. In only the basal layer of the thick skin, however, fodrin was seen intensely in the cytoplasm. As in vitro keratinocytes, a mouse cell line (Pam 212) cultured in low (0.06 mM) as well as standard (1.87 mM) Ca2+ was examined. In low Ca2+, fodrin was observed throughout the cytoplasm without marked accumulation irrespective of the cell density. The cytoplasmic labeling in low Ca2+ looked filamentous and became aggregated when cells were treated with cytochalasin B; at least some of the aggregates coexisted with those of F-actin. In contrast, fodrin distribution was not affected with colchicine. On the other hand, in standard Ca2+, the protein became concentrated along the cell periphery and less conspicuous in the cytoplasm as the cells reached confluency. When cells were transferred from low to standard Ca2+, the distribution of fodrin changed accordingly within 180min. The present results indicate that fodrin in the keratinocyte is likely to be associated with actin filaments and that it takes two different ways of distribution both in vivo and in vitro. The peripheral and the cytoplasmic labeling of in vivo and in vitro cells are likely to correspond. It may be that fodrin changes its localization according to the cell's proliferative activity

Surface Distribution of Pemphigus Antibody-Binding Substance(s) on Isolated Guinea Pig Epidermal Cells. An Immunoferritin Electron Microscopic Study

The surface distribution of pemphigus antibody-binding substance(s) on trypsinized, isolated guinea pig epidermal cells was studied by means of immunoferritin electron microscopy. Both fresh and 4% paraformaldehyde-fixed cells were incubated with pemphigus serum at 4°C for 15min and then labeled with ferritin-conjugated goat antihuman IgG at 4°C or 37°C for 15 min, respectively. In these cells clusters of ferritin particles were distributed randomly on the surface of nondesmosomal membranes and hemidesmosome. Fresh basal and lower spinous cells showed clusters of 10 to 20 ferritin particles at 4°C while they displayed various sizes of clusters of 10 to more than 50 particles, some of which were interpreted as patches, at 37°C. Fresh upper spinous and granular cells showed clusters of 10 to 20 particles at both 4°C and 37°C. Fixed cells displayed clusters of no more than 20 ferritin particles irrespective of the layers from which they derived and of labeling conditions. Occurrence of patches implied that aggregation of binding substance(s) was induced by pemphigus antibodies. It was suggested that pemphigus antibody-binding substance(s) were movable in the membranes of basal and lower spinous cells while they were less mobile or immobile in upper spinous and granular cells

Lamina Densa Malformation Involved in Histogenesis of Primary Localized Cutaneous Amyloidosis

Skin lesions of lichenoid amyloidosis and macular amyloidosis were immunohistochemically investigated using five monoclonal antibodies against basement membrane zone (BMZ) components. A hemidesmosomal component did not contribute to amyloid deposits, but components of the lamina densa and anchoring fibrils were associated with amyloid deposits in the uppermost dermis. Immunoelectron microscopy revealed that these BMZ components were not only aggregated in the BMZ and dermis, but were also involved in the individual amyloid islets. The lamina densa was disrupted in the interface areas just above the amyloid deposits, where cytoplasm of the basal cells directly faced the aggregate of amyloid filaments. Aggregates of some BMZ components were continuous to the amyloid islets from the lamina densa area. These findings suggest that a lamina densa malformation is involved in amyloid production in the interface of the BMZ, and support the secretion theory rather than the fibrillar body theory of amyloidogenesis in these types of primary localized cutaneous amyloidosis

Human Cutaneous Dendritic Cells Migrate Through Dermal Lymphatic Vessels in a Skin Organ Culture Model

The capacity to migrate from peripheral tissues, where antigen is encountered, to lymphoid organs, where the primary immune response is initiated, is crucial to the immunogenic function of dendritic cells (DC). The skin is a suitable tissue to study migration. DC were observed to gather in distinct nonrandom arrays (“cords”) in the dermis upon culture of murine whole skin explants. It is assumed that cords represent lymphatic vessels. Using a similar organ culture model with human split-thickness skin explants, we investigated migration pathways in human skin.We made the following observations. 1) Spontaneous emigration of Langerhans cells took place in skin cultured for 1–3 d. Nonrandom distribution patterns of strongly major histocompatibility complex class II-expressing DC (cords) occurred in cultured dermis. A variable, yet high (>50%) percentage of these DC coexpressed the Birbeck granule-associated antigen “Lag” Ultrastructurally, the cells corresponded to mature DC. 2) Electron microscopy proved that the dermal structures harboring the accumulations of DC (i.e., cords) were typical lymph vessels. Moreover, markers for blood endothelia (monoclonal antibody PAL-E, Factor VIII-related antigen) and markers for cords (strong major histocompatibility complex class II expression on nonrandomly arranged, hairy-appearing cells) were expressed in a mutually exclusive pattern. 3) On epidermal sheets we failed to detect gross changes in the levels of expression of adhesion molecules (CD44, CD54/ICAM-1, E-cadherin) on keratinocytes in the course of the culture period.The reactivity of a part of the DC in the dermal cords with Birbeck granule-specific monoclonal antibody “Lag” suggests that the migratory population is composed of both epidermal Langerhans cells and dermal DC. We conclude that this organ culture model may prove helpful in resolving pathways and mechanisms of DC migration

An Alanine to Proline Mutation in the 1A Rod Domain of the Keratin 10 Chain in Epidermolytic Hyperkeratosis

We report a mutation in a case of epidermolytic hyperkeratosis that results in a proline for alanine substitution in the residue position 12 of the 1A subdomain of the keratin 10 chain (codon 158). The disease phenotype is consistent with the inappropriate substitution of a proline near the beginning of the rod domain, because it is likely to seriously disrupt the structural organization of coiled-coil molecules within keratin intermediate filaments. Mutations/substitutions in this position have not been reported in any keratin disease. Position 12 is an alanine in all intermediate filament chains, and lies in the outer b heptad position of the coiled-coil. In vitro peptide interference assembly assays revealed that substitutions that alter residue size or charge at this position primarily interfere with keratin filament elongation

Sulfated Dextrans Enhance In Vitro Amplification of Bovine Spongiform Encephalopathy PrPSc and Enable Ultrasensitive Detection of Bovine PrPSc

Prions, infectious agents associated with prion diseases such as Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy (BSE) in cattle, and scrapie in sheep and goats, are primarily comprised of PrP(Sc), a protease-resistant misfolded isoform of the cellular prion protein PrP(C). Protein misfolding cyclic amplification (PMCA) is a highly sensitive technique used to detect minute amounts of scrapie PrP(Sc). However, the current PMCA technique has been unsuccessful in achieving good amplification in cattle. The detailed distribution of PrP(Sc) in BSE-affected cattle therefore remains unknown.We report here that PrP(Sc) derived from BSE-affected cattle can be amplified ultra-efficiently by PMCA in the presence of sulfated dextran compounds. This method is capable of amplifying very small amounts of PrP(Sc) from the saliva, palatine tonsils, lymph nodes, ileocecal region, and muscular tissues of BSE-affected cattle. Individual differences in the distribution of PrP(Sc) in spleen and cerebrospinal fluid samples were observed in terminal-stage animals. However, the presence of PrP(Sc) in blood was not substantiated in the BSE-affected cattle examined.The distribution of PrP(Sc) is not restricted to the nervous system and can spread to peripheral tissues in the terminal disease stage. The finding that PrP(Sc) could be amplified in the saliva of an asymptomatic animal suggests a potential usefulness of this technique for BSE diagnosis. This highly sensitive method also has other practical applications, including safety evaluation or safety assurance of products and byproducts manufactured from bovine source materials

Accumulation of L-type Bovine Prions in Peripheral Nerve Tissues

We recently reported the intraspecies transmission of L-type atypical bovine spongiform encephalopathy (BSE). To clarify the peripheral pathogenesis of L-type BSE, we studied prion distribution in nerve and lymphoid tissues obtained from experimentally challenged cattle. As with classical BSE prions, L-type BSE prions accumulated in central and peripheral nerve tissues

The Localization And Distribution Of Gram-Positive Cocci In Normal Skin And In Lesions Of Acne Vulgaris

The localization of gram-positive cocci in the normal skin and in the lesions of acne vulgaris was investigated using fluorescein-labeled antiserum raised to gram-positive, coagulase-negative cocci. The cocci were found in 10 of 19 specimens from normal facial skin and in 3 of 11 specimens from the normal skin of the rest of the body. The bacteria were found mostly in the openings of follicles, but in 6 of 10 facial skin specimens, they were also present deeply in the lumina of the dilated sebaceous follicles near the sebaceous glands. Cocci were found in 5 of 6 noninflammatory acne comedones. In inflammatory acne they were demonstrated not only in the follicular canals but also sparsely in the infiltrate surrounding the follicles