Expression profile of cornified envelope structural proteins and keratinocyte differentiation-regulating proteins during skin barrier repair.

BACKGROUND: Recent studies have emphasized the importance of heritable and acquired skin barrier abnormalities in common inflammatory diseases such as psoriasis and atopic dermatitis (AD). To date, no comprehensive studies on the effect of experimental barrier disruption on cornified envelope protein expression have been performed. OBJECTIVES: To analyse the effect of experimental skin barrier disruption on the expression of cornified envelope structural proteins and keratinocyte differentiation-regulating proteins. METHODS: We examined mRNA (day 1, 3 and 7) and protein (day 1, 2, 4 and 9) expression levels of structural proteins and regulatory molecules after sodium dodecyl sulphate (SDS) application on normal skin, and tape stripping of uninvolved epidermis of patients with psoriasis and AD and healthy controls. RESULTS: Upon tape stripping, several structural molecules were significantly downregulated (at the mRNA level as well as the protein level), including LCE5A, LCE2B, FLG, FLG2 and LOR, whereas others were upregulated: IVL, SPRR1, SPRR2, HRNR and most notably LCE3A. The epidermal crosslinking enzymes TGM1, TGM3 and TGM5 were all upregulated, whereas proteases involved in the desquamation process (CTSV, KLK5 and KLK7) were downregulated or unaffected. Most results were similar in SDS-instigated irritant contact dermatitis. There was no significant difference in response between normal epidermis and nonlesional skin of patients with psoriasis and AD. CONCLUSIONS: Skin barrier disruption induces a temporary barrier repair response composed of increased expression of several cornification-related proteins, and decreased expression of some structural and desquamation-related proteins.

Expression of apogens and engulfens during programmed cell death in the nervous system of the chick embryo.

Two categories of cell death related to antigens, apogens and engulfens, have been reported to be expressed by apoptotic cells and the cells involved in their engulfment in the immune system, and in mesenchymal tissue in the limb of the chick embryo (ROTTELLO et al., 1994). To determine whether these antigens are also expressed during the process of neuronal death, the distribution of immunoreactivity to both anti-apogen and anti-engulfen antibodies was examined in the spinal cord and the dorsal root ganglia of the chick embryo. Anti-apogen antibodies labeled a sub-population of the profiles of dying cells in regions where cell death was occurring. The extent of labeling by anti-apogens varied from 3% to 70% of the total number of dying profiles depending on the specific antibody used and the neuronal region examined. Immunoreactive labeling by the anti-engulfen antibodies mainly involved large cells that contained debris of dead cells. These results indicate that at least some dying neuronal cells express common antigens that are shared by dying mesenchymal cells during programmed cell death, and that phagocytotic cells of the immune system are involved in the engulfment of neuronal cells that have undergone programmed cell death.

A novel type of programmed neuronal death in the cervical spinal cord of the chick embryo.

We examined the massive early cell death that occurs in the ventral horn of the cervical spinal cord of the chick embryo between embryonic days 4 and 5 (E4 and E5). Studies with immunohistochemical, in situ hybridization, and retrograde-tracing methods revealed that many dying cells express Islet proteins and Lim-3 mRNA (motoneuron markers) and send their axons to the somatic region of the embryo before cell death. Together, these data strongly suggest that the dying cells are somatic motoneurons. Cervical motoneurons die by apoptosis and can be rescued by treatment with cycloheximide and actinomycin D. Counts by motoneuron numbers between E3.5 and E10 revealed that, in addition to cell death between E4 and E5, motoneuron death also occur between E6 and E10 in the cervical cord. Studies with [3H]thymidine autoradiography and morphological techniques revealed that in the early cell-death phase (E4-E5), genesis of motoneurons, axonal elongation, and innervation of muscles is still ongoing. However, studies with [3H]thymidine autoradiography also revealed that the cells dying between E4 and E5 become postmitotic before E3.5. Increased size of peripheral targets, treatment with neuromuscular blockade, and treatment with partially purified muscle or brain extracts and defined neurotropic agents, such as NGF, BDNF, neurotrophin-3, CNTF, bFGF, PDGF, S100-beta, activin, cholinergic differentiation factor/leukemia inhibitory factor, bone morphogenetic protein-2, IGF-I, interleukin-6, and TGF-beta 1, were all ineffective in rescuing motoneurons dying between E4 and E5. By contrast, motoneurons that undergo programmed cell death at later stages (E6-E10) in the cervical cord are target-dependent and respond to activity blockade and trophic factors. Experimental approaches revealed that early cell death also occurs in a notochord-induced ectopic supernumerary motoneuron column in the cervical cord. Transplantation of the cervical neural tube to other segmental regions failed to alter the early death of motoneurons, whereas transplantation of other segments to the cervical region failed to induce early motoneuron death. These results suggest that the mechanisms that regulate motoneuron death in the cervical spinal cord between E4 and E5 are independent of interactions with targets. Rather, this novel type of cell death seems to be determined by signals that either are cell-autonomous or are derived from other cells within the cervical neural tube.

Nestin-EGFP transgenic mice: visualization of the self-renewal and multipotency of CNS stem cells.

We generated transgenic mice carrying enhanced green fluorescent protein (EGFP) under the control of the nestin second-intronic enhancer (E/nestin:EGFP). Flow cytometry followed by in vitro assays revealed that in situ EGFP expression in the embryonic brain correlated with the mitotic index, the cogeneration of both neurons and glia, and the frequency of neurosphere formation in vitro. High-level EGFP expressors derived from embryos included a distinct subpopulation of cells that were self-renewable and multipotent, criteria that define neural stem cells (NSCs). Such cells were largely absent among lower-level or non-EGFP expressors, thereby permitting us to enrich for NSCs using EGFP expression level. In adults, although E/nestin:EGFP-positive cells included the NSC population, the frequency of neurosphere formation did not correlate directly with the level of EGFP expression. However, moderately EGFP-expressing cells in adults gained EGFP intensity when they formed neurospheres, suggesting embryonic and adult NSCs exist in different microenvironments in vivo.