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Notes: Abstract An important factor in determining species rarity is the geographic distribution of species. Estimates were made of the level of endemism of the flora of two sites in the southern Bahian wet forest zone. Estimates were made for endemism in the Atlantic forest biome and for the much more restricted area of southern Bahia and northern Espi´rito Santo and are derived from analyses of the distributions of the species known from each area. The species checklist for each area is based on identified specimens resulting from intensive collecting in a forest near Serra Grande (40km north of Ilhe´us) and the Una Biological Reserve (40km south of Ilhe´us). Slightly less than half of the species (45.2% at Una and 47.7% at Serra Grande) have widespread distributions and 7.4% at each site are disjunct between the coastal forests and Amazonia. In the Una Reserve, 44.1% of the species are endemic to the coastal forest and 28.1% endemic to southern Bahia and northern Espi´rito Santo. At Serra Grande, 41.6% of the species are endemic to the coastal forest and 26.5% endemic to southern Bahia and northern Espi´rito Santo.

Notes: Senile lentigo (SL) (lentigo senilis, aging spot, liver spot, solar lentigine) is a common component of photoaged skin and women could be affected by these non-aesthetic spots. SL is characterized by hyperpigmented macules which affects mostly face (forehead and cheeks) or scalp (in bald patients), forearms and dorsa of hands after age 50. Hodgson [1] in a comprehensive study among residents of a home for the aged has shown that the number of spots is clearly related to age and associated features of skin senility. Macules vary in color from yellow-light brown to black, the medium hue being dark brown, depending probably on underlying skin (photo)type. The coloration is usually homogeneous but the darker lesions have frequently a mottled appearance. Size and shape are much variable, the commonest lesions being round/oval with smooth outlines, but larger lesions are irregular in shape and measure up to several centimeters in diameter, and small patches may coalesce in larger ones especially on the dorsum of hands. The surface of SL is usually smooth in skin showing moderate senile changes but shows roughness and scaling in older subjects like surrounding skin in the context of diffuse actinic keratoses. Miescher et al. [2] have proposed a classification in three categories of lentigo senilis, namely the small macular type (kleinfleckige Typ, ‘senile ephelides’), the large macular type (grossfleckige Typ) and the leucomelanodermic type (Leukomelanoderm), which however overlaps and may correspond to chronological stages of the disease [3].〈section xml:id="abs1-2"〉〈title type="main"〉MethodsA systematic comparison between lesional versus perilesional skin using immunohistochemistry and electron microscopy was done to detect precursor lesions of SL and to determine whether melanocytes or keratinocytes were first affected in the evolution of lesions.〈section xml:id="abs1-3"〉〈title type="main"〉PatientsThe study was approved by the University Bordeaux Hospitals internal review board. Twelve patients recruited in the out- and inpatient clinics volunteered to participate, after giving informed consent. Seven females and five males aged 58–92 years were included in the study.〈section xml:id="abs1-4"〉〈title type="main"〉Biopsy procedure and sample preparationAfter local anesthesia with lidocaine, a 4 mm punch biopsy was done at the center of a typical lesion on the dorsum of one hand and at a control site situated immediately at the border from the lesion in clinically non-pigmented skin. Half of the biopsy was fixed in 4% formaldehyde and the rest was fixed in a 6.25% glutaraldehyde, 0.1 M cacodylate pH 7.4 solution.〈section xml:id="abs1-5"〉〈title type="main"〉Histopathologic studiesBiopsies were embedded in paraffin, cut in 5 μm sections and stained using hematoxylin–eosin and Fontana-Masson techniques to assess the general morphology of the epidermis and melanin distribution. An anti-MelanA antibody (Dako, Trappes, France) was used to stain melanocytes. As secondary antibody, we used the Envision Kit HRP+ (Dako, Trappes, France), which was revealed with aminoethylcarbamide (AEC). AEC was chosen because red staining facilitates the count of melanocytes in the pigmented basal layer. To analyze the dermis, we stained sections with the orcein stain to visualize elastic fiber, and with Masson's trichrome to visualize collagen fibers. An anti alpha smooth muscle actin monoclonal antibody (clone 1A4, Sigma, St Louis, MO, U.S.A.) was used to detect myofibroblasts. The secondary horseradish peroxidase (HRP) antibody was revealed with diaminobenzidine (DAB).〈section xml:id="abs1-6"〉〈title type="main"〉Electron microscopyBiopsies were embedded in Epon and cut in 0.6 μm sections. Sections were examined after post fixation in osmium tetroxide with a CM 10 (Philips, FEI Company, Eindhoven, The Netherlands) electron microscope.〈section xml:id="abs1-7"〉〈title type="main"〉Results〈section xml:id="abs1-8"〉〈title type="main"〉Epidermal morphology of lesional versus perilesional skin〈section xml:id="abs1-9"〉〈title type="main"〉Conventional microscopyAn accumulation of melanin was noted in epidermal basal layers together with an elongation of rete ridges. This phenomenon increased with the presumed age of the lesion, determined by the age of the patient, and SL could be classified histopathologically in three grades of severity (〈link href="#f1-13"〉Fig. 1). Observation of perilesional skin revealed the presence of cluster of cells accumulating pigment (〈link href="#f1-13"〉Fig. 1). When comparing lesional skin with perilesional skin, a significant increase was detected in the number of melanocytes by mm of stratum corneum with anti-melanA antibody. Melanocytes did not accumulate in the ridges, a finding which was confirmed by electron microscopy.〈figure xml:id="f1-13"〉1〈mediaResource alt="image" href="urn:x-wiley:01425463:ICS254_13_13:ICS_254_f1-13"/〉Histologic evolution of senile lentigo. Fontanna-Masson staining (A, B, D–F) and anti-melan-A staining (C). (A–C) Perilesional skin. Note in (B) and (C), clusters of keratinocytes retaining pigment capping the nucleus (arrow) and in C melanocyte (arrow head). (D–F) Lesional skin, (D) stage 1, (E) stage 2, and (F) stage 3 corresponding to a progressive accumulation of pigment at the tip of epidermal rete ridges of lesser thickness and increased length (scale bar: 10 μm).〈section xml:id="abs1-10"〉〈title type="main"〉Electron microscopyIn melanocytes, we observed melanosomes of normal size at all maturation stages. In lesional skin many melanocytes showed an activated phenotype, i.e. numerous mitochondria in the cytoplasm and melanosomes in migration within dendrites. In two perilesional skin samples we observed an increase number of mitochondria in melanocytes, whereas most other melanocytes looked poorly active. Melanocytes ‘hanging down’ in the dermis were occasionally noted in lesional and perilesional skin. A massive capping formed by melanosomial complexes, some of which of unusual large size, was found in basal keratinocytes (〈link href="#f2-13"〉Fig. 2).〈figure xml:id="f2-13"〉2〈mediaResource alt="image" href="urn:x-wiley:01425463:ICS254_13_13:ICS_254_f2-13"/〉Observation of lesional skin by electron microcopy. Melanosomes are present in dendrites. Some are unusually large and suggest complexation of single melanosomes (arrows), whereas in perinuclear cytoplasm, melenosomes are normal size. In Keratinocytes, melanosomial complexes form a massive capping of nuclei (D). Numerous mitochondria (arrows heads) can be seen in melanocytes. M, melanocyte; K, keratinocyte; and D, dendrite (scale bar: 1 μm).〈section xml:id="abs1-11"〉〈title type="main"〉Dermal morphology of lesional versus perilesional skin〈section xml:id="abs1-12"〉〈title type="main"〉Conventional microscopyAfter staining with Orcein, Masson's trichrome or AML, there was no difference detected between lesional and perilesional skin, which showed age-related elastotic changes (data not shown). In lesional and perilesional skin, beneath a superficial dermis showing fibrotic changes, a thick band of elastotic deposits was noted in the reticular dermis corresponding to closely packed and randomly orientated elastic fibers on hematoxylin–eosin and orcein stains.Immunohistochemistry using the anti alpha smooth muscle actin antibody showed in the dermis a low number of interstitial stained cell corresponding to myofibroblasts, without quantitative differences between lesional and perilesional skin.〈section xml:id="abs1-13"〉〈title type="main"〉Electron microscopyElectron microscopic examination of areas of solar elastosis in both cases showed aggregates of thick elastotic fibers. These elastotic fibers were composed in their thin periphery by electron dense tubular microfibrils and mainly in the center by elastin characterized by a fine granular matrix of low/medium electron density containing electron dense inclusions.〈section xml:id="abs1-14"〉〈title type="main"〉DiscussionSL responds to destruc

Notes: Background  In vitiligo, melanocytes are gradually lost in depigmented macules of the skin. The disappearance of melanocytes has, however, not been clearly observed and consequently the aetiology of the disease (autoimmune, neural, cytotoxic) is still elusive. The starting point of vitiligo macules is frequently determined by local conditions such as wounds and excoriations, but may also follow minor traumas such as pressure or repeated friction. This prominent feature is often neglected.Objectives  To clarify the biological consequences of repeated friction on the attachment and survival of melanocytes in non lesional vitiligo skin.Methods  Light reproducible skin friction was performed for 4 min on the volar forearm of 18 patients with extensive vitiligo and five controls with normal healthy skin. Biopsies from the test area and control skin were taken at 1, 4, 24 and 48 h following friction. Serial sections were examined with standard light microscopy, transmission electron microscopy, histochemistry and immunohistochemistry (dihydroxyphenylalanine, HMB-45, E-cadherin and an early apoptosis marker, M30 cytoDEATH antibody).Results  The observation of sections at 1 and 48 h after friction on vitiligo skin and at all time points in controls revealed no changes. In contrast, in vitiligo skin at 4 and 24 h after friction, several melanocytes had undergone detachment and were found in various suprabasal positions, including the stratum spinosum, granular layer, and within and outside the stratum corneum.Conclusions  Detachment and transepidermal elimination of melanocytes following minor mechanical trauma in non lesional vitiligo skin is probably the cause of depigmentation occurring in the isomorphic response (Koebner phenomenon). We propose that transepidermal elimination of melanocytes in vitiligo should be regarded as a possible mechanism of chronic loss of pigment cells, perhaps previously damaged by another process.