Melanocyte Density Research Articles

Compound blue nevus: a reappraisal of the concept in the genomic era.

We report a series of 21 compound blue nevi, a rare variant in the vast clinical and morphological spectrum of blue melanocytic proliferations. Clinically, they presented in young adults, with a slight female predominance. One-third were located on the dorsum of the foot. Morphologically, all cases displayed large dendritic melanocytes restricted to the deep layers of the epidermis. The compound component was central and evenly distributed. Melanocytic density ranged from scarce isolated cells to a confluent lentiginous architecture. In 12 of the 21 cases, junctional nests of small, bland, weakly pigmented melanocytes were associated. These nests became confluent in the most cellular cases. In all cases, a dermal component was immediately present underneath, mainly of cellular blue nevus-type. All cases were genetically confirmed to harbor either a GNAQ or GNA11 hotspot mutation. This study expands the morphological spectrum of blue nevi that should not be restricted to a strictly intradermal melanocytic proliferation.

Histochemical study of the distribution of epidermal melanoblasts and melanocytes in Asian human skin.

The classical study revealed the melanocyte density in the epidermis of many skin sites of humans. However, these data were obtained by counting melanocytes using dihydroxyphenylalanine (dopa)-treated epidermal sheets. Since rete ridges are developed well in human epidermis, there is a concern about the accuracy of these data. The accurate counting of the melanocyte density in the epidermis including the rete ridges should be performed using histological sections after the dopa treatment. Moreover, it is not known well how many melanoblasts are present in Asian epidermis. The aim of this study was to count the accurate number of melanocytes and melanoblasts. Normal skin sites of 9- to 77-year-old patients were fixed with buffered formalin and processed to the dopa and combined dopa-premelanin reactions. The numbers of cells positive to the dopa reaction (melanocytes) and to the combined dopa-premelanin reaction (melanoblasts and melanocytes) were scored. In the skin of arms, legs, back, and belly, similar density (approximately 110-120cells/0.1mm2 ) of melanocytes was observed, whereas in the skin of scalp, melanocyte density was much lower (approximately 70cells/0.1mm2 ). By contrast, the melanoblast density did not differ between skin sites (approximately 100cells/0.1mm2 ). These results suggest that the melanocyte density does not differ between skin sites except the scalp skin, and a certain number of melanoblasts are present in each skin site of Asian. Melanoblasts seem to be required for producing new melanocytes required to maintain epidermal homeostasis.

MART-1-labeled melanocyte density and distribution in actinic keratosis and squamous cell cancer in situ: Pagetoid melanocytes are a potential source of misdiagnosis as melanoma in situ.

Actinic keratosis (AK) and squamous cell carcinoma in-situ (SCCIS) within or near melanoma in situ (MIS) can complicate diagnosis due to overlapping clinical and microscopic features. This study aimed to describe basilar melanocyte density and pagetoid spread in AK and SCCIS for improved diagnostic accuracy. A total of 22 AK and 22 SCCIS biopsies containing a margin of uninvolved epidermis were immunostained with MART-1 (melanoma antigen recognized by T-cells 1). The basilar melanocyte:keratinocyte ratio and the number and distribution of pagetoid melanocytes were compared in AK, SCCIS, and uninvolved epidermis. An in-vitro human skin model was created to assess the impact of keratinocyte atypia on melanocyte distribution. The median basilar melanocyte:keratinocyte ratio in SCCIS (1:11.49) was lower than in uninvolved epidermis (1:5.59, P = 0.0011), and the ratio in AK (1:6.94) was similar to uninvolved epidermis (P = 0.987). Pagetoid melanocytes were absent in perilesional skin but common in AK (21/22, P < 0.0001) and SCCIS (22/22, P < 0.0001). Pagetoid melanocytes at or above the mid-spinous layer were more common in SCCIS (21/22) vs AK (7/22, P < 0.0001). Pagetoid melanocytes were present in the in-vitro skin model made with neoplastic but not normal keratinocytes. Pagetoid melanocytes in AK and SCCIS should be interpreted with caution to avoid overdiagnosis of MIS.

Evaluation of large clinically atypical vulvar pigmentation with RCM: atypical melanosis or early melanoma?

Vulvar melanosis can occasionally be clinically challenging by mimicking an early melanoma. To report our experience of initial evaluation and follow-up in this peculiar subset of vulvar melanosis using reflectance confocal microscopy (RCM). We retrospectively evaluated 18 consecutive cases referred for atypical vulvar pigmentation or for which melanoma was considered and that underwent both RCM examination and histopathological assessment. In 13 cases with available dermoscopic pictures, RCM classification was compared to dermoscopic diagnosis, and in all cases, the density of melanocytes was evaluated on biopsies using MelanA immunostaining. Among the 18 atypical pigmented lesions, 17 vulvar melanosis and one melanoma were histologically determined. RCM concluded a benign vulvar melanosis in 10 of 17 cases, whereas dermoscopy did so in three of 12 cases. RCM identified the only early malignant lentiginous melanoma. In several cases of vulvar melanosis, RCM could identify foci of melanocytic hyperplasia in an otherwise benign pattern. In this clinically and dermoscopically challenging subset of vulvar pigmentations, RCM appears relevant for initial extensive evaluation, especially to target initial biopsy sampling, and to perform non-invasive monitoring of foci of melanocytic hyperplasia.

Evaluation of MITF, SOX10, MART-1, and R21 Immunostaining for the Diagnosis of Residual Melanoma In Situ on Chronically Sun-Damaged Skin.

Melanocytic immunostains can assist in margin evaluation of melanoma in situ (MIS) excisions; however, their accuracy and reliability relative to hematoxylin & eosin (H&E) is yet to be determined. The objective of this study was to evaluate the sensitivity, specificity, and concordance of 4 melanocyte-specific immunostains for diagnosing MIS occurring on chronically sun-damaged skin. Serial permanent sections from representative areas of negative margin and residual tumor were stained using H&E, MITF, MART-1, SOX10, and R21 and examined in a blinded fashion. The study set included 100 digital microscopy images from 10 cases of MIS excisions from the face. Two board-certified dermatopathologists, 4 fellowship-trained Mohs surgeons, 2 Mohs fellows, and 2 dermatology residents independently reviewed the 100 images. The average melanocyte density was 11 versus 28 melanocytes per 0.5 mm for chronically sun-damaged skin versus residual MIS on H&E, respectively. Statistically significantly higher melanocyte densities were observed using MITF, MART-1, and SOX10 on negative margins. The sensitivity and interobserver concordance was highest using MITF and SOX10. The intraobserver agreement on 4 duplicate images was 85%. In conclusion, the nuclear immunostains (MITF and SOX10) show the most promise for improving the diagnosis of MIS in chronically sun-damaged skin.

Melanoblasts but not melanocytes decrease in number in human epidermis of idiopathic guttate hypomelanosis

Idiopathic guttate hypomelanosis (IGH) is a common disease that appears mainly in trunk, arms, and legs. IGH is a benign leukoderma characterized by multiple, hypo- or depigmented discrete macules. Although melanocytes have been reported to decrease in number in the IGH macules, changes in melanoblast number are not known. Moreover, satisfactory therapies for this disease have not been established. In this study, we investigated the presence of melanoblasts in the IGH macules, and tried to treat the IGH macules by mini-punch grafting using a machine (mMG) in combination with narrow-band ultraviolet B (NB-UVB) therapy. Mini-punch biopsies of the lesional skin of five patients (11–72-year-old men and woman) were fixed and processed to dopa and combined dopa-premelanin reactions to detect melanocytes and melanoblasts. Although similar melanocyte density was observed in the lesional epidermis compared with the normal epidermis, melanoblast density was dramatically decreased. Moreover, epidermal melanin accumulation was greatly reduced in the lesional skin. For the treatment of the IGH macules, numerous mini-punch biopsies were removed from both recipient lesional skin and donor normal scalp skin, and then the donor biopsies were placed into the holes of the lesional skin. After 1 week, we started to expose NB-UVB to the lesional skin and continued the exposures. Excellent color-matched repigmentation was soon obtained. These results suggest that melanoblast proliferation and/or differentiation in addition to the melanosome transfer are defected in IGH, and the mMG and NB-UVB can support the excellent color-matched repigmentation.

Melanocytes Pattern in the Normal Nail, With Special Reference to Nail Bed Melanocytes.

Previous studies of the density of melanocytes in the normal nail bed have had conflicting results. This is unfortunate because knowing the normal values might help the difficult distinction between a benign subungual melanotic macule and an early melanoma in situ. Five specimens of normal nail unit were analyzed. On hematoxylin and eosin-stained sections the melanocytes were undetectable. We defined the melanocyte count (MC) as the number of melanocytes per 1-mm stretch of nail epithelium. The mean MC for nail matrix was 6.86 with a range of 4-14. The melanocytes were irregularly scattered in the basal and suprabasilar layer of the matrix epithelium. Abundant and uneven cytoplasmic dendrites were focally observed in the matrix. The MC for the nail bed ranged from 0 to 5 with a mean of 0.43. The melanocytes were restricted to the basal layer with thin cytoplasmic dendrites. Two cases showed a complete absence of melanocytes in the nail bed. In the ventral portion of the proximal nail fold, called the eponychium, the MC ranged between 0 and 5/mm with a mean of 2.27/mm. In conclusion, we discovered foci in normal nail beds, in which the melanocytic density can be relatively high and reach the level seen in the matrix. HMB45 is more sensitive than Microphtalmia-associated transcription factor (MITF) for the evaluation of intraepithelial melanocytic density of the nail unit. If MITF is used alone in the nail bed, its weak sensitivity may result in a false-negative interpretation and may be wrongly reassuring in the evaluation of early melanomas. On hematoxylin and eosin sections, basal and suprabasal nail keratinocytes are sometimes crowded, showing oval or elongated dark-staining nucleus and a clear cytoplasm and mimics a melanocytic proliferation. On HMB45 or Melan A staining, the morphology and the distribution of the dendrites of matrical melanocytes can mimic the dendritic pattern usually described in acral melanoma. Therefore, the interpretation of nail melanocytic atypia must be prudent.

Comparison of Melanocyte Density Counts in Topical Imiquimod-Treated Skin Surrounding Lentigo Maligna vs Control Biopsy Specimens

This study examines melanocyte density counts in imiquimod-treated lentigo maligna and negative control biopsy specimens to determine if there was a measurable difference in melanocyte density.

Reference values for skin microanatomy: A systematic review and meta-analysis of ex vivo studies

Few studies have characterized reference values of normal human skin microanatomy parameters. To quantify histologic measurements of epidermal thickness, melanocyte density, hair follicle density, and eccrine gland density as a function of age and anatomic site. We searched the PubMed, Embase, Web of Science, and Cochrane databases for articles published through May 25, 2017. Two reviewers independently screened 2016 articles; 327 relevant articles and 151 additional articles found via forward or reference citations underwent full-text review by 1 of 4 reviewers for relevance, data extraction, and critical appraisal. Weighted averages, meta-analysis, and meta-regression were used in statistical analysis. A total of 56 articles were included; when all anatomic locations were used, the overall estimates for epidermal thickness, melanocyte density, hair follicle density, and eccrine gland density were 99.75μm (95% confidence interval [CI], 83.25-116.25), 955.05cells/mm2 (95% CI. 880.89-1029.21), 1.40 hairs/mm2 (95% CI. 0.91-1.89), and 1.28 glands/mm2 (95% CI. 0.91-1.64), respectively. There was significant data heterogeneity across studies, possibly because of differences in histological techniques and absence of standardized microanatomy definitions. We established summary estimates for normal human skin microanatomy parameters.

Histopathologic and Immunohistochemical Correlates of Confocal Descriptors in Pigmented Facial Macules on Photodamaged Skin

Pigmented facial macules on photodamaged skin are a clinical, dermoscopic, and histopathologic challenge. To clinically and dermoscopically characterize, by means of reflectance confocal microscopy (RCM), ambiguous pigmented facial macules and establish a correlation between RCM, histopathologic, and immunohistochemical findings. A prospective study of ambiguous pigmented facial macules on photodamaged skin was conducted in a tertiary referral center for dermatology between January 1, 2009, and December 31, 2015. Sixty-one patients with 63 ambiguous pigmented facial macules and 12 control photodamaged facial areas were included in the study. Melanocyte density in 1-mm basal layers was determined in skin biopsy specimens from all lesions stained with hematoxylin-eosin and immunohistochemical markers (melan-A, microphthalmia-associated transcription factor, and SRY-related HMG-box gene 10). Dermoscopic, RCM images, and histopathologic preparations were systematically evaluated for the presence of lentigo maligna (LM) criteria. Confocal evaluation was blinded to clinical and dermoscopic diagnosis. Sensitivity and specificity of RCM for LM diagnosis and κ value to establish correlations between dermoscopy, RCM, and histopathology were performed. Sensitivity and specificity of RCM for LM diagnosis. Of the 61 patients included in the study, 31 (51%) were women; mean (SD) age was 71.8 (13.1) years. Twenty-four of the 63 (38%) lesions were diagnosed as LM or LM melanoma (LMM) and 39 (62%) as benign pigmented lesions. Reflectance confocal microscopy enhanced the diagnosis of pigmented facial macules with 91.7% sensitivity and 86.8% specificity. Multivariate analysis showed 2 dermoscopic and 2 confocal features associated with LM or LMM: (1) asymmetric follicular pigmentation and targetlike structures, and (2) round, large pagetoid cells and follicular localization of atypical cells, respectively. Continuous proliferation of atypical melanocytes was found in 21 (88%) LM or LMM and in 3 (77%) benign lesions. Asymmetric pigmented follicular openings by dermoscopy correlated with follicular localization of pagetoid cells by RCM (κ = 0.499, P < .001). The presence of 3 or more atypical cells at the dermal-epidermal junction (DEJ) by RCM correlated with hyperplasia of melanocytes in hematoxylin-eosin sections (κ = 0.422, P < .001). Reflectance confocal microscopy improves LM diagnosis in photodamaged skin with good histopathologic correlation although false-positive and false-negative cases exist. False-positives obtained with RCM in photodamaged skin are due to the presence of basal melanocyte hyperplasia and intraepidermal Langerhans cells. Histopathologic features of these lesions sometimes are not enough for a definite diagnosis and immunohistochemical studies may be required.

Immunohistochemical evaluation of epidermal proliferation, differentiation and melanocytic density in symmetrical acrokeratoderma.

Symmetrical acrokeratoderma (SAK) is characterized by brown to black hyperkeratotic patches on acral regions. Although epidermal hyperkeratosis and acanthosis are consistent pathological changes, the nature of epidermal hyperplasia is unknown. To evaluate epidermal proliferation and differentiation and melanocytic density in skin lesions of SAK. Expression of keratin 10 (K10), K14, K16, involucrin, filaggrin, Ki-67, and Melan-A was detected by immunohistochemistry in eight patients with SAK, seven patients with ichthyosis vulgaris (IV) and six healthy controls (HCs). Expression of K14, K16, involucrin and filaggrin was upregulated in patients with SAK compared with patients with IV and the HCs (P < 0.01-0.05), but K10 expression was similar for the three groups (P > 0.05). Numbers of Ki-67+ and Melan-A+ cells were higher in patients with SAK than in patients with IV and the HCs (P < 0.05). These results demonstrate that excessive keratinocyte proliferation and abnormal differentiation contribute to epidermal hyperplasia, while melanocytic proliferation is responsible for the pigmented lesions in SAK.

Testing of viable human skin cell dilution cultures as an approach to validating microsampling.

Skin biopsies are a valuable technique in the diagnosis of cutaneous inflammatory and neoplastic conditions. We were interested to test the minimal size or equivalent volume by dilution of proteolytically disassociated skin tissue required to allow the isolation and propagation of cutaneous cells, for freezing, storage and biochemical analysis. It was possible to propagate with 100% efficiency fibroblast and melanocytic cells from a 0.1 to 0.5mm3 equivalent neonatal foreskin sample using a combination of DispaseII and CollagenaseIV. The smallest tissue dilution that allowed melanocytic cell culture was 0.01mm3, which equated to approximately 16 cells based on the average skin density of melanocytes. However, passaging of cells to create frozen stocks was achieved routinely only from 1mm3 skin, equating to 1560 cells. Tissue-specific antigen expression of these cultures was tested by western blot of total protein extracts. There was no pigmentation antigen expression in fibroblast cultures; however, smooth muscle actin protein expression was high in fibroblast but absent from melanocytic cell strains. Melanocytic cells expressed pigmentation antigens and E-cadherin, but these were not detected in fibroblasts. Moreover, maturation of these melanocytic cells resulted in a decrease of Dopachrome Tautomerase antigen expression and induction of Tyrosinase protein consistent with the differentiation potential seen in cell cultures derived routinely from large sections of skin tissue.

Immuno-Histochemical and Quantitative Study of Melanocytes and Melanin Granules in Oral Epithelial Dysplasia.

Oral Epithelial dysplasia (OED) is a potentially malignant disorder that is characterized by the presence of architectural and cytological changes. One of the prime factors responsible for the development of these lesions is the usage of tobacco. A variety of factors provide protective mechanism in order to prevent the effects of chemotoxic agents including tobacco products of which, melanin pigmentation is one of the vital elements. Role of melanocytes in progression of OED has remained unclear, so the present study was done to evaluate density of melanocyte and melanin granules in different grades of epithelial dysplasia and to correlate both findings with different grades of epithelial dysplasia. The study included 60 OED cases, of which three histopathogical sections were prepared from each block. The sections were stained with Hematoxylin and Eosin, Masson Fontana and Human Melanoma Black (HMB-45), an immunohistochemical stain. Quantification of melanin granules was evaluated under 40X magnification using arbitrary scale with micrometer square as, 0= Absence of melanin granules, 1= Rare and scattered melanin granules, 2= Dense but not aggregated melanin granules, 3= Dense and aggregated melanin granules. Density of melanocytes was evaluated under 10X magnification. Five consecutive fields were evaluated for melanocytes and melanin granules starting from the field of highest density. There was an insignificant increase in number of melanocytes and melanin granules in mild and moderate dysplasia compared to normal but significant reduction was observed in severe dysplasia. The decrease in number of melanocytes and melanin granules was proportional to severity of epithelial dysplasia. This could be due to chronic irritation by chemical products leading to death of melanocytes.

Acquired blue nevus of the nail bed

Blue nevi are benign proliferations of melaninproducingdendritic melanocytes located in thedermis. These nevi tend to occur mostly on the skin,predominantly on the head and neck, dorsal aspectsof the distal extremities, and the sacral area, butcan also occasionally appear on mucosal surfaces.Blue nevi of the nail apparatus are uncommon. Themajority originate in the nail matrix where there is ahigher density of melanocytes. Herein we report ona 47-year-old man with a rare common blue nevus ofthe nail bed, an area with low melanocyte density. Athorough review of the English language literaturefound no documented cases of acquired blue nevioriginating in the nailbed of the toe.

Effect of Uveal Melanocytes on Choroidal Morphology in Rhesus Macaques and Humans on Enhanced-Depth Imaging Optical Coherence Tomography

PurposeTo compare cross-sectional choroidal morphology in rhesus macaque and human eyes using enhanced-depth imaging optical coherence tomography (EDI-OCT) and histologic analysis.MethodsEnhanced-depth imaging–OCT images from 25 rhesus macaque and 30 human eyes were evaluated for choriocapillaris and choroidal–scleral junction (CSJ) visibility in the central macula based on OCT reflectivity profiles, and compared with age-matched histologic sections. Semiautomated segmentation of the choriocapillaris and CSJ was used to measure choriocapillary and choroidal thickness, respectively. Multivariate regression was performed to determine the association of age, refractive error, and race with choriocapillaris and CSJ visibility.ResultsRhesus macaques exhibit a distinct hyporeflective choriocapillaris layer on EDI-OCT, while the CSJ cannot be visualized. In contrast, humans show variable reflectivities of the choriocapillaris, with a distinct CSJ seen in many subjects. Histologic sections demonstrate large, darkly pigmented melanocytes that are densely distributed in the macaque choroid, while melanocytes in humans are smaller, less pigmented, and variably distributed. Optical coherence tomography reflectivity patterns of the choroid appear to correspond to the density, size, and pigmentation of choroidal melanocytes. Mean choriocapillary thickness was similar between the two species (19.3 ± 3.4 vs. 19.8 ± 3.4 μm, P = 0.615), but choroidal thickness may be lower in macaques than in humans (191.2 ± 43.0 vs. 266.8 ± 78.0 μm, P < 0.001). Racial differences in uveal pigmentation also appear to affect the visibility of the choriocapillaris and CSJ on EDI-OCT.ConclusionsPigmented uveal melanocytes affect choroidal morphology on EDI-OCT in rhesus macaque and human eyes. Racial differences in pigmentation may affect choriocapillaris and CSJ visibility, and may influence the accuracy of choroidal thickness measurements.

Lentigo maligna - anatomic location as a potential risk factor for recurrences after non-surgical treatment.

BackgroundA higher incidence of lentigo maligna (LM) recurrences on the nose was previously observed in our cohort after non‐surgical treatment.ObjectivesTo determine histological parameters that might be related to the previously observed higher incidence of LM recurrences on the nose after non‐surgical treatment.MethodsWe randomly selected 22 surgical specimens of LM on the nose and 22 on the cheek. Histopathological analysis was performed on haematoxylin and eosin stained and microphthalmia transcription factor immunohistochemically stained slides. The number of pilosebaceous units (PSU) per mm, maximum depth of atypical melanocytes along the skin appendages and maximum depth of the PSU itself were determined.ResultsThe nose had a significantly higher density of PSU than the cheek. The atypical melanocytes extended deeper along the PSU on the nose with a mean (SD) depth of 1.29 mm (0.48) vs. a mean depth of 0.72 mm (0.30) on the cheek (P < 0.001). The maximum depth of the PSU on the nose was greater than on the cheek, mean (SD) depth of 2.28 mm (0.41) vs. 1.65 mm (0.82) (P = 0.003).ConclusionsThe higher recurrence risk of LM on the nose after non‐surgical treatment that we previously observed in our cohort is most likely based on a higher density of atypical melanocytes and also their deeper extension into the follicles. These results shed more light on our previous findings and learn that anatomical location is relevant for the risk of recurrence of LM after non‐surgical treatment.

The Influence of Pigment Transfer on the Risk of Developing Melanoma: The Significance of the Melanocyte ‘Amputation Cycle’

It has been shown that cancer incidence is not only a function of the size of the population at risk but is strongly associated with the turnover rate of the tissue concerned. There is a strong negative correlation between melanoma incidence and the degree of skin pigmentation, and yet the melanocyte density is the same for all races. The proposal advanced in this communication is that the probability of undergoing malignant change is critically dependent on the melanocyte turnover and that this is regulated by the pigmentation process. In melanocytes, the division rate is influenced by the process of pigment donation, probably by a mechanism whereby the continual cytoplasmic loss due to cytocrine transfer of melanosomes (termed the ‚Amputation Cycle‚) inhibits replication. Consequently the turnover of melanocyte stem cells in heavily pigmented epidermis will be diminished, and this is held to account for the strong negative correlation between the degree of skin pigmentation and melanoma incidence.

Assessment of Melanocyte Density in Anorectal Mucosa for the Evaluation of Surgical Margins in Primary Anorectal Melanoma.

Accurate histopathologic evaluation of surgical resection margins for anorectal melanoma (AM) is diagnostically challenging but essential to clinical management. We studied intraepithelial melanocyte density and growth pattern in anorectal mucosa and BRAF V600E mutation status in AM compared to controls. Histomorphology and melanocytic immunostains, microphthalmia transcription factor (MITF) and human melanoma black 45 (HMB45), were evaluated. Utility of VE1 immunostaining for determination of BRAF V600E mutation status was studied. Immunostains aid in the distinction between "trailing" melanoma in situ (MIS) and benign melanocyte hyperplasia (BMH), by facilitating assessment of melanocyte density, and evaluation of nuclear atypia and growth pattern. While respective melanocyte densities overlapped, "trailing" MIS could be distinguished by melanocyte nuclear atypia and near confluent growth, compared to the banal cytology and scattered growth of BMH. In the histopathologic assessment of AM resections, MITF and HMB45 immunostains aid in distinguishing between "trailing" MIS and BMH, by highlighting melanocyte density, nuclear atypia, and growth pattern, with the latter two being reliable features. VE1 showed nonspecific immunopositivity in anorectal glandular epithelium, a potential diagnostic pitfall when assessing BRAF mutation status.

Expression of zTOR-associated microRNAs in zebrafish embryo treated with rapamycin

AimsMicroRNAs (miRNAs) are vital in modulating lifespan and various biological processes including vascular function. The pivotal roles of mammalian target of rapamycin (mTOR) in regulating senescence and angiogenesis have been extensively described. However, the roles of its orthologue, zebrafish target of rapamycin (zTOR) in senescence and angiogenesis remain to be unravelled. In the present study, we aimed to investigate the role of zTOR and identify miRNAs associated with senescence and angiogenesis. Main methodsZebrafish embryos were treated with rapamycin and the inhibition of zTOR and its downstream proteins were validated by immunoblotting. Following the treatment, melanocyte density was quantitated, and senescence and angiogenic responses were determined by senescence-associated beta-galactosidase (SA-β-gal) and endogenous alkaline phosphatase (ALP) staining, respectively. Relative expression of microRNAs were determined by quantitative RT-PCR. Key findingsRapamycin (400nM) suppressed zTOR pathway by down-regulating the phosphorylation of zTOR-associated proteins such as P70S6K and S6K at both 4h post-fertilisation (hpf) and 8hpf while 4E-BP1 was only down-regulated at 8hpf when compared to their respective vehicle controls. Treatment with rapamycin also resulted in significant suppression of melanocyte development and senescence-associated beta-galactosidase (SA-β-gal) activity, and perturbed the development of intersegmental vessels (ISVs) of zebrafish embryos. In addition, the expressions of dre-miR-9-5p and -3p, dre-miR-25-3p and dre-miR-124-3p were significantly up-regulated in embryos treated with rapamycin from 4hpf. SignificanceOur findings suggest the involvement of zTOR in embryonic senescence and angiogenesis which could be potentially mediated by selected miRNAs.

Clinicopathological Features and Immunohistochemical Alterations of Keratinocyte Proliferation, Melanocyte Density, Smooth Muscle Hyperplasia and Nerve Fiber Distribution in Becker's Nevus.

BackgroundAlthough Becker's nevus (BN) is a relatively common disease, the systematic studies of clinicopathological and immunohistochemical results are poorly reported.ObjectiveTo investigate the clinicopathological features and immunohistochemical alterations of keratinocyte proliferation, melanocyte density, smooth muscle hyperplasia and nerve fiber distribution in BN.MethodsClinical and pathological data were collected in 60 newly-diagnosed BN cases. Immunohistochemical stain of Ki-67, Melan-A, keratin 15, smooth muscle actin and protein gene product 9.5 was performed in 21 cases.ResultsThe median diagnostic and onset age was 17 and 12 years, respectively. Skin lesions usually appeared on the upper trunk and upper limbs. The pathological features included the rete ridge elongation and fusion and basal hyperpigmentation. Epidermal Ki-67, Melan-A and keratin 15 expression and dermal nerve fiber length were significantly higher in lesional and perilesional skin than in normal skin (p<0.05~0.01), while smooth muscle actin expression was upregulated only in skin lesion (p<0.05).ConclusionAlthough the clinical diagnosis of BN is often straightforward, histopathology is helpful to differentiate from other pigmentary disorders. The hyperproliferation of keratinocytes, melanocytes, arrector pili muscle and dermal nerve fibers could be involved in the pathogenesis of BN.