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Aims Treatment decisions in patients with scimitar syndrome (SS) are often challenging, especially in patients with isolated SS who are often asymptomatic and who might be diagnosed accidentally. We queried a large multi-institutional registry of SS patients to evaluate the natural history of this condition and to determine the efficacy of surgical treatment in terms of survival and clinical status. Methods and results We collected data on 485 SS patients from 51 institutions; 279 (57%) patients were treated surgically (STPs) and 206 (43%) were clinically monitored (CMPs). Median age at last follow-up was 11.6 years (interquartile range 4-22 years). Overall survival probability at 30 years of age was 88% [85-92% confidence intervals (CI)] and was lower in patients with associated congenital heart disease (CHD) (P

Altered expression of microRNAs (miRNAs) has been detected in cancer, suggesting that these small non-coding RNAs can act as oncogenes or tumor suppressor genes. In the present study, we investigated the expression of miRNA-17-5p, miRNA-18a, miRNA-20a, miRNA-92a, miRNA-146a, miRNA-146b and miRNA-155 by real-time quantitative RT-PCR in a panel of melanocyte cultures and melanoma cell lines and explored the possible role of miRNA-155 in melanoma cell proliferation and survival. The analyzed miRNAs were selected on the basis of previous studies strongly supporting their involvement in cancer development and/or progression. We found that miRNA-17-5p, miRNA-18a, miRNA-20a, and miRNA-92a were overexpressed, whereas miRNA-146a, miRNA-146b and miRNA-155 were down-regulated in the majority of melanoma cell lines with respect to melanocytes. Ectopic expression of miRNA-155 significantly inhibited proliferation in 12 of 13 melanoma cell lines with reduced levels of this miRNA and induced apoptosis in 4 out of 4 cell lines analyzed. In conclusion, our data further support the finding of altered miRNA expression in melanoma cells and establish for the first time that miRNA-155 is a negative regulator of melanoma cell proliferation and survival.

Altered expression of microRNAs (miRNAs) has been detected in cancer, suggesting that these small non-coding RNAs can act as oncogenes or tumor suppressor genes. In the present study, we investigated the expression of miRNA-17-5p, miRNA-18a, miRNA-20a, miRNA-92a, miRNA-146a, miRNA-146b and miRNA-155 by real-time quantitative RT-PCR in a panel of melanocyte cultures and melanoma cell lines and explored the possible role of miRNA-155 in melanoma cell proliferation and survival. The analyzed miRNAs were selected on the basis of previous studies strongly supporting their involvement in cancer development and/or progression. We found that miRNA-17-5p, miRNA-18a, miRNA-20a, and miRNA-92a were overexpressed, whereas miRNA-146a, miRNA-146b and miRNA-155 were down-regulated in the majority of melanoma cell lines with respect to melanocytes. Ectopic expression of miRNA-155 significantly inhibited proliferation in 12 of 13 melanoma cell lines with reduced levels of this miRNA and induced apoptosis in 4 out of 4 cell lines analyzed. In conclusion, our data further support the finding of altered miRNA expression in melanoma cells and establish for the first time that miRNA-155 is a negative regulator of melanoma cell proliferation and survival.

Human epidermal keratinocytes differentiate in vitro into a stratified epithelium suitable for grafting on burned patients. In this paper, we show that differentiated melanocytes are present in the cultured epithelium. In particular, we have found that i) melanocytes proliferate in the same culture conditions that allow keratinocyte growth, ii) during the culture the ratio between keratinocytes and melanocytes tends to remain constant, iii) melanocytes organize into the basal layer of the cultured epithelium independently of the presence of dermis, develop dendritic arborizations with melanosome-containing processes and transfer melanosomes into keratinocyte cytoplasm.

An intronic point mutation was identified in the ocular albinism type 1 (OA1) gene (HUGO symbol, GPR143) in a family with the X-linked form of ocular albinism. Interestingly, the mutation creates a new acceptor splice site in intron 7 of the OA1 gene. In addition to low levels of normally spliced mRNA product of the OA1 gene, the patient samples contained also an aberrantly spliced mRNA with a 165 bp fragment of intron 7 (from position +750 to +914) inserted between exons 7 and 8. The abnormal transcript contained a premature stop codon and was unstable, as revealed by Northern blot analysis. We defined that mutation NC_000023.8:g.25288G>A generated a consensus binding motif for the splicing factor enhancer ASF/SF2, which most likely favored transcription of the aberrant mRNA. Furthermore, it activated a cryptic donor-splice site causing the inclusion between exons 7 and 8 of the 165 bp intronic fragment. Thus, the aberrant splicing is most likely explained by the generation of a de novo splicing enhancer motif. Finally, to rescue OA1 expression in the patient's melanocytes, we designed an antisense morpholino modified oligonucleotide complementary to the mutant sequence. The morpholino oligonucleotide (MO) was able to rescue OA1 expression and restore the OA1 protein level in the patient's melanocytes through skipping of the aberrant inclusion. The use of MO demonstrated that the lack of OA1 was caused by the generation of a new splice site. Furthermore, this technique will lead to new approaches to correct splice site mutations that cause human diseases. Hum Mutat 27(5):420–426, 2006. Published 2006 Wiley-Liss, Inc.

Melanocytes represent the second most important cell type in the skin and are primarily responsible for the pigmentation of skin, hair, and eyes. Their function may be affected in a number of inherited and acquired disorders, characterized by hyperpigmentation or hypopigmentation, consequent aesthetic problems, and increased susceptibility to sun-mediated skin damage and photocarcinogenesis. Nevertheless, the possibility of genetically manipulating human melanocytes has been hampered so far by a number of limitations, including their resistance to retroviral infection. To address the problem of human melanocyte transduction, we generated a melanocyte culture from a patient affected with ocular albinism type 1 (OA1), an X-linked pigmentation disorder, characterized by severe reduction of visual acuity, retinal hypopigmentation, and the presence of macromelanosomes in skin melanocytes and retinal pigment epithelium (RPE). The cultured patient melanocytes displayed a significant impairment in replication ability and showed complete absence of endogenous OA1 protein, thus representing a suitable model for setting up an efficient gene transfer procedure. To correct the genetic defect in these cells, we used a retroviral vector carrying the OA1 cDNA and exploited a melanocyte-keratinocyte coculturing approach. Despite their lower replication rate with respect to wildtype cells, the patient melanocytes were efficiently transduced and readily selected in vitro, and were found to express, process, and properly sort large amounts of recombinant OA1 protein. These results indicate the feasibility of efficiently and stably transducing in vitro not only normal neonatal, but also mutant adult, human melanocytes with nonmitogenic genes.

Keratinocyte adhesion to basal lamina and lateral interactions among basal epidermal cells are mediated, besides other molecules, by integrin receptors that are sorted to defined membrane domains. The hemidesmosome-associated integrin alpha 6 beta 4 is sharply localized to the basal surface of basal cells while alpha 2 beta 1 and alpha 3 beta 1 are enriched laterally. This integrin sorting pattern is perfectly reproducible in vitro by cultured keratinocytes and takes place progressively in primary or secondary culture in the presence of 1.8 mM Ca2+. The polarized topography of integrins is gradually lost with higher passage numbers and between passage 5 and passage 7 there is a complete pericellular redistribution of the above integrins. Along with the decreased basal adhesive value of alpha 6 beta 4 there is a marked increase in the number of focal contacts in high-passage keratinocyte colonies. A similar loss of polarized topography of integrins occurs under low-Ca2+ culture conditions. Increasing the number of culture passages beyond the fifth induces the appearance of the fibronectin receptor alpha 5 beta 1 on the surface of keratinocytes, particularly at intercellular junctions and in some focal contacts. The receptor alpha 5 beta 1 is not detectably exposed by low-passage cells. We propose that forcing keratinocytes into more frequent cell cycles by continuous passaging may perturb the polarized topography of integrins and the adhesion mechanisms of keratinocytes. Then, low-passage keratinocytes are, in our opinion, the most reliable in vitro models for studying the physiology of epidermal cells.