Andrea Paradisi

Differential roles of p63 isoforms in epidermal development : selective genetic complementation in p63 null mice

Epidermal development requires the transcription factor p63, as p63−/− mice are born dead, without skin. The gene expresses two proteins, one with an amino-terminal transactivation domain (TAp63) and one without (ΔNp63), although their relative contribution to epidermal development is unknown. To address this issue, we reintroduced TAp63α and/or ΔNp63α under the K5 promoter into p63−/− mice by in vivo genetic complementation. Whereas p63−/− and p63−/−;TA mice showed extremely rare patches of poorly differentiated keratinocytes, p63−/−;ΔN mice showed significant epidermal basal layer formation. Double TAp63α/ΔNp63α complementation showed greater patches of differentiated skin; at the ultrastructural level, there was clear reformation of a distinct basal membrane and hemidesmosomes. At the molecular level, ΔNp63 regulated expression of genes characteristic of the basal layer (K14), interacting (by Chip, luc assay) with the third p53 consensus site. Conversely, TAp63 transcribed the upper layers genes (Ets-1, K1, transglutaminases, involucrin). Therefore, the two p63 isoforms appear to play distinct cooperative roles in epidermal formation.

Dermatofibrosarcoma protuberans: Wide local excision vs. Mohs micrographic surgery

BACKGROUND Dermatofibrosarcoma protuberans (DFSP) is an uncommon tumor of the skin with high rates of local recurrence. It is debated whether Mohs micrographic surgery (MMS) involves lower recurrence rates than wide local excision (WLE). Recent preliminary reports indicate more consistently favorable cure rates with MMS. We report comparative observational data on 41 patients who underwent MMS and 38 who underwent WLE. Their data were then pooled with those available in the medical literature to obtain more precise estimates of recurrence rates with MMS and WLE. METHODS The records of 79 patients with DFSP who underwent WLE (n=38) or MMS (n=41) in 1990-2005 were reviewed retrospectively. The primary endpoint was tumor recurrence rate. The PubMed database was searched for DFSP case series treated with WLE or MMS, and the recurrence proportions reported for the two separate procedures were pooled. RESULTS Five of the 38 WLE patients (follow-up=4.8 years) had recurrences (13.2%, 95% CI 4.4-28.1%) as opposed to none (95% CI 0-8.6%) of the 41 MMS patients (follow-up=5.4 years). Pooling of these data with those from the literature yielded 6/463 recurrences for MMS (1.3%, 95% CI 0.5-2.8%) and 288/1394 recurrences for WLE (20.7%, 95% CI 18.6-22.9%). The relative risk of recurrence for WLE vs. MMS patients was 15.9 (95% CI 7.2-35.5). CONCLUSIONS Significantly lower recurrence rates were recorded in our patients subjected to MMS compared with those treated with WLE. The pooled data also indicated a clear advantage of MMS. There is inconclusive evidence for any advantage of MMS in non-primary cases, while MMS was most effective in treating head and neck tumors. These data may be useful to guide clinicians in the choice of the more appropriate surgical treatment for DFSP patients.

Anandamide regulates keratinocyte differentiation by inducing DNA methylation in a CB1 receptor-dependent manner

Anandamide (arachidonoylethanolamide, AEA) belongs to an important class of endogenous lipids including amides and esters of long chain polyunsaturated fatty acids, collectively termed “endocannabinoids.” Recently we have shown that AEA inhibits differentiation of human keratinocytes, by binding to type-1 cannabinoid receptors (CB1R). To further characterize the molecular mechanisms responsible for this effect, we investigated the expression of epidermal differentiation-related genes after AEA treatment. We observed that keratin 1 and 10, transglutaminase 5 and involucrin are transcriptionally down-regulated by AEA. Most importantly, we found that AEA is able to decrease differentiating gene expression by increasing DNA methylation in human keratinocytes, through a p38, and to a lesser extent p42/44, mitogen-activated protein kinase-dependent pathway triggered by CB1R. An effect of AEA on DNA methylation because of CB1R-mediated increase of methyltransferase activity is described here for the first time, and we believe that the importance of this effect clearly extends beyond the regulation of skin differentiation. In fact, the modulation of DNA methylation by endocannabinoids may affect the expression of a number of genes that regulate many cell functions in response to these substances.

Etanercept therapy for toxic epidermal necrolysis

BACKGROUND Toxic epidermal necrolysis (TEN) is a severe and potentially lethal drug reaction for which no standard treatment is available. OBJECTIVE To describe a case series of patients with TEN treated with a single dose of etanercept. METHODS We observed 10 consecutive patients with TEN. For each patient, we recorded the presence of comorbidities and all the drugs recently started (ie, in the last month). In all cases, 50 mg of etanercept was administered in a single subcutaneous injection. The clinical severity of disease was computed using the SCORe of Toxic Epidermal Necrosis (SCORTEN) scale. Using the probabilities of death linked to each level of SCORTEN score, we calculated the expected probability of death in our patients. Healing was defined as complete reepithelialization, and a time to healing curve was then obtained using the Kaplan-Meier method. RESULTS All patients promptly responded to treatment, reaching complete reepithelialization without complications or side effects. The median time to healing was 8.5 days. LIMITATIONS This is a small, uncontrolled case series. CONCLUSION These preliminary results suggest the possibility that tumor necrosis factor-alfa may be an effective target for control of TEN, a dangerous skin condition for which no effective cure has yet been found.

Quality of life in patients with epidermolysis bullosa

Background  Epidermolysis bullosa (EB) is a rare, inherited group of disorders characterized by blistering of the skin following friction or mechanical trauma. EB has a clinical and socioeconomic impact on patients and their families.

Transglutaminase 5 is regulated by guanine–adenine nucleotides

Transglutaminases (TGases) are Ca2+-dependent enzymes capable of catalysing transamidation of glutamine residues to form intermolecular isopeptide bonds. Nine distinct TGases have been described in mammals, and two of them (types 2 and 3) are regulated by GTP/ATP. TGase2 hydrolyses GTP and is therefore a bifunctional enzyme. In the present study, we report that TGase5 is also regulated by nucleotides. We have identified the putative TGase5 GTP-binding pocket by comparative amino acid sequence alignment and homology-derived three-dimensional modelling. GTP and ATP inhibit TGase5 cross-linking activity in vitro, and Ca2+ is capable of completely reversing this inhibition. In addition, TGase5 mRNA is not restricted to epidermal tissue, but is also present in different adult and foetal tissues, suggesting a role for TGase5 outside the epidermis. These results reveal the reciprocal actions of Ca2+ and nucleotides with respect to TGase5 activity. Taken together, these results indicate that TGases are a complex family of enzymes regulated by calcium, with at least three of them, namely TGase2, TGase3 and TGase5, also being regulated by ATP and GTP.

Markedly reduced incidence of melanoma and nonmelanoma skin cancer in a nonconcurrent cohort of 10,040 patients with vitiligo

BACKGROUND Genetic findings suggesting a lower susceptibility to melanoma in patients with vitiligo are supported by recent clinical studies. Nonmelanoma skin cancer (NMSC) has also been studied, but mainly in small samples, and with conflicting results. OBJECTIVE We sought to study the relative risk (RR) of melanoma and NMSC in patients with vitiligo compared with that in patients seen for vascular surgery. METHODS The frequency of melanoma and NMSC was compared between patients with vitiligo and patients seen for vascular surgery. Occurrence of skin cancer was compared by computing RR and modeled using multiple logistic regression. RESULTS Overall, the crude RR for melanoma was 0.24 (95% confidence interval [CI] 0.13-0.45) in patients with vitiligo compared with those with a nondermatologic condition (occurrence 1.1‰, 95% CI 0.5‰-2.0‰ in patients with vitiligo and occurrence 4.5‰, 95% CI 3.8‰-5.4‰ in the control cohort). The crude RR for NMSC was 0.19 (95% CI 0.14-0.17) and the occurrence was 3.8‰ (95% CI 2.7‰-5.2‰) among patients with vitiligo and 19.6‰ (95% CI 18.0‰-21.4‰) in control subjects. Patients with vitiligo who underwent phototherapy had a markedly higher risk of both cancers. CONCLUSIONS In our large study, patients with vitiligo have a decreased risk of developing skin neoplasms, even considering that a larger proportion in this patient group is exposed to higher levels of ultraviolet radiation.

Death fold domain interaction in apoptosis

The death fold is a structurally defined motif characterized by six to seven tightly coiled a-helices in a ‘Greek key fold’ (see Figure 1). It is formed by several defined protein-interaction domains, all of which are found on proteins with known or suspected roles in apoptosis and other signaling pathways. Death folds have the interesting feature that they bind to another death fold-containing domain via the same type of protein-interaction domain (homotypic interaction). The four known death-fold domains are the caspase recruitment domain (CARD), the death domain (DD), the death effector domain (DED), and, as recently predicted, the PYRIN domain. The homotypic interactions that characterize the death folds appear to be highly specific, with only two (or in some cases, a few) partners capable of interacting. In every known case, the binding partners have homologous domain (DD– DD, DED–DED, CARD–CARD) and there are no established interactions across groups. The most primitive role for homotypic interactions is obviously that of self-assembly – proteins with such domains have the potential to assemble into larger multisubunit structures composed only of that protein. However, there are no examples of a death-fold interaction in which the domain binds to itself (i.e., to an identical sequence or to another molecule). This suggests that early in evolution the original death fold diverged to ensure homotypic interactions only between slightly different deathfold domains from different molecules. These molecules then came to have very different functions that may nevertheless be related. One danger in the interpretation of structural domains is what we can call the ‘functional homology trap.’ Although significant homology can suggest a conservation of function, and this can be useful in designing experiments, we simply cannot assume these relations. While several of the deathfold proteins have established roles in apoptosis, immune defense, and/or NF-kB signaling, many of these proteins have unknown or questionable functions. It is probably a mistake to assume that they necessarily have similar physiological roles. CARD-containing proteins are found throughout the animal kingdom and may be present in plants, fungi, and prokaryotes. In proteins with known roles in apoptosis, CARD domains are present on several mammalian procaspases (caspase-1, -2, -4, -5, -9, -12, -13), the CED-3 caspase in Cernorabdis elegans, the Dronc caspase in Drosophila melanogaster, and on adapter molecules involved in caspase activation (RAIDD-caspase-2, Apaf-1-procaspase-9, CED-4-CED-3, ARK-Dronc). CARD-containing proteins that may inhibit or perhaps activate caspases (in particular, caspase-1) include ICEBERG, Pseudo-ICE, Cop, and Cardiak. In mammals, CARD domain-containing proteins have a wide range of functions (apoptosis, cytokine processing, immune defense, NF-kB activation), while in insects and nematodes, the CARDs appear to be (so far) restricted to proteins involved in apoptosis. Probably the best-characterized CARD–CARD interaction is that between Apaf-1 and caspase-9 that activates caspase9. This interaction is mediated by a CARD present on both Apaf-1 and procaspase-9. A wealth of structural information is in fact available through the determination of solution and crystal structure of the isolated Apaf-1 CARD and the crystal structure of the complex with the procaspase-9 CARD. Together with complementary mutagenesis data, this provides a detailed view of the molecular basis governing the regulation of the central machinery of apoptosis, with implications for possible therapeutical interventions. The CARD domain of Apaf-1 is formed by a bundle of six (or seven) tightly packed a-helices., closely resembling the overall structure of the RAIDD CARD that interacts with caspases 2 and 9. In the RAIDD CARD only six a-helices are present with Apaf-1 helices H1a and H1b combined into a single helix H1. In the Apaf-1/procaspase-9 complex helices H2 and H3 of Apaf-1 CARD form a convex acidic surface that recognizes a complementary basic concave surface of caspase-9 CARD (formed by H1a, H1b, H4), with residues Y24, D27, S31, D32, Q40, N73, (Apaf-1) and R11, R13, R52, R56 (procaspase-9) providing specificity in the interaction. Besides these electrostatic interactions, additional hydrophobic contacts are present in the center of the interface thus explaining why the association is refractory to high ionic strength. DDs are a death fold found predominantly in the vertebrates, although represented throughout the animals. Several cytokine receptors in the TNF receptor family contain DDs, including the death receptors (TNFR1, CD95, TRAMP/DR3, and the TRAIL receptors). Structurally related DDs are also found on other TNFR family members such as p75 CNTR. Adapter molecules bearing DDs bind to these receptors via specific homotypic interactions (TRADD binds TNFR1, FADD binds CD95, DR3, and the TRAIL receptors). TRADD and FADD also bind each other via DD–DD interaction, but only when TRADD is bound to TNFR1. DD proteins function in both apoptosis and NF-kB signaling in mammals, but apparently only the latter in Drosophila. Cell Death and Differentiation (2003) 10, 10–12 & 2003 Nature Publishing Group All rights reserved 1350-9047/03

Acute generalized exanthematous pustulosis induced by hydroxychloroquine: Three cases and a review of the literature

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Safety of etanercept in patients with psoriasis and hepatitis C virus assessed by liver histopathology: preliminary data.

INTRODUCTION Acute generalized exanthematous pustulosis (AGEP) is a clinical reaction pattern that is principally drug induced and is characterized by acute, extensive formation of nonfollicular sterile pustules on an erythematous and edematous substrate. Hydroxychloroquine (HHCQ), an antimalarial drug widely used to treat rheumatic and dermatologic diseases, has been described as an uncommon cause of AGEP. OBJECTIVES This article reports 3 cases of HCQ-induced AGEP and reviews similar cases in the published literature. CASE SUMMARIES The first case involved a 36-year-old woman with a 10-year history of rheumatoid arthritis and Sjögrens syndrome who had begun a 25-day course of HCQ 100 mg BID due to lack of response to a corticosteroid, with a skin reaction developing 21 days into the new treatment. In the second case, a 70-year-old man with poorly controlled rheumatoid arthritis had begun a course of oral HCQ 100 mg BID 20 days before development of AGEP. The final case involved a 79-year-old woman with polymyalgia rheumatica who had been receiving HCQ 100 mg BID as a steroid-sparing agent for 22 days, with rash developing 20 days after the initiation of HCQ. Sixteen cases of HCQ-induced AGEP were identified in the literature, including some that may have been reported under a different name but were consistent with a clinical diagnosis of AGEP. The US Food and Drug Administration has mandated a change to the labeling for HCQ to include AGEP among potential adverse dermatologic reactions to the drug. CONCLUSIONS This article reports 3 cases of AGEP related to administration of HCQ. HCQ-induced AGEP is a rare but severe, extensive, and acute reaction. No specific therapy is available, and correct diagnosis generally leads to spontaneous resolution once the causative drug has been withdrawn.