Isolation and Analysis of Lipid Rafts from Neural Cells and Tissues.

Lipid rafts are membrane areas characterized by the clustering of selected membrane lipids, as the result of their phase separation forming a liquid-ordered phase floating in the lipid-disordered bulk membrane. van Meer and Simons hypothesized the existence of lipid rafts to explain the differential composition of the apical and basolateral domains of polarized epithelial cells and proposed that association of given proteins with lipid rafts along the traffic route might represent an important mechanism for protein sorting. However, great attention was paid to the lipid raft theory after Simons and Ikonen highlighted the enrichment of several proteins involved in signal transduction in "detergent-insoluble, glycolipid-enriched complexes," and postulated that lipid rafts might serve as hubs in regulating intracellular signaling. Most notably, the feature of detergent-insolubility was incorporated in the definition of lipid rafts used in 1997 by these authors. "Lipid rafts" and "detergent-resistant membranes" became almost synonymous after the publication, in 1992, of the seminal paper by Brown and Rose, describing the separation of a low-density, Triton X-100-insoluble fraction from epithelial cells, enriched in GSL and apical GPI-anchored proteins and depleted of basolateral membrane marker proteins. This paper provided a working definition of lipid rafts and a putative biochemical method for their separation. More than 2000 papers have been published using "the Triton method." Evidences obtained by the use of alternative biochemical methods for the isolation of lipid rafts and of methods enabling to analyze the dynamics of lipid rafts in intact cells highlighted the several limitations of the Triton X-100 method. On the other hand, the main findings obtained by this method have not been confuted, and the method is still widely used.In this chapter, we will discuss the most relevant methodological aspects related to the preparation of detergent-resistant membrane fractions, with a special focus on neural cells and tissues.

Paradoxical Psoriasis Induced by Anti-TNFα Treatment: Evaluation of Disease-Specific Clinical and Genetic Markers.

Paradoxical psoriasis (PP) may occur during treatment with anti-tumor necrosis factor-alpha (TNF-α) drugs in various chronic immune-mediated diseases, mainly inflammatory bowel diseases (IBD) and psoriasis. In this study, clinical and genetic characteristics of PP arising in IBD and psoriatic patients were investigated to identify disease-specific markers of the paradoxical effect. A total of 161 IBD and psoriatic patients treated with anti-TNF-α drugs were included in the study. Of these patients, 39 developed PP. All patients were characterized for the main clinical-pathologic characteristics and genotyped for six candidate single nucleotide polymorphisms (SNPs) selected for their possible role in PP susceptibility. In IBD patients, the onset of PP was associated with female sex, presence of comorbidities, and use of adalimumab. IBD patients with PP had a higher frequency of the TNF-α rs1799964 rare allele (p = 0.006) compared with cases without the paradoxical effect, and a lower frequency of the human leucocyte antigen (HLA)-Cw06 rs10484554 rare allele (p = 0.03) compared with psoriatic patients with PP. Overall, these findings point to specific clinical and genetic characteristics of IBD patients with PP and provide data showing that genetic variability may be related to the paradoxical effect of anti-TNF-α drugs with possible implications into clinical practice.

Erythema ab igne induced by laptop computer: an emerging disease among adolescents?

Erythema ab igne (EAI) was a very common disease in the past, when it occurred mainly among people who worked with fire, or in people who had used heat sources in contact with the skin for warming purposes for long time. In the last decades, with the introduction of central heating in the buildings, EAI incidence was remarkably decreased in Western Countries, and it was found almost exclusively among elderly, and in people affected by defects in thermoregulation or alteration of periphery circulation. Recently, a new slight increase of EAI prevalence has been observed, although with some new features. Here, we describe three cases of adolescents who presented with brownish, reticulated patch on the anterior surface of their thighs. An accurate medical questioning revealed that the patients used to place the lower surface of laptop computer on the extensor side of their thighs in a cross-legged position for many hours (about 6-8 hours) every days. In particular, the patients supported the laptop computer always on the same leg. Laptop computer-induced EAI was diagnosed. Only a few cases of laptop computer-induced EAI have been reported in the literature. Although EAI is poorly symptomatic and it generally evolve to complete remission after an early discontinuance of heat source exposure, chronic lesions of EAI have been regarded as precancerous lesions. Therefore, it is important to implement diagnosis and prevention measures of this disease. Dermatologists should consider new causal agents for old diseases.

MiRNAs as Potential Prognostic Biomarkers for Metastasis in Thin and Thick Primary Cutaneous Melanomas.

BACKGROUND/AIM: The identification of novel prognostic biomarkers for melanoma metastasis is essential to improve patient outcomes. To this aim, we characterized miRNA expression profiles in relation to metastasis in melanoma and correlated miRNAs expression with clinical-pathological factors. MATERIALS AND METHODS: MiR-145-5p, miR-150-5p, miR-182-5p, miR-203-3p, miR-205-5p and miR-211-5p expression levels were analyzed in primary cutaneous melanomas, including thin and thick melanomas, and in melanoma metastases by quantitative Real-Time PCR. RESULTS: A significantly lower miR-205-5p expression was found in metastases compared to primary melanomas. Furthermore, a progressive down-regulation of miR-205-5p expression was observed from loco-regional to distant metastasis. Significantly lower miR-145-5p and miR-203-3p expression levels were found in cases with Breslow thickness >1 mm, high Clark level, ulceration and mitotic rate ≥1/mm2 Conclusion: Our findings point to miR-205-5p as potential biomarker of distant metastases and to miR-145-5p and miR-203-3p as markers of aggressiveness in melanoma.

Telangiectasia macularis eruptiva perstans: a neglected type of mastocytosis with exclusively cutaneous involvement? A case series.

Telangiectasia macularis eruptiva perstans (TMEP) is a rare form of cutaneous mastocytosis (CM). Although TMEP has been traditionally thought to be restricted to the skin, a recent retrospective multicentric study established a diagnosis with systemic involvement of mastocytosis in 47% patients affected by TMEP and aggressive systemic mastocytosis in 9%. To evaluate systemic involvement in patients affected by TMEP. We conducted a retrospective monocentric study among patients affected by TMEP visited in our dermatology clinic. Data regarding gender, age at diagnosis, duration of the disease before diagnosis, topography, clinical features, presence of extra-cutaneous symptoms, serum tryptase levels, and histopathological and bone marrow biopsy features were analysed. Among 119 patients classified with mastocytosis, eight patients (six males, two females) with TMEP and one female patient affected by mastocytosis in the skin, a TMEP variant, were retrospectively studied. The mean diagnostic delay was two years (range: 8-26 months). In two patients (25%), bone marrow involvement was identified and osteoporosis and hepatosplenomegaly were also found. The two patients with systemic involvement exhibited a statistically significant increase in serum tryptase levels (p < 0.05). The detection of KIT gene mutation in skin specimens revealed a somatic mutation, KITD816 V, only in these two patients. TMEP is a rare form of CM, often neglected. A correct and early diagnosis of TMEP is important to rule out systemic involvement of the disease. Detection of serum tryptase levels may be a useful, rapid, and non-invasive marker of systemic involvement.

Monitoring chemotherapy-induced alopecia with trichoscopy.

BACKGROUND: Chemotherapy-induced alopecia (CIA) ranks among the psychologically most devastating effects of cancer treatment for oncological patients, with an overall incidence of 65%. Nowadays trichoscopy is largely employed in the diagnosis of alopecia, but no description of CIA trichoscopic pattern is present in literature. AIMS: We want to create an organic description of CIA trichoscopic aspects. METHODS: Oncological patients candidate to chemotherapy drugs, afferent to our trichological outpatient were studied. Anamnesis, clinical exam, clinical global photography, pull test, trichogram, and trichoscopy were conducted at the different moments of therapeutic treatment. RESULTS: A definite trichoscopic pattern in the different phases of treatment was observed. After the first 3 weeks of chemotherapy rare and scattered black dots, broken hairs, flame hairs and pohl pinkus appeared. At the end of chemotherapy besides the features described above, numerous thin hair in regrowth were detected, together to rare terminal hair, scattered black dots and circle hair. Three months after chemotherapy a progressive increase of follicular units and elongation of the existing hair were visible. CONCLUSIONS: We propose an description of CIA trichoscopic pattern and its evolution during the different phases of chemotherapy.

Case Report: Cetuximab use in advanced cutaneous squamous cell carcinoma resistant to chemotherapy.

We present the case of a 60-year-old man with unresectable cutaneous squamous cell carcinoma (cSCC) of the sternal area, which was not amenable to radiation therapy. The treatment history of this patient is remarkable as the disease had progressed through all lines of conventional therapy established in the literature. We decided to initiate treatment with epidermal growth factor receptor (EGFR) inhibitor cetuximab and we reassessed the patient after 12 weeks with a whole-body CT scan, documenting stability in the size and radiologic features of the disease. Cetuximab, like all current treatments for advanced cSCC, is administered off-label and proved effective in preventing further progression of disease in our patient.

Genetic counselling and high-penetrance susceptibility gene analysis reveal the novel CDKN2A p.D84V (c.251A>T) mutation in melanoma-prone families from Italy.

Genetic susceptibility to primary cutaneous melanoma (PCM) may account for up to 12% of PCMs, presenting as the familial atypical mole/multiple melanoma syndrome (FAMMM), an autosomal dominant condition with incomplete penetrance and variable expressivity, characterized by PCM in at least two relatives and/or more than one PCMs in the same patient. To identify individuals at high genetic risk of PCM, from 1 January 2012 to 31 December 2015, we offered genetic counselling and molecular analysis of the two high-penetrance FAMMM susceptibility genes, cyclin-dependent kinase inhibitor 2A (CDKN2A) and cyclin-dependent kinase 4 (CDK4), to 92 consecutive, unrelated patients with FAMMM. Age at diagnosis and number of PCMs were obtained from medical records; the number of PCMs and affected relatives were recorded for each family. The diagnostic work-up consisted of genetic counselling and cascade genetic testing in patients and further extension to relatives of those identified as mutation carriers. All exons and exon/intron boundaries of CDKN2A and CDK4 genes were screened by direct bidirectional sequencing. We identified CDKN2A mutations in 19 of the 92 unrelated patients (20.6%) and in 14 additional, clinically healthy relatives. Eleven of these latter subsequently underwent excision of dysplastic nevi, but none developed PCM during a median follow-up of 37.3 months. In three patients from unrelated families, the novel CDKN2A p.D84V (c.251A>T) mutation was observed, associated with PCM in each pedigree. Genetic screening of FAMMM patients and their relatives can contribute towards specific primary and secondary prevention programmes for individuals at high genetic risk of PCM. The novel CDKN2A p.D84V (c.251A>T) mutation adds to the known mutations associated with FAMMM.

Narrow-band UVB phototherapy and psoralen-ultraviolet A photochemotherapy in the treatment of cutaneous mastocytosis: a study in 20 patients.

BACKGROUND: In mastocytosis, the skin is almost invariably involved, and cutaneous symptoms deeply affect patients' quality of life. METHODS: A retrospective observational analysis of patients affected by cutaneous mastocytosis (CM) and indolent systemic mastocytosis (ISM) treated with phototherapy/photochemotherapy (PUVA or NB-UVB) has been conducted. For each patient, total numbers of PUVA or NB-UVB exposures, the cumulative UV dose (J/cm2 ), serum tryptase profile, and pruritus, before and after treatment, according to the visual analogue scale (VAS) were considered. Skin lesions of each patient were assessed, before and after treatment, according to a cutaneous scale score. RESULTS: Twenty patients affected by CM and ISM were studied; in particular, 10 patients received NB-UVB therapy, and other 10 patients received PUVA. A statistically significant mean reduction of pruritus in both groups (P < 0.01) was observed. The number of treatments necessary to obtain symptom relief was significantly lower in the PUVA group, but the mean exposure dose was significantly higher, if compared to the NB-UVB group. Serum tryptase levels showed a downward trend. The cutaneous score improved in both groups. LIMITATIONS: This study was a retrospective study with a small sample size and without a control group. CONCLUSION: This work provides evidence that both NB-UVB and PUVA represent a safe and useful second-line therapy of the cutaneous symptoms in mastocytosis.

Adult-onset tufted angiomas associated with an arteriovenous malformation in a renal transplant recipient: case report and review of the literature.

Tufted angioma (TA) is a rare benign vascular neoplasm characterized histopathologically by the proliferation of endothelial cells arranged in lobules in the dermis and subcutaneous fat. To date, about 200 cases have been reported, most of which are of Japanese ethnicity. TA predominantly affects children and young adults, developing in 80% of patients younger than 10 years. A white 72-year-old renal transplant recipient presented with 2 asymptomatic dusky red papules on his right leg. The lesions appeared 5 years after the start of immunosuppressive treatment. Histopathologic examination showed a proliferation of poorly canalized capillary-sized vascular structures with typical "cannonball" pattern in the dermis and subcutaneous fat. Eccrine glands were also evident focally in the stroma of capillary lobules. On immunohistochemistry, endothelial cells in the vascular tufts stained positive for CD31 and CD34 but were negative for factor VIII-related antigen, human herpes virus 8, and podoplanin (clone D2-40); α-smooth muscle actin stained pericytes disposed in a single layer in capillary-sized vessels and in 2-3 or more layers in vessels of larger size, respectively. The microscopic findings were suggestive of TA. In the deep dermis, venules with smooth muscle wall and arterioles, as shown by Van Gieson staining, normally not found at that level, were present and appeared surrounded by capillary lobules. Onset of TA in adulthood is rare and may be associated with pregnancy, varicella zoster virus infection, and pharmacological immunosuppression. A case of acquired adult-onset TA associated with an arteriovenous malformation in an elderly transplanted patient is described.

Incomplete penetrance of GLMN gene c.395-1G>C mutation in a family with glomuvenous malformations.

Glomuvenous malformations (GVMs, OMIM 138000) are hamartomas presenting in childhood as multiple, bluish, soft papules and nodules that tend to grow slowly in size and number with age. They are caused by autosomal dominant mutations in glomulin (GLMN) gene; penetrance varies from 80% at 20 to about 100% at age 30 years. We report on the c.395-1G>C mutation of GLMN gene in two siblings showing variable penetrance.

Glomuvenous malformations with smooth muscle and eccrine glands: unusual histopathologic features in a familial setting.

Glomuvenous malformations (OMIM 138000) are hamartomas presenting in childhood as multiple, bluish papules and nodules in the skin, which are characterized histopathologically by irregular vascular spaces surrounded by typical glomus cells. Glomuvenous malformations are caused by autosomal dominant mutations of the GLMN gene. A 34-year-old woman and her 16-year-old son presented with bluish papules and nodules since childhood. Biopsy specimens from both patients showed histopathologic features of glomuvenous malformations, unusually in consistent and close association with smooth muscle, hair follicles and eccrine glands. Sequencing of the GLMN gene revealed the p.C36X (c.108C>A) mutation in germline DNA from both patients. This is probably the first report describing the hamartomatous features of familial glomuvenous malformations consistently associated with a prominent smooth muscle component and eccrine glands.

Insulin receptor and glucose transporters mRNA expression throughout the menstrual cycle in human endometrium: a physiological and cyclical condition of tissue insulin resistance.

The expression of insulin receptor (IR), together with that of glucose transporters 1 and 4 (GLUT1-4) and of Insulin Growth Factor-I and -II (IGF-I,-II) in the endometrium of healthy and young women in both phases of menstrual cycle was assessed. Sixteen out of 20 healthy and normal menstruating volunteers were studied. Endometrial samplings were performed in every subject, twice in the same cycle, during the follicular and luteal phase respectively. The mRNA expression of IR, GLUT1-4, IGF-I and -II were evaluated by real-time quantitative RT-PCR and immunostaining reactions. Our results indicate that IR, GLUT1-4, IGF-I and -II mRNAs were expressed in both phases of the endometrial cycle: GLUT4 and IGF-I mRNA expression were significantly higher in the follicular phase and localized at the epithelial and stromal cell level, respectively, whereas IR, GLUT1 and IGF-II mRNA expression were mostly present in the secretory phase and mainly localized at the stromal level. An inverse tendency of IR and GLUT4 mRNA expression was respectively observed from follicular to luteal phase. In conclusion our data suggest that IR, glucose transporters and IGFs are significantly and differently expressed at the endometrial level throughout the menstrual cycle and that human endometrium cyclically undergoes through a transitory condition from normal to an insulin-resistance state.