The multiplex PCR/HD analysis is associated with a high diagnostic accuracy (92.7%) in CTCL patients. The finding of a clonal T-cell rearrangement is more closely associated with the histological pattern (in particular with the density and extent of the cell infiltrate) rather than with the MF cutaneous T-score or immunophenotype.

No abstract

The quantitative reduction of Tregs, together with that of TGF-beta and IL-10 serum levels, may be responsible for the loss of tolerance observed in both SSc and morphoea.

Cutaneous T-cell lymphomas are a heterogeneous group of lymphomas where the tumor population emerges within a multiple subclone pattern ("clonal heterogeneity"). PCR analysis has been shown to be useful in the diagnosis of mycosis fungoides (MF) and Sézary Syndrome (SS). Focusing the attention on clonal heterogeneity, the efficacy of the multiplex/heteroduplex (HD) PCR and the GeneScan (GS) capillary electrophoresis analysis was compared in the early diagnosis of MF/SS, using a multiple sample approach. Indeed, GS demonstrated TCRgamma gene rearrangement (GR) in all the 57 SS (100%) and in 123/146 (84%) of the MF samples, whereas the multiplex/HD PCR was less sensitive. An increase in clonality was observed in connection with both a worsening of the cutaneous disease (79% T1/T2; 100% T3/T4) and an increase in the histopathological score (HS< 5, 76%; HS > or = 5, 94%). Clonal heterogeneity with adjunctive reproducible skin TCRgamma-GRs was also observed. "Clonal instability," with different GRs, was present in a small percentage of patients. Therefore, it can be concluded that GS analysis in TCRgamma-GR is able to improve diagnosis in MF/SS patients and the multiple sample approach is helpful for a correct interpretation of clonal patterns in skin lesions, especially in early-stage MF and in SS skin/blood samples.

Background: Psoriasis is sustained by pro-inflammatory CD4+ T helper cells mainly belonging to the Th1, Th17 and Th22 lineage. Objective: To identify whether treatment with the anti-tumour-necrosis-factor antagonist etanercept is able to induce significant modulations in transcription factor and cytokine mRNA gene expressions related to the different T cell immune response polarization (Th1, Th2, Th17 and regulatory T cells, T_reg) and to correlate them with clinical response. Methods: The study population included 19 psoriasis patients treated with etanercept and 19 healthy subjects. Blood samples were collected at baseline and every 4 weeks during treatment. Taqman quantitative real-time polymerase chain reaction was applied to analyse the expression of: Stat-4, T-bet, IL-12p35 and IFN-γ (Th1-related); GATA-3, IL-4 (Th2-related); Stat-3, RORγt, IL-23p19 (Th17-related); Foxp3, IL-2 (T_reg-related). Flow cytometry was applied to analyse CD4+CD25+^brightFoxp3+ cells in peripheral blood. Results: Upregulation of Th1 and Th17 and downregulation of T_reg subsets was found at baseline. The response to etanercept could be associated with a significant reversal of the Th1/Th17 activation, and a concomitant upregulation of Th2 and T_reg subsets. Conclusion: Our data may contribute to a better understanding of the mechanisms underlying the achievement of clinical response in psoriasis and could be helpful for the identification of early predictive markers of response.

Vascular endothelial growth factor (VEGF) is known to play a crucial role in the growth and metastatization of solid tumours. In cancer patients, high VEGF serum levels correlate with tumour status and prognosis, but to date few data have been reported concerning VEGF in melanoma patients. In the present study, immunoenzymatic and reverse transcription-polymerase chain reaction (RT-PCR) techniques were used to detect VEGF-165 serum levels and the presence of tyrosinase mRNA, respectively, in the peripheral blood of a cohort of 155 melanoma patients at different clinical stages (30 stage I, 40 stage II, 40 stage III and 45 stage IV; AJCC classification). Data were compared with both the extent of the disease and the clinical course. The aim was to assess the relationship between VEGF serum levels, the presence of detectable circulating melanoma cells and melanoma progression. A significant increase in VEGF serum levels was found in melanoma patients, in particular in those with metastatic disease; a higher incidence of relapses was found in stage I-III disease-free patients who showed an increase in VEGF during follow-up. VEGF serum levels were significantly higher in patients with detectable circulating melanoma cells than in those with negative tyrosinase mRNA expression. The finding of both an increase in VEGF and the presence of detectable melanoma cells during follow-up was associated with a relapse rate of 81%. The relapse rate was significantly lower when either of the two parameters were present separately. Multivariate analysis of both overall survival and time-to-progression selected baseline tyrosinase expression in peripheral blood but not VEGF serum levels as an independent prognostic factor.

Bullous pemphigoid (BP) is the most frequent autoimmune bullous skin disease, characterised by autoantibodies against the hemidesmosome complex. Recently, regulatory T cells (Tregs) have been implicated in the development of several autoimmune diseases; few data are available in BP, failing to demonstrate a role of this subset in disease pathogenesis. The aim of this study was to investigate the expression and phenotypes of different Tregs (CD4+ CD25brightFOXP3+ and CD8+ CD28-cells) in BP to clarify whether the depletion of this subset constitutes one mechanism of tolerance loss. The CD4+ CD25bright-FOXP3 and CD8+ CD28-circulating subsets were determined by flow-cytometry in 26 untreated BP patients and compared with a group of age-and sex-matched healthy controls (HC, n = 30). Absolute and percentage values of the CD4+ CD25brightFOXP3+ cells were significantly reduced in BP compared with HC (median CD25bright-FOXP3+ expression within CD4+ cells: 1.8 vs. 3.5%, p = 0.002); conversely, BP patients were characterised by a significant expansion of the CD25brightFOXP3-''activated'' T-cell subset. CCR4 and CD62L were expressed on the majority of CD4+ CD25brightFOXP3+ cells (75.2 and 82.3%, respectively). No differences in the CD8+ CD28-subset were found between BP and HC. This is the first report showing a significant reduction of circulating CD4+ CD25brightFOXP3+ Treg frequency in BP patients.

Background: Regulatory T-cell (T_reg) modulation is one of the potential mechanisms of anti-tumour-necrosis-factor biological agents. However, literature data on psoriasis patients are lacking. Objective: To analyse the circulating CD4+CD25^brightFOXP3+ subset in 30 patients with psoriasis vulgaris/arthropathic psoriasis treated with biologicals and to investigate its relationship with the clinical response. Methods: The CD25^brightFOXP3+ expression within the CD4+ subset was determined by multi-parameter flow cytometry at baseline and during treatment. FOXP3 mRNA expression was analysed by real-time reverse transcription PCR. Results: A response was obtained in 16/17 patients (91.1%) with increased CD25^brightFOXP3+ values and in only 3/11 patients (27.3%) who showed a CD25^brightFOXP3+ decrease during biological treatment (p = 0.0001). Responders showed significantly higher values than did non-responders as from the first 2 months of treatment (p = 0.0032). A significantly higher posttreatment expression of mRNA FOXP3 was observed in responders compared to non-responders. Conclusion: Biological drugs induce a circulating T_reg up-regulation in a significant percentage of patients; such an increase is an early predictive marker of response.