Alessandra Comessatti

T-cell receptor gamma gene rearrangement by multiplex polymerase chain reaction/heteroduplex analysis in patients with cutaneous T-cell lymphoma (mycosis fungoides/Sézary syndrome) and benign inflammatory disease: correlation with clinical, histological and immunophenotypical findings.

Background  A dominant T‐cell clone can be detected by polymerase chain reaction (PCR) in 40–90% of cutaneous samples from patients with cutaneous T‐cell lymphoma (CTCL).

Regulatory T cells in the skin lesions and blood of patients with systemic sclerosis and morphoea

Background  Systemic sclerosis (SSc) and morphoea are connective tissue diseases characterized by fibrosis of the skin. Although to date their pathogenesis has not been clearly defined, it is thought that autoimmunity may play a role in the development of the skin lesions observed in both these diseases. As regulatory T cells (Tregs) play a key role in the modulation of immune responses, it has recently been suggested that Treg impairment may lead to the development of autoimmune diseases.

VEGF-165 serum levels and tyrosinase expression in melanoma patients: correlation with the clinical course.

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.

Th1, Th2, Th17 and regulatory T cell pattern in psoriatic patients: modulation of cytokines and gene targets induced by etanercept treatment and correlation with clinical response.

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, Treg) 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 (Treg-related). Flow cytometry was applied to analyse CD4+CD25+brightFoxp3+ cells in peripheral blood. Results: Upregulation of Th1 and Th17 and downregulation of Treg 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 Treg 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.

Circulating CD4+CD25 bright FOXP3+ T cells are up-regulated by biological therapies and correlate with the clinical response in psoriasis patients.

Background: Regulatory T-cell (Treg) 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+CD25brightFOXP3+ subset in 30 patients with psoriasis vulgaris/arthropathic psoriasis treated with biologicals and to investigate its relationship with the clinical response. Methods: The CD25brightFOXP3+ 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 CD25brightFOXP3+ values and in only 3/11 patients (27.3%) who showed a CD25brightFOXP3+ 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 Treg up-regulation in a significant percentage of patients; such an increase is an early predictive marker of response.

Epstein-Barr Virus in Cutaneous T-Cell Lymphomas: Evaluation of the Viral Presence and Significance in Skin and Peripheral Blood

The importance of viral agents in the development of cutaneous T-cell lymphomas (CTCL) is still debated. For this purpose, we retrospectively evaluated the Epstein-Barr virus (EBV) presence in Sézary syndrome (SS), mycosis fungoides (MF), inflammatory dermatoses (ID), and healthy donors (HD) using different approaches: EBV-DNA was quantified in skin biopsies and peripheral blood using real-time PCR, EBV-encoded small RNA (EBER) transcripts were detected by in situ hybridization (ISH), and latent membrane protein1-2 antigens were detected by immunohistochemistry. Skin biopsies were EBV-DNA-positive in 8/30 (27%) SS, 7/71 (10%) MF, and 2/18 (11%) ID patients and in none of the 25 normal skin samples. Positive mRNA (EBER) signals, always confined to cerebriform T lymphocytes, were found in 5/30 SS patients (17%), whereas signals in all MF and ID patients were negative. The presence of EBV-DNA in skin and blood samples was associated with a significantly lower survival in MF/SS patients. In evaluating EBV serological status, most (>70%) SS, MF, and ID patients showed a serological reactivation demonstrated by the presence of anti-EA IgG. In conclusion, although the finding of EBV-DNA in CTCL does not prove its etiopathogenetic role and may be related instead to immunosuppression, our study demonstrates that it has prognostic relevance.

Collagenase digestion and mechanical disaggregation as a method to extract and immunophenotype tumour lymphocytes in cutaneous T-cell lymphomas.

Various enzymatic or mechanical methods have been proposed in the past to dissociate cells from different solid tissues. An automated mechanical disaggregation device (Medimachine™) has recently been proposed. Unfortunately, most of these techniques are associated with a high cellular damage and a low cell recovery and are difficult to apply to skin biopsies. In this paper, we propose a combined enzymatic and mechanical method based on Medimachine™, useful for the isolation of skin infiltrating T‐lymphocytes from small cutaneous biopsies. As this method is easy and allows for a more correct qualitative and quantitative cytofluorimetric analysis of the lymphocyte subsets, it may be useful in the immunophenotyping of cutaneous T‐cell lymphomas.

Expression Pattern of Chemokine Receptors and Chemokine Release in Inflammatory Erythroderma and Sézary Syndrome

Background: Erythroderma can be caused by inflammatory dermatoses or cutaneous T-cell lymphoma. Even if chemokines and their receptors are involved in the skin-selective lymphocyte recruitment, their role in inflammatory erythroderma is yet unclear. Objective: To evaluate the chemokine release (TARC, MDC, IP-10) and to define the expression pattern of Th1- (CCR5, CXCR3) and Th2-related (CCR4) chemokine receptors in inflammatory erythroderma and Sézary syndrome (SS). Materials and Methods: Flow cytometry has been carried out on both circulating and skin-infiltrating T lymphocytes; serum chemokine levels have been evaluated using ELISA techniques. Results: CCR4, CCR5 and CXCR3 were expressed on about 40% of peripheral blood lymphocytes and on the majority of skin-infiltrating lymphocytes in the inflammatory erythroderma patients, whereas the leukemic CD4+CD26– subpopulation in SS was characterized by a high CCR4 expression without a concurrent increase in CCR5 or CXCR3. TARC, MDC and IP-10 serum levels were significantly increased in both erythrodermic and SS patients. Conclusions: Our results confirm that SS is a Th2 disorder with a selective expression of CCR4, whereas inflammatory erythroderma shares an overexpression of both Th1- and Th2-related chemokine receptors, suggesting an activation of different pathways driving reactive lymphocytes to the skin.

Heterogeneity of Circulating CD4+ Memory T-Cell Subsets in Erythrodermic Patients: CD27 Analysis Can Help to Distinguish Cutaneous T-Cell Lymphomas from Inflammatory Erythroderma

Background: Chronic dermatoses, as well as Sézary syndrome (SS), the erythrodermic and leukaemic cutaneous T-cell lymphoma, display a T-cell memory pattern. Recent findings suggest that different memory T-cell subsets can be recognized based on CD27 and CD45RO/RA expression. No data are reported as to CD27 expression in SS. Objectives: To evaluate different memory T-cell subsets, i.e. central memory (TCM), effector memory (TEM) and terminally differentiated cells in SS and inflammatory erythroderma (IE). Materials and Methods: Forty SS and 137 IE patients were included. CD27 and CD45RO/CD45RA expression was analysed by flow cytometry on peripheral blood lymphocytes and immunohistochemistry.Results: A significantly higher expression of the CD4+CD27+CD45RA– TCM subset was observed in SS whilst IE patients were characterized by increased CD4+CD27–CD45RA– TEM levels. The Vβ-restricted population was homogeneously CD4+CD26–CD27+ in the SS subjects. Conclusions: SS and IE are characterized by a different memory T-cell subset expression; CD27 expression could be used as an additional diagnostic tool in the differential diagnosis.

Human herpesvirus 7 detection by quantitative real time polymerase chain reaction in primary cutaneous T-cell lymphomas and healthy subjects: lack of a pathogenic role.

Background  Primary cutaneous T‐cell lymphomas (CTCLs) are a heterogeneous group of lymphomas where the tumour population emerges within a multiple subclone pattern. Mycosis fungoides (MF) and Sézary syndrome (SS) are characterized by the expansion of clonal CD4+/CD45RO+ memory T cells. Lymphomatoid papulosis (LyP) is a chronic, lymphoproliferative disorder included in the CD30+ primary CTCL spectrum. Several studies have suggested a role of viral infection for super‐antigenic activation of T lymphocytes; however, evidence of their association with CTCLs is still lacking. Human herpesvirus (HHV) 7 is a CD4+ T‐lymphotropic herpesvirus; its restricted cellular tropism and the ability to induce cytokine production in infected cells could make it an important pathogenic cofactor in lymphoproliferative disorders.