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Sezary syndrome (SS) is a rare and aggressive variant of Cutaneous T-Cell Lymphoma characterized by neoplastic distribution mainly involving blood, skin, and lymph-node. Although a role of the skin microenvironment in SS pathogenesis has long been hypothesized, its function in vivo is poorly characterized. To deepen this aspect, here we compared skin to blood-derived SS cells concurrently obtained from SS patients highlighting a greater proliferation-index and a PI3K/AKT/mTORC1 pathway activation level, particularly of mTOR protein, in skin-derived-SS cells. We proved that SDF-1 and CCL21 chemokines, both overexpressed in SS tissues, induce mTORC1 signaling activation, cell proliferation and Ki67 up-regulation in a SS-derived cell line and primary-SS cells. In a cohort of 43 SS cases, we observed recurrent copy number variations (CNV) of members belonging to this cascade, namely: loss of LKB1 (48%), PTEN (39%) and PDCD4 (35%) and gains of P70S6K (30%). These alterations represent druggable targets unraveling new therapeutic treatments as metformin here evaluated in vitro. Moreover, CNV of PTEN, PDCD4, and P70S6K, evaluated individually or in combination, are associated with reduced survival of SS patients. These data shed light on effects in vivo of skin-SS cells interaction underlying the prognostic and therapeutic relevance of mTORC1 pathway in SS.

Introduction: Cutaneous T-cell Lymphomas (CTCLs) are extranodal non-Hodgkin’s lymphomas deriving from neoplastic transformation of T lymphocytes that home to the skin. Mycosis fungoides (MF) is the most diffuse form, usually having an indolent course but presenting in some cases as an aggressive disease with a poor prognosis. Sézary syndrome (SS) is a rare and aggressive variant, with erythroderma, leukemia, and lymph node involvement. Despite recent advances, CTCLs are still incurable cancers and the clinical symptoms are highly disabling. Among the landscape of genomic alterations identified in CTCL, we and others have described alterations of the PI3K pathway members (loss of PTEN, LKB1 and PDCD4, and gain of p70S6K). As a consequence, several SS patients show hyper-activation of PI3K/AKT/mTOR pathway. Hence, inhibition of this pathway represents a potential therapeutic choice against CTCL. Objective: In this work, we investigated the therapeutic potential of the dual-PI3K/mTOR inhibitor PF-04691502 (hereafter PF-502) against CTCL. Materials and Methods: PF-502 therapeutic efficacy was evaluated in four CTCL cell lines and in patient-derived SS cells, for its ability to inhibit cell growth, to induce apoptosis and to inhibit chemotaxis toward the chemokine SDF-1. Moreover, to evaluate PF-502 therapeutic effects in vivo we used a xenograft mouse model, consisting of nude mice transplanted with the CTCL-derived HH cell line. Results: PF-502 inhibited the cell growth of all the CTCL cell lines (0.07-0.32 µM, IC50 range), and of tumor cells derived from six SS patients (2.3 µM, mean IC50). The mechanism of action of PF-502 in CTCL cell lines was a combination of apoptosis induction and G1 block, while in SS patients it was mostly based on a strong apoptosis induction. Of note, PF-502 showed an only modest effect in T-lymphocytes from 3 healthy donors. PF-502 inhibited also cell migration toward the chemokine SDF-1, in both the systems. This result might have an important clinical implication because we have recently demonstrated that malignant lymphocytes are activated by the skin microenvironment. Thus, blocking the homing to the skin could affect the disease course. Finally, PF-502 has a robust antitumor activity in the xenograft mouse model, by reducing tumor volume (400 mm3 in treated mice vs 936 mm3 in controls) as well as tumor weight (0.2 g in treated mice vs 0.56 g in controls). Moreover, the Kaplan-Meier curve, in these mice, revealed that PF-502 treatment prolongs survival (P Conclusions: Our results strongly support the therapeutic potential of targeting PI3K/AKT/mTOR pathway in CTCL. Citation Format: Antonella Bresin, Cristina Cristofoletti, Francesca Monzo, Elisabetta Caprini, Mauro Helmer Citterich, Alessandra Frezzolini, Alessandro Monopoli, Roberto Benucci, Maria Cantonetti, Enrico Scala, Giandomenico Russo, Maria Grazia Narducci. The PI3K/mTOR dual inhibitor PF-04691502 shows antitumor activity in Sezary cells and in a xenograft mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3912.

Cutting edge experiments and thorough investigations have pointed out that radial components affect the needle lift of diesel nozzles. So far, the effects of such needle “off-axis” have been investigated within the nozzle and immediately downstream from the hole outlet. Here, the focus has been extended to the spray ambient, far outside a multi-hole VCO (Valve Covered Orifice) nozzle. A reference off-axis configuration of the needle has been defined and used to investigate its effects on the spray, in terms of hole-to-hole differences. Indeed, the spray alterations due to the needle position have been addressed for those factors, such as the velocity-coefficient CV and the area-coefficient CA, able to describe the nozzle flow behavior under needle off-axis. The investigation has been based on 3D-CFD (three-dimensional computational fluid dynamics) campaigns. The modeling of diesel nozzle flow has been interfaced to the Eulerian–Eulerian near-nozzle spray simulation, initializing the break-up model on the basis of the transient flow conditions at each hole outlet section. The dense spray simulation has been on-line coupled to the Eulerian–Lagrangian modeling of the dilute spray region. Quantitative results on each fuel spray have been provided (in terms of penetration and Sauter Mean Diameter). The range of variability within the spray characteristics are expected to vary has been found and reported, providing reference information for lumped parameter models and other related investigations.

Toll-like receptors (TLRs) are a key part of the innate immune system that detect pathogen-associated molecular patterns (PAMPs) of microorganisms and their stimulation results in the activation of signaling pathways leading to the modulation of inflammatory and immune responses. Since psoriasis is a complex, inflammatory and immune skin disease, characterized by an abnormal immune response and increased proliferation of keratinocytes, with an increased production of proinflammatory cytokines, TLRs could play an important role in the pathogenesis of the disease. We propose to assess the modulation of TLR expression on psoriatic skin of patients treated with Adalimumab and systemic conventional therapies. We therefore recruited fifteen patients: ten were treated with adalimumab and five with systemic conventional therapies; their clinical conditions were analyzed by PASI index and skin biopsies were evaluated for TLR1 and TLR2 expression by immunohistochemistry assays. Our data suggest adalimumab is not only able to improve the clinical condition of psoriatic patients, but also to modulate TLR1 and TLR2 expression involved in psoriasis, as in healthy skin. Adalimumab is a most promising biological drug able to orchestrate immune and inflammatory responses in psoriatic lesions, recovering TLR expression on basal keratinocytes and improving clinical conditions of psoriatic patients, with no evident side effects.

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are considered to be severity variants of the same disease, which is almost always associated with drug intake. In contrast, erythema multiforme (EM) is a disorder regarded as only rarely caused by drugs. Keratinocyte apoptosis has been shown to play an important part in the pathogenesis of SJS and TEN, whilst its role in EM remains controversial. To determine the expression of apoptosis-associated molecules Fas, Fas ligand (FasL), Bcl-2 and Bax in the above disorders, an immunohistochemical analysis was performed. We studied both lesional skin from thirty patients having drug-induced EM and 5 cases classified within the SJS/TEN spectrum and normal skin samples. We found a keratinocyte overexpression of Fas antigen, an important molecule mediating apoptosis, not only in SJS and TEN but also in EM. Another noteworthy finding was the strong expression of Bcl-2, a protein known as blocking apoptosis, along the basal layer and in the dermal infiltrate both in SJS/TEN and in EM. Taken together, these findings suggest that Fas-dependent keratinocyte apoptosis may play a part in the pathogenesis of both SJS/TEN and EM. Fas-mediated cell death may be partially suppressed by the Bcl-2 protein. Copyright © by Biolife, s.a.s.

Summary Background. The basophil activation test (BAT) has been recently described as a useful in vitro tool for diagnosis of allergy to Anisakis species in patients with acute urticaria. Aim. To evaluate the relationship between sensitization to Anisakis simplex and chronic urticaria (CU), using flow cytometry analysis of in vitro BAT. Methods. A. simplex sensitization was evaluated in patients with CU (n = 57) and in atopic (n = 22) and healthy controls (n = 20) by means of skin prick test (SPT), specific IgE and Anisakis-induced BAT using a triple-labelled strategy with anti-CD123, anti-human leucocyte antigen DR and anti-CD63 antibodies. During a follow-up period of 6 months in 10 patients with CU who accepted a fish-free dietary regimen, the diagnostic performance of the in vivo and in vitro methods was calculated, and changes in specific IgE and BAT were evaluated with respect to clinical response. Results. A significant association between CU and A. simplex sensitization was found, with an overall prevalence of 75.4% in patients with CU (43/57) compared with 18% (4/22) and 10% (2/20) of the atopic and healthy controls, respectively (P 13%) had the highest sensitivity and specificity, with significantly better ability than specific IgE testing for the identification of A. simplex sensitization in patients with CU. During the 6-month follow-up, clinical improvement was seen in all patients, and specific IgE and BAT results decreased to normal values in 6/10 (60%) and 10/10 (100%) patients, respectively. Conclusions. BAT can be considered a reliable new in vitro method to evaluate A. simplex hypersensitivity in patients with CU, supplementing standardized procedures in both diagnosis and follow-up.

Sezary Syndrome (SS) is an aggressive variant of cutaneous T cell lymphoma (CTCL) characterized by an accumulation of malignant T lymphocytes - the Sezary (SS) cells - in the skin, lymph nodes and blood. Until now, most studies on the characterization of this lymphoma have been performed on circulating SS cells because of the easy availability of tumor material from this compartment. For this reason, the role of skin microenvironment has not been characterized although it might play a fundamental role in the pathogenesis and in the disease progression of this neoplasia. According to this view, we recently demonstrated that PTEN downregulation, due to genetic loss and microRNA deregulation, commonly observed in SS patients, mainly activates AKT in skin-resident respect to blood-derived SS cells. This data underline how microenvironment signals amplify genetic abnormalities. Based on these observations, we investigated the effect of the skin microenvironment on SS cells in vivo. Comparing skin and blood-derived SS cells concurrently obtained from SS patients, we demonstrate that skin milieu promotes proliferation and PI3K/AKT/mTORC1 activation, a pathway that we prove to have multiple copy number (CN) alterations in our cohort of forty-three patients, thus indicating these genetic lesions as valid therapeutic targets to treat SS. At the end, we also demonstrate that the progressive increase of these CN alterations is associated to the shortening of overall survival of SS patients. Citation Format: Cristina Cristofoletti, Antonella Bresin, Mario Picozza, Maria Cristina Picchio, Francesca Passarelli, Francesca Monzo, Mauro Helmer Citterich, Alessandro Monopoli, Roberto Benucci, Maria Cantonetti, Alessandra Frezzolini, Enrico Scala, Elisabetta Caprini, Giandomenico Russo, Maria Grazia Narducci. The role of PI3 kinase pathway in the the skin of Sezary syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 761.

Background: No data exist as to Th2 chemokines in erythema multiforme (EM) and Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN). Objective: To evaluate thymus- and activation-regulated chemokine (TARC), macrophage-derived chemokine (MDC) and regulated upon activation, normal T-lymphocyte-expressed and secreted chemokine (RANTES) expression in EM and SJS/TEN and to correlate with the serum levels of the Th1 promoter interleukin (IL)-12 and soluble Fas ligand (sFasL). Materials and Methods: IL-12, sFasL, TARC, MDC and RANTES expression were analyzed by ELISA techniques in 31 untreated EM (n = 24) or SJS/TEN (n = 7) patients and in 28 healthy donors (HD). Results: EM and SJS/TEN exhibited significantly higher levels of TARC, IL-12 and sFasL with respect to HD. TARC upregulation paralleled both the IL-12 (p = 0.0225) and sFasL increase (p = 0.0194). Conclusions: Our results support a role of TARC in the pathophysiology of EM/SJS/TEN and confirm the coexistence of a Th2 response in addition to the predominant Th1 profile.

Summary Background Erythema multiforme (EM) and Stevens–Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) are caused by a dysregulation of cellular immunity. Objectives To evaluate further the potential role of certain cytokines and chemokine receptors in cutaneous lesions of patients affected by EM and SJS/TEN and to establish whether such diseases are polarized preferentially towards a T-helper (Th) 1 or Th2 pattern. Methods Biopsy specimens from eight patients with EM, six with SJS/TEN and three healthy controls were stained for immunohistochemical examination using the alkaline phosphatase–antialkaline phosphatase method. The monoclonal antibodies used included those to tumour necrosis factor (TNF)-a, interferon (IFN)-c, interleukin (IL)-2, IL-5, IL-13, receptor 3 for C-C chemokines (CCR3), receptor 3 for C-x-C chemokines (CXCR3) and CXCR4. Results The SJS/TEN specimens showed significantly higher expression of all the cytokines and chemokine receptors (except CXCR3) tested than the EM specimens. Both lesional series showed significantly higher expression of all the receptors tested than the healthy control specimens, with the sole exception of a lower expression of CCR3 in EM specimens. Comparison between molecules associated with a Th1 or Th2 response revealed a predominance of Th1 response in EM and no significant imbalance between Th1 and Th2 in SJS/TEN. Conclusions We have provided further evidence that TNF-a is strongly expressed in SJS/TEN lesions and therefore it may be involved in the epidermal necrosis featured in such diseases. IFN-c may play an important role both in EM and SJS/ TEN. IL-2, IL-5 and IL-13 may contribute to the cutaneous immunoinflammation in these diseases. Chemokine receptors may be involved strongly in the recruitment of inflammatory cells in lesional skin. In our cases we found a sharp polarization towards a Th1 pattern in EM, while the SJS/TEN lesions showed a mixed Th1/Th2 pattern.

We have previously described proopiomelanocortin (POMC) gene-expression in human normal cultured dermal fibroblasts, and its dose- and time-dependent modulation by transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha). The aim of the work described here was to investigate POMC-derived peptide release in vitro by cultured fibroblasts following incubation with different concentrations of both TNF-alpha and TGF-beta for 24 hours (1, 5, and 10 ng/ml). The effect of simultaneous addition of both TNF-alpha and TGF-beta (10 ng/ml) was also evaluated. Culture supernatants of human skin fibroblasts were collected to detect adrenocorticotropin hormone (ACTH), alpha-melanotropin (alpha-MSH), and beta-endorphin (beta-EP) levels by specific immunoenzymatic assay. We investigated the in vitro histamine-releasing activity of the POMC-derived peptides, alpha-MSH and beta-EP, on human foreskin mast cells. Detection of cleavage products in supernatants from cultured normal human dermal fibroblasts indicated intracellular processing by POMC protein. We were able to measure detectable levels of all peptides in basal conditions. TNF-alpha addition resulted in an increase in beta-EP and ACTH levels. TGF-beta-stimulated fibroblasts showed no alteration in beta-EP and alpha-MSH levels, whereas ACTH release was significantly enhanced. Both alpha-MSH and beta-EP induced histamine release from human foreskin mast cells in vitro with beta-EP-induced histamine levels as high as those observed with the calcium ionophore, ionomycin. Our data document fibroblast POMC-derived peptide release and modulation by cytokines, suggesting that they have a possible role in extracellular matrix deposit regulation and skin inflammation.

Summary Background Erythema multiforme (EM) and Stevens–Johnson syndrome (SJS) ⁄toxic epidermal necrolysis (TEN) are determined by a dysregulation of cellular immunity. Objectives To evaluate the effector role of cellular immunity and the involvement of the CD40 ⁄CD40 ligand (CD40L) system in the pathogenesis of EM and SJS ⁄ TEN. Methods Biopsy specimens from eight patients with EM and six with SJS ⁄ TEN were stained for immunohistochemical examination using the alkaline phosphatase ⁄antialkaline phosphatase method. The monoclonal antibodies used included those to CD1a, CD4, CD8, CD40, CD40L, CD68, Fas, Fas ligand (FasL) and myeloperoxidase. Results The cellular infiltrate in both EM and SJS ⁄ TEN lesions was composed mainly of T lymphocytes and CD68+ macrophages. We also detected large amounts of neutrophils. Fas and FasL were very highly expressed in SJS and TEN, but weakly in EM. CD40 staining was strong in all tissue sections; there were numerous CD40L+ cells in SJS ⁄ TEN but much fewer in EM. Conclusions Activated T lymphocytes and macrophages, but also neutrophils, are presumably the main triggers of mucocutaneous damage in the SJS ⁄ TEN disease spectrum. The Fas ⁄ FasL system is significantly expressed in SJS ⁄ TEN lesions, but not in EM, where this apoptotic pathway presumably does not play a pivotal role in the epidermal damage. We suggest that the CD40 ⁄CD40L system may represent an important pathway of induction of SJS ⁄ TEN lesions, while in EM it would contribute to the immunoinflammation only as a second-line mechanism.