Search

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.

Lupus nephritis (LN) may represent a diagnostic problem, particularly in pediatric patients that present with typical histological lesions but do not fulfill the American Rheumatism Association (ARA) criteria for the diagnosis of systemic lupus erythematosus (SLE). Based on the well-described deposition of immunoglobulins (Ig) and complement at the dermoepithelial junction in SLE, we hypothesized that skin biopsies may help in the diagnosis of LN. To test this hypothesis, we carried out a pilot study, performing a skin biopsy in 22 patients with LN and 13 patients with lupus-like lesions, regardless of the time elapsed from onset of renal disease. The latter group of patients was further divided into a purely renal group, designated as isolated full-house nephropathy (FHN), and a dubious cases group, presenting with additional clinical and biological features consistent with SLE but insufficient for diagnosing SLE. None of the 6 isolated FHN patients had positive skin immunofluorescence. Conversely, 5/7 patients in the dubious cases group (p < 0.02) and 13/22 in the LN group (p < 0.002) had positive staining for C1q, and 5/7 patients in the dubious cases group (p < 0.02) and 16/22 patients in the LN group (p < 0.001) had positive staining for IgM. No correlation was observed with the time elapsed from the initial diagnosis. These data suggest that skin biopsies may help distinguishing LN from isolated FHN. In addition, they identify an intermediate group of patients with evidence of systemic involvement despite the absence of a sufficient number of ARA criteria to be labeled as SLE.

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.

Background: Multiple drug allergy syndrome is a clinical condition characterized by reactions against more than one different class of, both pharmacologically and structurally, unrelated drugs. Scanty data are available to date about a multiple drug delayed hypersensitivity syndrome. Our aim was to report the case of a delayed reaction to both β-methasone (β-MT) and penicillin-G (pen-G) occurring in the same patient, and analyse β-MT- and pen-G-specific T-cell Lines (TCLs) with regard to their specificity, phenotype and cytokine profile. Methods: We generated two drug-specific TCLs from biopsies at the site of positive intradermal reactions, and analysed their immunophenotype, T-cell receptor Vβ (TCR-Vβ) domains expression and cytokine profile. Results: We demonstrated the specificity of the T cells isolated from positive intradermal test reactions to pen-G and β-MT through the strict dose-dependent proliferation in response to drug-pulsed autologous antigen presenting cells. Fluorescence activated cell sorter (FACS) analysis revealed a predominance of CD4+ cells in the inflammatory cell infiltrate of intradermal test with β-MT, while a predominance of CD8+ T cells in the site of delayed reaction to pen-G was found. The drug specific CD4+ and CD8+ T cells were heterogeneous, with regard to TCR-Vβ usage. CD8+ pen-G-TCL displayed a preferential T helper 2 (Th2) profile, while a substantially heterogeneous pattern of cytokine production characterized specific β-MT TCL. Conclusion: The study describes the coexistence in the same patient of a delayed hypersensitivity to both penicillin G and β-MT, driven, respectively, by pen-G-specificTh2-skewed CD8+ and β-MT specificTh0 CD4+ T cells. This case further support the existence of a multiple drug allergy syndrome also for delayed hypersensitivity.

Cell migration is mediated by a group of chemotactic cytokines called chemokines: low molecular weight molecules that have been shown as important leukocyte chemical attractants to sites of inflammation and infection. Eotaxin-1, also called CCL11, was first described in 1994, as a highly specific eosinophils chemokine. Many cell types including lymphocytes, macrophages, bronchial smooth muscle cells, endothelial cells and eosinophils, are able to produce this chemokine, predominantly after cytokine stimulation, however little is known about its expression in human skin in vivo. Eotaxin-1 also regulates the chemiotaxis and, in some conditions, activation of basophils, mast cells and T lymphocytes. Chemokine receptors are named from their ligand families, thus the CC chemokine eotaxin-1 binds to the CCR3 receptor which is expressed on eosinophis, mast cells, Th2 type lymphocytes and even on keratinocytes. It seems that eotaxin-1 is one of the most important cytokines involved in tissue inflammation playing a central role in the pathogenesis of allergic airway diseases (asthma and rhinitis), in inflammatory bowel disease and gastrointestinal allergic hypersensitivity and recently it has been proposed as a therapeutical target for these conditions. Our group has studied the role of eotaxin-1 in the pathogenesis of two skin conditions: dermatitis herpetiformis and AIDS-associated eosinophilic folliculitis, demonstrating that this chemokine, together with Th2 type cytokines (IL-13 and IL-4) is important in cell recruitment, inflammation and tissue damage; moreover eotaxin has proven to paly an important role in other skin conditions such as, bullous pemphigoid, pemphigoid gestationis, atopic dermatitis and allergic drug reactions Recent advances in the understanding of eotaxin-1-mediated mechanisms of chemotaxis in allergic and inflammatory conditions may predict that therapeutic antagonism is achievable. This paper will focus on the role that eotaxin and its receptor play in the pathogenetical mechanism in a number of dermatologic diseases, some of which, like atopic dermatitis, may benefit from the introduction of novel and more selective therapeutic options.

Background: Chemokines play a central role in atopic eczema/dermatitis syndrome (AEDS). Interleukin 16 (IL-16) has been described as a main cytokine involved in CD4+ cell recruitment during inflammation. Recently the influx of CD4+ lymphocytes has been related to the up-regulation of IL-16 in AEDS skin lesions. Circulating β-chemokines (Eotaxin and RANTES) and IL-16 were investigated in children with AEDS to correlate their presence with the severity of the disease. We also measured serum levels of soluble CD30 (sCD30), a marker of Th2 immune responses related to AEDS disease activity. Methods: Serum levels of eotaxin, RANTES, IL-16 and sCD30 were measured by immunoenzymatic assay in paediatric patients with pure AEDS (pAEDS, n = 39); the severity of the disease was graded by SCORAD. Fifteen children with AEDS in presence of respiratory allergy (AEDS+A), 15 with allergic asthma (A) and 20 age-matched healthy donors were investigated as control groups. Results: When compared to normals, high amounts of Eotaxin and IL-16 were detected in sera of pAEDS (P = 0.002; P

BACKGROUND Physical urticaria (PU) includes a heterogeneous group of urticarias whose etiopathogenic aspects are still obscure and whose therapeutical management is often difficult. We have previously demonstrated the efficacy of a sequential treatment with nimesulide, a unique nonsteroidal anti-inflammatory drug, and ketotifen in various forms of PU. METHODS The expression of some inflammatory parameters was evaluated in 10 patients affected with some forms of PU. In particular, serum levels of interleukin (IL)-4, IL-1beta, tumor necrosis factor (TNF)-alpha, adhesion molecules (sELAM, sICAM-1 and sVCAM), soluble receptors (sIL-2R, sCD30, sCD23) and IgE were assessed. Moreover, the cutaneous expression of IL-1beta, TNF-alpha and ICAM-1 was studied on biopsy specimens taken from nonlesional skin of patients. The same parameters were further evaluated in both skin and serum following treatment with nimesulide and ketotifen, in order to better understand the possible effects of these agents on the inflammatory network of PU. RESULTS Before therapy we could detect significantly higher serum levels of IL-1beta and TNF-alpha (P < 0.001) and of circulating adhesion molecules in comparison with controls (sELAM, sICAM: P < 0.001; sVCAM: P < 0.02); after treatment, a significant reduction of each was observed (P < 0.05). Simultaneously, a high expression of IL-1beta, TNF-alpha and ICAM-1 was detected in all skin specimens at the baseline, with a relevant decrease following therapy. CONCLUSIONS These results confirm the therapeutical value of treatment with nimesulide and ketotifen in PU and suggest that these agents are able to reduce the release and the expression of some inflammatory molecules that are up-regulated in PU.

Several published studies suggest the involvement of immune and inflammatory factors in psoriasis. We recently demonstrated that the number of circulating ICAM-1 + lymphocytes and the levels beta 2-microglobulin are useful parameters in monitoring the activity of the disease. In this study we investigated serum levels of SCCr-Ag in 24 patients with psoriasis in order to determine whether this antigen is a marker of disease activity. Our results demonstrated high serum levels of SCCr-Ag, IL-2, sIL-2R, sCD4, sCD8, sICAM-1 and beta 2-microglobulin in the acute phase of psoriasis. Furthermore, we found a positive correlation of SCC with TBSA, PASI score, sICAM-1 and beta 2-microglobulin. These data demonstrate that serum levels of SCCr-Ag depend on the severity of the disease and correlate with both immunological and inflammatory markers of disease activity. We suggest that expression of SCCr-Ag may be induced by cytokines in the microenvironment of psoriatic lesions, suggesting that SCC-Ag may play a role in the inflammatory process.

In this study we analysed the in vitro effect of recombinant interferon gamma on cytotoxic activities mediated by both lymphoid and polymorphonuclear cells from 16 patients with myelodysplastic syndromes. Our results indicate the inability of interferon to restore the defective natural killer activity, natural killer cells and lectin-induced cytotoxicity. On the contrary we detected a boosting effect on the depressed polymorphonuclear cell cytotoxic activities. In our view, the ability of interferon to potentiate polymorphonuclear cell lytic efficiency could support an alternative defensive pathway against either neoplastic or infectious agents.

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.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.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 (P0.0001). BAT (cut-off13%) 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.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.

Background Treatment of pemphigus vulgaris can be challenging. Systemic steroids associated with other immunosuppressant agents are the mainstay of therapy and have dramatically reduced morbidity and mortality from pemphigus vulgaris. In some patients, however, these agents are not able to control the disease or have severe adverse effects. Rituximab (MabThera; Roche, Basel, Switzerland), a chimeric monoclonal anti-CD20 antibody, induces depletion of B cells in vivo and has shown efficacy in patients with refractory antibody-mediated autoimmune disorders. We report 10 cases of pemphigus vulgaris and 2 cases of pemphigus foliaceous treated with rituximab—to our knowledge the largest series of patients so far—and review the existing literature on the topic. Observation The 12 patients were selected for treatment with the anti-CD20 antibody. Rituximab was administered intravenously at a dosage of 375 mg/m 2 once weekly for 4 weeks. The treatment was well tolerated, and all 12 patients showed a good clinical response during an 18-month follow-up period, along with a consensual decline of the serum antidesmoglein titers. No infectious complications were observed. Conclusions Rituximab is able to induce a prolonged clinical remission in patients with both pemphigus vulgaris and pemphigus foliaceous after a single course of 4 treatments. The preliminary experiences worldwide make rituximab a promising therapeutic option for patients with autoimmune diseases. The high costs and the limited knowledge of long-term adverse effects, however, limit its use to selected patients with treatment-resistant or life-threatening disease.

Background: Functional autoantibodies against the α-chain of the high-affinity IgE receptor (FcɛRIα) identify a subset of patients with chronic urticaria (CU) due to autoreactivity, as assessed by an in vivo positive response to autologous serum skin test (ASST). We performed a study to standardize the serum-induced basophil activation assay by flow cytometry (FCM) using a new tricolour method, assessing the diagnostic performance of this test in discriminating between ASST+ and ASST− CU patients. Methods: Sera of 64 CU patients (22 ASST+ CU and 42 ASST− CU) and 10 healthy subjects were tested for their ability to induce basophil CD63 expression when incubated with whole blood of both atopic (DA) and non-atopic donors (DNA). Using a triple-labelled strategy with anti-CD123, anti-HLA-DR and anti-CD63 antibodies, CD63+ basophils were identified on a selected population of CD123+ HLA-DR- cells. In 3 ASST+ CU patients who underwent cyclosporine therapy, the assay was performed before and after treatment. Results: The ASST+ CU sera resulted in a significant higher induction of basophil CD63 expression compared with ASST− CU and healthy donors sera; when whole blood from DA was used, sensitivity and specificity of the assay were 95.5% and 90.5% respectively. ASST+ CU serum activity was significantly decreased during cyclosporine A treatment, in parallel with clinical remission. Conclusions: Chronic urticaria serum-induced CD63 expression assay performed on DA whole blood by means of our tricolour FCM method could be the most useful tool for identification of a subset of patients with autoimmune CU and may become a promising tool also for monitoring treatment efficacy.

SUMMARYSystemic sclerosis (SSc) is a connective tissue disorder characterized by excessive collagen deposition in the skin and internal organs. Several cytokines and chemokines have been implicated in the induction of fibrosis, but a definitive relationship between specific cytokines and organ involvement has not been established yet. Serum samples, PBMC and T cell lines (TCL) obtained from 54 patients affected by SSc and 20 healthy donors (HD) were examined by ELISA for Interferon-γ (IFN-γ ), interleukin (IL)-4, IL-6, IL-10, IL-18, Transforming growth factor (TGF)-β1, Tumour necrosis factor (TNF)-α, sCD30, Macrophage derived chemokine (MDC), Monocyte chemoattractant protein (MCP)-1, Macrophage inflammatory protein (MIP)-1α and Regulated on activation normal T-cell expressed and secreted (RANTES). In all the SSc serum samples, we found significantly increased levels of IL6, TNFα and MCP-1 but reduced amounts of γ-IFN and MDC. IL6, IL10, IL18, MIP-1α and TNFα measured in supernatants from PHA-stimulated PBMC and IL6, MCP-1 and RANTES in supernatants from stimulated TCL were also increased in patients. MDC was decreased in all the biological SSc sources studied. TGF-β1, IL10, and sCD30 were produced at a significantly lower level by SSc TCL. Serum IL6 and sCD30 levels were significantly increased in dc-SSc patients compared to lc-SSc as were levels of MCP-1 produced by PBMC and IL10 from TCL. We observed a strict relationship between pulmonary fibrosis and IL10, MCP-1 (both from TCL) and serum IL6. Kidney involvement was related to serum MCP-1 levels and IL18 production from PBMC. Oesophageal involvement correlated with MDC production from PBMC and IL10 synthesis by TCL. We showed that IL-6, IL-10, MDC and MCP-1 are variably associated with internal organ involvement and allow the discrimination between limited and diffuse forms of the disease.

SUMMARYCutaneous infiltration of activated CD4+ T cells and eosinophils is an early event in blister formation during bullous pemphigoid (BP), suggesting that the trafficking of circulating leucocytes through the sites of inflammation, their activation and cytokine release is crucial in the pathogenesis of the disease. IL-16 is a major chemotactic factor able to recruit CD4+ cells in the skin during inflammation and to induce the expression of functional high-affinity interleukin (IL)-2 receptors, thus contributing to cellular activation and proliferation. We performed a study in order to evaluate the presence of IL-16 in skin samples and sera and blister fluids of patients affected with BP in active phase of the disease (n = 39), compared with healthy donors studied as control group. Ten patients were also evaluated before and after steroid therapy. Our results demonstrated that IL-16 was expressed strongly by keratinocytes and by dermal infiltrating CD4+ T lymphocytes in lesional skin of BP patients. High levels of IL-16 were detected in sera and blisters of BP, significantly higher in respect to healthy donors. When patients were investigated for the presence of eosinophil cationic protein (ECP) and soluble CD30 (sCD30) to reveal signs of eosinophils and Th2-cells activation, we found a positive correlation between IL-16 serum levels and both ECP and sCD30, suggesting that IL-16 is involved in Th2 lymphocytes and eosinophils recruitment during BP.

The aim of this study was to investigate the imbalance between Th-1 and Th-2 cytokines in systemic lupus erythematosus patients (SLE) and to asses if any of these cytokines could be related to disease activity.Twenty SLE patients and 20 healthy individuals were investigated. Blood samples were collected to evaluate, using ELISA method, serum levels of a wide array of cytokines including: Th-1 type cytokines (Interleukin (IL)-12, Interferon (IFN)-gamma), Th-2 cytokines (IL-4, IL-10), pro-inflammatory cytokines (tumor necrosis factor (TNF)-alpha, IL-1beta and IL-18). Disease activity was assessed using the SLE Disease Activity Index (SLEDAI). Data were evaluated using the Mann-Whitney and Spearman's rank tests.The SLE patients group had a higher IL-4, IL-10, IL-12 and IL-18 serum concentration compared to the normal control group. IL-18 was negatively correlated with IL-4 and positively correlated with IFN-gamma. No serum cytokine level was correlated with disease activity except for IL-18, which was found strongly correlated with "active disease", defined as SLEDAI8 points. IL-18 showed no correlation with pro-inflammatory cytokines.Our results show that Th-1 as well Th-2 cytokines can be elevated in SLE patients suggesting that lupus is a complex disease that may be supported by different cytokine patterns in different time-points. Only IL-18 has been found to be disease-activity related. The role of IL-18 in the pathogenesis of SLE might be important through apoptosis-mediating properties.

Several skin infiltrating inflammatory cells, such as eosinophils, neutrophils and activated T lymphocytes, are involved in bullous pemphigoid (BP) blister formation. The presence of CD4+ T cells able to produce IL-4 and IL-5 suggests Th2 involvement in the disease. The role of eotaxin in the recruitment of eosinophils into inflammatory sites has been recently described and the specific eotaxin receptor, CCR3, has been documented to be expressed on eosinophils, basophils, and Th2 cells. In this study, we analyzed by immunohistochemistry the expression of both eotaxin and CCR3 in lesional skin from patients with active BP (n = 10) and control subjects affected with pemphigus vulgaris (PV) (n = 3); furthermore eotaxin concentration in BP sera and blister fluids was also evaluated by enzyme-linked immunosorbent assay (ELISA), in comparison to sera from PV and normal donors (n = 10) and to suction blisters from 3 healthy volunteers. A strong immunostaining for eotaxin and CCR3 in BP skin specimens in lesional and, to a lesser extent, in perilesional skin was observed. CCR3 expression was documented on both eosinophils and T cells infiltrating skin lesions. Eotaxin serum levels were significantly higher in BP patients when compared to healthy donors (p = 0.003) and PV patients (p = 0.01). The highest eotaxin concentration was detected in BP blister fluids, in respect to both corresponding BP sera and blister fluids from normal donors (p = 0.003). These results account for the role of eotaxin in the recruitment of activated cells at inflammatory sites during BP and the expression of CCR3 on infiltrating T lymphocytes further supports the involvement of Th2 cells in the pathogenesis of BP.

Atopic dermatitis (AD) is a chronic inflammatory skin disease frequently associated with asthma, rhinitis, and food allergy. Lymphocytes producing Th2-type cytokines (such as interleukin [IL]-3, IL-4, and IL-5) have been thought to have a key role in the pathogenesis of the disease. We have recently demonstrated that elevated serum levels of the soluble form of CD30 (sCD30), an activation marker of Th2-cell clones, correlates with disease activity in pediatric patients suffering from AD. Clinical trials have demonstrated that cyclosporin A (CyA) treatment resulted in significant improvement of clinical symptoms in patients affected with AD. In this study, we evaluated the role of CyA in modulating sCD30 release in a group of adult patients affected by severe AD treated with CyA at the dosage of 3.5 mg/kg body weight for 12 weeks. Our results demonstrated, in parallel with an improvement of clinical symptoms, a significant reduction of serum levels of both IL-4 and sCD30, thus suggesting that CyA can prevent the activation of Th2 cells observed in AD.

Atopic dermatitis (AD) is a chronic, inflammatory skin disease in which a pathogenetic role of Th2 cells has been supposed. This study investigated the presence of soluble CD30 (sCD30), an activation marker of T-cell clones able to produce Th2-type cytokines, in sera from pediatric patients affected by AD (n = 25) with no symptoms of asthma or rhinitis. The severity of the disease was graded by both the SCORAD and Costa et al. clinical scoring systems. Serum levels of sCD30 were significantly higher in patients with AD in respect to both normal donors (n = 20) and urticaria patients (n = 10), and a positive correlation between serum sCD30 and clinical score was found (r = 0.508; P = 0.01) when AD patients were evaluated by Costa et al.'s method. Furthermore, a significant association (r = 0.443; P = 0.027) between sCD30 and serum levels of the soluble interleukin (IL)-2 receptor (sIL-2R) was observed in AD. The presence of high amounts of sCD30 in atopic patients seems to confirm the role of this molecule as an activation marker useful for in vivo evaluation of a Th2 immune response, and the correlation observed with both clinical score and sIL-2R levels indicates the role of sCD30 as an additional marker of disease activity in pediatric patients with AD.

The immune system involvement in psoriasis has been documented by the presence of activated T-cells both in peripheral blood and in psoriatic skin lesions and by the intervention of cytokines in the inflammatory process. On this basis, we have undertaken a study in order to examine, in addition to activation markers such as CD25 and CD54 (ICAM-1) on peripheral blood mononuclear cells (PBMNCs) surface, serum levels of soluble interleukin (IL)-2 receptor (sIL-2R), soluble ICAM-1 (sICAM-1), soluble CD4 (sCD4), soluble CD8 (sCD8), beta 2-microglobulin and fibronectin (FN) in psoriatic patients analyzed both in acute and remission phase obtained by topical therapy alone. Our results show that PBMNCs expressing IL-2 receptor (CD25) were increased both in percentage and absolute number in respect to controls, and were not modified after remission. On the contrary, the significantly higher number of CD54+ lymphocytes evaluated in acute psoriasis, showed a reduction during the remission phase, even if the values persisted higher than controls. Serum levels of sIL-2R, sICAM-1, sCD4, sCD8 and beta 2-microglobulin were significantly higher than controls both in acute and remission phase; only FN levels were found to be lower, in patients evaluated both in acute psoriasis and after therapy, in respect to normal donors. On the whole, these results seem to indicate the persistence of both cellular and soluble activation markers even in psoriasis remission phase; in this light, we can suppose that topical therapy alone is not able to efficiently down-regulate activation mechanisms involved in the pathogenesis of the disease.

We describe the clinical and laboratory findings of a young man with mycosis fungoides. The disease was associated, since the early stages, with autoimmune purpura. Interferon alfa (IFN-alpha) administration improved this patient's condition, both the purpuric eruption and patchy cutaneous lesions, thus suggesting T-cell abnormalities may be responsible for the development of the disease.

To assess the role of platelets and lymphocyte-related immunological mechanisms in livedo vasculopathy (LV) and cutaneous small vessel vasculitis (CSVV). Livedo vasculopathy is thought to be related to the thrombotic occlusion of small and medium-sized dermal vessels. Cutaneous small vessel vasculitis comprises a heterogeneous group of disorders in which the main pathogenetic events could be modulated by circulating cytokines.Case series study of 2 groups of patients affected respectively with LV and CSVV.A large clinical and research institute for the study and treatment of cutaneous diseases.Consecutive patients with clinically and histologically proved idiopathic LV (n = 8) and CSVV (n = 20) were studied and compared with healthy donors (n = 20). Patients with potentially correlated systemic diseases were excluded.Surface expression of platelet P-selectin and circulating level of interleukin (IL) 1beta, tumor necrosis factor alpha (TNF-alpha), IL-8, IL-2, and soluble IL-2 receptor.The IL-2 and soluble IL-2 receptor levels were significantly higher in serum samples from patients with both LV (1.24 +/- 0.46 IU/mL [mean +/- SD] vs 0.46 +/- 0.24 IU/mL, P.001; 899 +/- 368 IU/mL vs 628 +/- 132 IU/mL, P.02) and CSVV (0.91 +/- 0.57 IU/mL, P.02; 1087 +/- 451 IU/mL, P.001) than in those from the healthy controls. The serum levels of IL-1beta, TNF-alpha, and IL-8 were higher in patients with CSVV than in controls (7.53 +/- 6.7 pg/mL vs 4.58 +/- 2.72 pg/mL; 23.7 +/- 12.6 pg/mL vs 10.82 +/- 2.46 pg/mL, P.001; 37.8 +/- 46 pg/mL vs 8.25 +/- 3.53 pg/mL, P.02, respectively). No significant difference in the serum levels of IL-1beta (7.2 +/- 4.9 pg/mL), TNF-alpha (12.9 +/- 3.47 pg/mL), and IL-8 (5.9 +/- 4.13 pg/mL) was observed in patients with LV compared with controls. An increased expression of platelet P-selectin was also detected in patients with LV in comparison with controls and patients with CSVV. The mean +/- SD percentage of positive cells for P-selectin was 43% +/- 5% in the patients with LV, 5.1% +/- 2% in the controls (P.001), and 5.3% +/- 2% in the patients with CSVV (P.001).Taken together, these data demonstrate that different pathogenetic mechanisms are operative in LV and CSVV. In fact, platelet and lymphocyte activation is present in LV, whereas the levels of inflammatory mediators are in a normal range. In CSVV, the high serum levels of proinflammatory cytokines suggest that they are actively involved in the pathogenesis of cutaneous vasculitis.