Quantity, distribution and immunophenotypical modification of dendritic cells upon biological treatments in psoriasis.

Psoriasis is an immune-mediated disease which affects a large world population. It has long been considered a dermatological disorder in which keratinocytes and lymphocytes play a relevant pathogenic role. The aim of our study is to more closely observe and better define the role of dendritic cells (DCs) in psoriasis. We made a comparative analysis of the antigenic profile and the number, by immunohistochemical and electron microscopical study, of skin biopsy samples from psoriatic patients before and 4 months after biological treatments. Our results demonstrate an abundant distribution of activated DCs in lesional skin of psoriatic plaques and a marked decrease after biological therapies [a decrease of 70% for Langerhans cells (LCs) and mature myeloid dendritic cells (mDCs) and of 50% for plasmocytoid dendritic cells (pDCs)]. Both previous reports and the results of the present study support an underlying persistent immune response involving DCs in the onset and persistence of psoriasis. As DCs play a pivotal role in pathogenesis of psoriasis by presenting antigens via major histocompatibility complex class (MHC) II molecules, the present study supports the view that biological therapies are also effective in clearing psoriatic lesions as well as in reducing the number of DCs

Efalizumab-induced severe thrombocytopenia can be resolved

Efalizumab is a monoclonal a humanized recombinant IgG1 monoclonal antibody which targets the CD11a, the alpha-subunit of LFA-1 (lymphocyte function-associated antigen-1). It acts by blocking the T-lymphocyte pathogenetic mechanisms of psoriasis. Thrombocytopenia is an adverse event that occurs during therapy. Thrombocytopenia can be mild and can occur quite early during treatment, together with leukocytosis. Both adverse events tend to normalize with ongoing therapy, or, in cases worsening, with therapy suspension. There have been multiple reports of thrombocytopenia associated with efalizumab therapy for the treatment of psoriasis. The general recommendation is to check platelet counts monthly for the first 3 months of efalizumab therapy, then every 3 months for the duration of therapy. According to our experience on a wide range of patients, it is useful to check platelets every month for the first 6 months of therapy. We report a case of efalizumab-associated thrombocytopenia that occurred after 16 weeks of therapy together with clinical worsening of skin lesions. The peculiarity of our case is the absence of signs and symptoms linked to thrombocytopenia and the quick return to normal platelet count without corticosteroid therapy

Modelling terrestrial route networks to understand inter-polity interactions (southern Etruria, 950-500 BC)

Ancient regional routes were vital for interactions between settlements and deeply influenced the development of past societies and their "complexification". At the same time, since any transportation infrastructure needs some level of inter-settlement cooperation to be established, they can also be regarded as an epiphenomenon of social interactions at the regional scale. Here, we propose to analyze ancient pathway networks to understand the organization of cities and villages located in a certain territory, attempting to clarify whether such organization existed and if so, how it functioned. To address such a question, we chose a quantitative approach. Adopting network science as a general framework, by means of formal models, we try to identify how the collective effort that produced the terrestrial infrastructure was directed and organized. We selected a paradigmatic case study: Iron Age southern Etruria, a very well-studied context, with detailed archaeological information about settlement patterns and an established tradition of studies on terrestrial transportation routes, perfectly suitable for testing new techniques. The results of the modelling suggest that a balanced coordinated decision-making process was shaping the route network in Etruria, a scenario which correlates well with the picture elaborated by different scholars using a more traditional technique.Comment: 29 pages, 6 figures. This version: extends and corrects text, adds 1 explanatory figure, develops conclusion

Structural and ultrastructural evaluation of the effects induced by IL-22 alone or in combination with psoriatic cytokines in an ex-vivo human skin model

L-22 is a pro-inflammatory cytokine playing a crucial role in the pathogenesis of psoriasis, an autoimmune chronic inflammatory skin disease. The immunological acti- vation during the progression of the psoriatic lesion is driven by IL-22 together with other cytokines, such as (TNF)-alpha and interleukin (IL)-17 [1]. The aim of our study was to evaluate the early, direct, and specific effect of IL-22 alone or in combination with TNF-alpha and IL-17 by immunofluorescence on i) the molecular composition of intercellular junctions (desmocollin (DSC)1, E-cadherin, and occludin), ii) keratin(K) 10 and 17 expression, iii) keratinocyte proliferation, and, by transmission electron micros- copy (TEM), on the ultrastructural morphology of the skin. An innovative model of human skin culture standardized in our laboratory, in which a psoriatic microenviron- ment was reproduced, was used [2]. Skin explants obtained from plastic surgery of healthy 20-40 year-old women (n = 7) after informed consent were cultured overnight in Dulbecco’s modified Eagle’s medium, divided before adding IL-22 or a combination of the three cytokines, and harvested 24, 48, and 72 hours after cytokine incubation.Interestingly, keratinocyte proliferation was inhibited after exposure to the combi- nation of cytokines while was not affected by IL-22 incubation. In both experimental groups, starting from T24, occludin immunostaining was non homogeneously distrib- uted, K10 immunostaining gradually decreased in scattered clusters in the spinous layer, while K17 expression was induced and progressively increased with time in the suprabasal layers of epidermis. By TEM, after IL-22 incubation we observed keratin aggregates in the perinuclear cytoplasm of cells, while the combination of the three cytokines induced an enlargement of intercellular spaces.Altogether, our results suggest that IL-22 mainly affects keratinocyte terminal dif- ferentiation, whereas, for inducing an impairment in cell proliferation, a more com- plex psoriatic-like microenvironment is needed.

A proinflammatory microenvironment induces NFkB activation and beta-defensin expression through specific Toll Like Receptors in a 3D human skin model

Psoriasis is an autoimmune skin disease characterized by the formation and the progression of silvery plaques on the extensory surfaces of our body. Proinflammatory cytokines as Tumor Necrosis Factor (TNF)-alpha, interleukin (IL)-17, IL-22 and IL-23 represent for the normal skin a psoriatic microenvironment. In the 3D human skin model standardized in our lab in the last decade, we were able to dissect the events in which each cytokine exerts a specific effect, e.g. keratinocyte proliferation, Langerhans cell activation, cytoskeleton arrangement, and, more recently, the epidermal expression of Toll like Receptors (TLRs) 2, 7, 9. Several experimental studies reported that in psoriasis TLRs are expressed and their activation triggers i) NFkB translocation from the cytoplasm to the nucleus and ii) the release of beta defensins (HBDs). The present study was aimed at investigating the intracellular NFkB activation and HBD1 and HBD2 expression induced by a cytokine mix (TNF-alpha, IL-17, IL-22, IL-23) by indirect immunofluorescence. Bioptic samples of normal human skin were obtained after aesthetic surgery of young healthy informed women (n=7). After overnight incubation to reduce mechanical and termical stress, skin fragments were incubated in a Transwell system for 5 (T5), 24 (T24), and 48 (T48) hours with the cytokine mix. Parallel control samples were carried out and each patient was represented at all time points. In controls at all time-points NFkB was localized only in the cytoplasm, while, starting from T5, scattered basal nuclei were observed in the cytokine-incubated samples. At later time points, in the upper spinous and granular layers, NFkB nuclear immunostaining was evident. HBD2 expression was affected after cytokine mix exposure, while HBD1 distribution was similar to controls. Thanks to this simple but effective model, a deep knowledge of the early events occurring in the normal epidermis exposed to cytokines can be achieved, excluding the contribution of the blood and lymphatic vessels herein absent. This basic research can thus represent an important tool for targeting and counteracting single phenomenon leading to the formation/progression with the most innovative biological drugs

Adalimumab in the management of psoriasis and psoriatic arthritis: Results from a Delphi investigation

Background and Objectives: Psoriasis (PsO) and psoriatic arthritis (PsA) are often undertreated and require a multidisciplinary approach. In recent years, patent expiration has allowed the introduction of tumor necrosis factor inhibitor (antiTNF) biosimilars, which have stimulated a significant increase in the use of biological therapies. This article reports the f indings of a multidisciplinary approach to achieve a consensus on the use of adalimumab in patients with PsO or PsA. Methods: A voting panel of 36 Italian dermatologists and rheumatologists were chosen by eight Italian clinicians (the Board), to provide a consensus on the real-world management of PsO and PsA with adalimumab using the Delphi Method, comprising three survey rounds. Twelve statements were defined by the Board and submitted to the panel (rating scale 1–7). Results: Clinicians reached a wide consensus on the effectiveness (score 6–7: 67%) and long-term efficacy (6–7: 100%) of adalimumab in all clinical forms of PsO and PsA, including pediatric patients (6–7: 85%). Considering cost-effectiveness and safety, adalimumab is suggested as a first-line treatment in patients with enthesitis, predominant peripheral arthritis, axial involvement or associated inflammatory bowel disease (IBD) or uveitis. Adalimumab can be also considered after failure of etanercept (6–7: 94%). Conclusion: Results from this Delphi study clearly show an overall consensus on the use of adalimumab in the management of PsO and PsA, particularly as first-choice for specific subpopulations (uveitis, IBD, hidradenitis suppurativa). Considering the cost-effectiveness of biosimilars within Italy, adalimumab may represent an effective and safe first-line treatment for patients with moderate-to-severe PsO or PsA, and a valid choice for switching after failure

Effect of TNF-alpha and IL-17 on TLR expression and Langerhans cells phenotype in a three-dimensional model of normal human skin: a morphological study

Toll-like receptors (TLRs) are essential for innate immunity and contribute to create the skin barrier. Their abnormal stimulation is involved in the development of several dermatological diseases, among which psoriasis. Tumor Necrosis Factor (TNF)-alpha and interleukin (IL)-17 play a pivotal role in the pathogenesis of psoriatic plaques and their proinflammatory activity can affect Langerhans cell (LC) phenotype. In a well characterized three-dimensional model of organotypic cultures of normal human skin [1-3] we evaluated the effect of TNF-alpha and IL-17 on the expression of TLR2 and 9 by immunofluorescence, on the ultrastructural morphology of keratinocytes and LCs by transmission electron microscopy (TEM). Human skin explants (n=7) were cultured at the air-liquid interface overnight in a Transwell system and exposed to 50 ng/ml IL-17 or 100 ng/ml TNF-alpha or a combination of both cytokines. Samples were harvested 24 (T24) and 48h (T48) after cytokines incubation. After incubation with IL-17 and IL-17+TNF-alpha, TLR2 immunostaining was not detectable in the basal layer, differently from controls and TNF-alpha-treated samples. Conversely, TLR9 expression was progressively induced in granular keratinocytes in all cytokine-exposed groups. By TEM, enlargements of intercellular spaces were evident especially and, after IL-17 treatment, LCs showed an activated phenotype. At T24 LCs number increased indicating that TNF-alpha and IL-17+TNF-alpha exert a chemoattractant activity, while at T48 only IL-17+TNF-alpha maintained this effect on trapping LCs in epidermis. TNF-alpha and IL-17 differently affect LCs behaviour and TLR expression, with a specific contribution to the inflammatory loop underlying the lesion formation. These results suggest that the simultaneous inhibition of the effect of different cytokines with a defined role in the pathogenesis of psoriasis could improve psoriasis treatment

Langerhans cells and Toll Like Receptors: how do they act and react in an in vitro psoriatic microenvironment?

Tumor Necrosis Factor (TNF)-α, interleukin (IL)-17, IL-22 and IL-23 are involved in the psoriasis pathogenesis and represent a strong proinflammatory stimulus. Both epidermal keratinocytes (KCs) and Langerhans cells (LCs) early respond promoting an early epidermal response [1, 2]. Human skin can count on the cellular response supported by LCs and on innate immunity through the expression of Toll-like Receptors (TLRs) [4]. We aimed at investigate whether the exposure of normal human skin to a combination of TNF-α, IL-17, IL-22, and IL-23 (cytokine mix) affected i) LCs immunophenotype, ii) expression of TLR2 and TLR9 and iii) KC proliferation. Human skin samples were obtained after plastic surgery (n = 5) and exposed to the cytokine mix in a Transwell system at air-liquid interface, with a parallel control group. Samples were harvested 24 and 48 hours after cytokine stimulation, processed in parallel for immunofluorescence or ultrastructural analysis. A decrease of cell proliferation was evident in samples exposed to cytokine mix for 24 hours and this phenomenon was more and more evident later. TLR2 immunopositivity progressively disappeared in the basal layer after cytokine mix exposure compared to the control group, while TLR9 expression was induced in scattered granular keratinocytes. By TEM, LCs showed an activated phenotype. In conclusion, these results suggest that, in a microenvironment mimicking the psoriatic plaque, epidermis early stimulates two important lines of defense, thus proposing that a therapeutic intervention in this direction can interfere with the formation/progression of the psoriatic plaque