Marta Carretero

Inhibition of Xenografted Human Melanoma Growth and Prevention of Metastasis Development by Dual Antiangiogenic/Antitumor Activities of Pigment Epithelium-Derived Factor

Human melanoma mortality is associated with the growth of metastasis in selected organs including the lungs, liver, and brain. In this study, we examined the consequences of overexpression of pigment epithelium-derived factor (PEDF), a neurotrophic factor and potent angiogenesis inhibitor, on both melanoma primary tumor growth and metastasis development. PEDF overexpression by melanoma cells greatly inhibited subcutaneous tumor formation and completely prevented lung and liver metastasis in immunocompromised mice after tail vein injection of metastatic human melanoma cell lines. Whereas the effects of PEDF on primary tumor xenografts appear mostly associated with inhibition of the angiogenic tumor response, abrogation of melanoma metastasis appears to depend on direct PEDF effects on both migration and survival of melanoma cells. PEDF-mediated inhibition of melanoma metastases could thus have a major impact on existing therapies for melanoma.

Assessment of Optimal Virus-Mediated Growth Factor Gene Delivery for Human Cutaneous Wound Healing Enhancement

Using a recently described skin-humanized model based on the engraftment of human bioengineered skin equivalents onto immunodeficient mice, we compared the efficacy of different in vivo gene transfer strategies aimed at delivering growth factors to promote skin wound healing. The approaches involving transient delivery of keratinocyte growth factor (KGF) to wounds performed in the engrafted human skin included (1) KGF gene transfer by intradermal adenoviral injection; (2) KGF gene transfer by adenoviral vector immobilized in a fibrin carrier; and (3) KGF-adenoviral gene-transferred human fibroblasts embedded in a fibrin matrix. All delivery systems achieved KGF protein overproduction at the wound site, with a concomitant re-epithelialization enhancement. However, although direct gene delivery strategies exhibited variability in terms of the number of successfully transduced humanized mice, the use of genetically modified fibroblast-containing matrix as an in situ protein bioreactor was highly reproducible, leading to a significant improvement of the overall healing process. This latter approach appeared to be the most reliable means to deliver growth factors to wounds and also avoided the potential danger of scoring cases of faulty administration as therapeutic failures and direct exposure to viral vectors. The combined use of cell and gene therapy appears a robust tool to aid healing in a clinical context.

Modeling normal and pathological processes through skin tissue engineering.

Skin tissue engineering emerged as an experimental regenerative therapy motivated primarily by the critical need for early permanent coverage of extensive burn injuries in patients with insufficient sources of autologous skin for grafting. With time, the approach evolved toward a wider range of applications including disease modeling. We have established a skin‐humanized mouse model system consisting in bioengineered human‐skin‐engrafted immunodeficient mice. This new model allows to performing regenerative medicine, gene therapy, genomics, and pathology studies in a human context on homogeneous samples. Starting from skin cells (keratinocytes and fibroblasts) isolated from normal donor skin or patients biopsies, we have been able to deconstruct‐reconstruct several inherited skin disorders including genodermatoses and cancer‐prone diseases in a large number of skin humanized mice. In addition, the model allows conducting studies in normal human skin to gain further insight into physiological processes such as wound healing or UV‐responses.

Development of a Bioengineered Skin-Humanized Mouse Model for Psoriasis: Dissecting Epidermal-Lymphocyte Interacting Pathways

Over the past few years, whole skin xenotransplantation models that mimic different aspects of psoriasis have become available. However, these models are strongly constrained by the lack of skin donor availability and homogeneity. We present in this study a bioengineering-based skin-humanized mouse model for psoriasis, either in an autologous version using samples derived from psoriatic patients or, more importantly, in an allogeneic context, starting from skin biopsies and blood samples from unrelated healthy donors. After engraftment, the regenerated human skin presents the typical architecture of normal human skin but, in both cases, immunological reconstitution through intradermal injection in the regenerated skin using in vitro-differentiated T1 subpopulations as well as recombinant IL-17 and IL-22 Th17 cytokines, together with removal of the stratum corneum barrier by a mild abrasive treatment, leads to the rapid conversion of the skin into a bona fide psoriatic phenotype. Major hallmarks of psoriasis were confirmed by the evaluation of specific epidermal differentiation and proliferation markers as well as the mesenchymal milieu, including angiogenesis and infiltrate. Our bioengineered skin-based system represents a robust platform to reliably assess the molecular and cellular mechanisms underlying the complex interdependence between epidermal cells and the immune system. The system may also prove suitable to assess preclinical studies that test the efficacy of novel therapeutic treatments and to predict individual patient response to therapy.

Targeted silencing of DEFB4 in a bioengineered skin‐humanized mouse model for psoriasis: development of siRNA SECosome‐based novel therapies

Psoriasis is a complex inflammatory skin disease that presents a wide variety of clinical manifestations. Human β defensin‐2 (hBD‐2) is highly up‐regulated in psoriatic lesions and has been defined as a biomarker for disease activity. We explored the potential benefits of targeting hBD‐2 by topical application of DEFB4‐siRNA‐containing SECosomes in a bioengineered skin‐humanized mouse model for psoriasis. A significant improvement in the psoriatic phenotype was observed by histological examination, with a normalization of the skin architecture and a reduction in the number and size of blood vessels in the dermal compartment. Treatment leads to the recovery of transglutaminase activity, filaggrin expression and stratum corneum appearance to the levels similar to those found in normal regenerated human skin. The availability of a reliable skin‐humanized mouse model for psoriasis in conjunction with the use of the SECosome technology may provide a valuable preclinical tool for identifying potential therapeutic targets for this disease.

Identifying targets for topical RNAi therapeutics in psoriasis: assessment of a new in vitro psoriasis model.

Diseases of the skin are amenable to RNAi-based therapies and targeting key components in the pathophysiology of psoriasis using RNAi may represent a successful new therapeutic strategy. We aimed to develop a straightforward and highly reproducible in vitro psoriasis model useful to study the effects of gene knockdown by RNAi and to identify new targets for topical RNAi therapeutics. We evaluated the use of keratinocytes derived from psoriatic plaques and normal human keratinocytes (NHKs). To induce a psoriatic phenotype in NHKs, combinations of pro-inflammatory cytokines (IL-1α, IL-17A, IL-6 and TNF-α) were tested. The model based on NHK met our needs of a reliable and predictive preclinical model, and this model was further selected for gene expression analyses, comprising a panel of 55 psoriasis-associated genes and five micro-RNAs (miRNAs). Gene silencing studies were conducted by using small interfering RNAs (siRNAs) and miRNA inhibitors directed against potential target genes such as CAMP and DEFB4 and miRNAs such as miR-203. We describe a robust and highly reproducible in vitro psoriasis model that recapitulates expression of a large panel of genes and miRNAs relevant to the pathogenesis of psoriasis. Furthermore, we show that our model is a powerful first step model system for testing and screening RNAi-based therapeutics.

Applicability of bioengineered human skin: from preclinical skin humanized mouse models to clinical regenerative therapies.

Ongoing progress in the field of regenerative medicine, in combination with the development of tissue-engineered skin products, has opened new possibilities for the treatment of certain diseases in which current treatments are aimed at alleviating symptoms but are not able to get a permanent cure. Our laboratory has developed a fibrin-based bioengineered human skin that has been successfully used for permanent regenerative therapies in different situations in the clinic. Moreover, we have been able to stably regenerate human skin by orthotopic grafting of this skin equivalent onto the back of immunodeficient mice. The so-called skin-humanized mouse model system has permitted us to model several monogenic skin diseases, when keratinocytes and fibroblasts harboring the genetic defect were used. In most cases different gene therapy approaches for ex vivo correction of cells have proved effective in reverting the phenotype using this model. More importantly, the feasibility of the system has allowed us to generate a skin humanized mouse model for psoriasis, a common chronic inflammatory disease where the immune component has a pivotal role in the pathogenesis. Establishing reliable humanized animal models for skin diseases is necessary to gain a deeper knowledge of the pathogenesis and to develop novel therapeutic strategies. In this sense, the skin humanized mouse model developed in our laboratory meets the needs of this field of research.RNA interference (RNAi) has rapidly advanced to become a powerful genetic tool and holds promise to revolutionizing agriculture by providing a strategy for controlling a wide array of crop pests. Numerous studies document RNAi efficacy in achieving silencing in viruses, insects, nematodes and weeds parasitizing crops. In general, host derived pest resistance through RNAi is achieved by genetically transforming host plants with double stranded RNA constructs targeted at essential parasite genes leading to generation of small interfering RNAs (siRNAs). Small interfering RNAs formed in the host are then delivered to the parasite and transported to target cells. Delivery can be oral - worms and insects, viral infections, viruses - or through a vascular connections - parasitic plants, while delivery to target cells is by cell to cell systemic movement of the silencing signal. Despite the overall optimism in generating pest resistant crops through RNAi-mediated silencing, some hurdles have recently begun to emerge. Presently, the main challenge is delivery of sufficient siRNAs, in the right cells, and at the right time to mount; a strong, durable, and broad-spectrum posttranscriptional gene silencing (PTGS) signal. This review highlights the novel strategies available for improving host derived RNAi resistance in downstream applied agriculture.

Differential Features between Chronic Skin Inflammatory Diseases Revealed in Skin-Humanized Psoriasis and Atopic Dermatitis Mouse Models

Psoriasis and atopic dermatitis are chronic and relapsing inflammatory diseases of the skin affecting a large number of patients worldwide. Psoriasis is characterized by a T helper type 1 and/or T helper type 17 immunological response, whereas acute atopic dermatitis lesions exhibit T helper type 2-dominant inflammation. Current single gene and signaling pathways-based models of inflammatory skin diseases are incomplete. Previous work allowed us to model psoriasis in skin-humanized mice through proper combinations of inflammatory cell components and disruption of barrier function. Herein, we describe and characterize an animal model for atopic dermatitis using similar bioengineered-based approaches, by intradermal injection of human T helper type 2 lymphocytes in regenerated human skin after partial removal of stratum corneum. In this work, we have extensively compared this model with the previous and an improved version of the psoriasis model, in which T helper type 1 and/or T helper type 17 lymphocytes replace exogenous cytokines. Comparative expression analyses revealed marked differences in specific epidermal proliferation and differentiation markers and immune-related molecules, including antimicrobial peptides. Likewise, the composition of the dermal inflammatory infiltrate presented important differences. The availability of accurate and reliable animal models for these diseases will contribute to the understanding of the pathogenesis and provide valuable tools for drug development and testing.

Gene therapy based in antimicrobial peptides and proinflammatory cytokine prevents reactivation of experimental latent tuberculosis.

Mycobacterium tuberculosis (Mtb) latent infection can lead to reactivation. The design of new strategies to prevent it is an important subject. B6D2F1 mice were infected intratracheally with a low dose of Mtb H37Rv to induce chronic infection. After 7 months, mice were treated with one dose of recombinant adenoviruses encoding TNFα, β defensin-3 and LL37. Immunosupression was induced 1 month later with corticosterone. In comparison with the control group, mice treated with adenoviruses showed significantly less bacterial load and pneumonia, the adenoviruses encoding TNFα and LL37 being the most efficient. Gene therapy based in a proinflammatory cytokine or antimicrobial peptides is a potentially useful system to prevent reactivation of latent tuberculosis.

“Hope This Helps!” An Analysis of Expressive Speech Acts in Online Task-Oriented Interaction by University Students

This paper explores the presence of expressive speech acts in a corpus of e-forum history logs derived from the online collaborative writing activity of three groups of undergraduate and postgraduate students in a tertiary education setting. The macro category of Expressives has been less frequently studied than others such as Directives or Commissives, and even nowadays its in-depth study tends to concentrate on specific subtypes such as Compliments. In computer mediated exchanges, the implicit disembodiment must ensure an outstanding role for expressive uses of language, since non-verbal means are not available as in face-to-face conversation. The study includes a qualitative and quantitative analysis which covers the similarities and differences found across the subcorpora corresponding to each of the three groups of students involved, in terms of subtypes of Expressives and their linguistic realisations. The results suggest that Expressives play a crucial role as rapport building devices in the online interaction, smoothing and complementing transactional language. The analysis also suggests that the variables of linguistic proficiency, group size, age, multiculturality, and method of assessment may have a bearing on the form and use of Expressives in online written interaction in blended learning environments.