A humanized mouse model of HPV-associated pathology driven by E7 expression

Human papillomavirus (HPV) is the causative agent of human cervical cancer and has been associated with oropharyngeal squamous cell carcinoma development. Although prophylactic vaccines have been developed, there is a need to develop new targeted therapies for individuals affected with malignant infected lesions in these locations, which must be tested in appropriate models. Cutaneous beta HPV types appear to be involved in skin carcinogenesis. Virus oncogenicity is partly achieved by inactivation of retinoblastoma protein family members by the viral E7 gene. Here we show that the E7 protein of cutaneous beta HPV5 binds pRb and promotes its degradation. In addition, we described an in vivo model of HPV-associated disease in which artificial human skin prepared using primary keratinocytes engineered to express the E7 protein is engrafted onto nude mice. Expression of E7 in the transplants was stably maintained for up to 6 months, inducing the appearance of lesions that, in the case of HPV16 E7, histologically resembled human anogenital lesions caused by oncogenic HPVs. Moreover, it was confirmed through biomarker expression analysis via immunodetection and/or quantitative PCR from mRNA and miRNA that the 16E7-modified engrafted skin shares molecular features with human HPV-associated pretumoral and tumoral lesions. Finally, our findings indicate a decrease of the in vitro capacity of HPV5 E7 to reduce pRb levels in vivo, possibly explaining the phenotypical differences when compared with 16E7-grafts. Our model seems to be a valuable platform for basic research into HPV oncogenesis and preclinical testing of HPV-associated antitumor therapies.This work was supported by Comunidad Autonoma de Madrid (Oncocycle S2006/BIO-0232), by Ministerio de Ciencia e Innovacion (ISCIII-RETIC RD06/0020 and SAF2008-00121), and by Fundación Sandra Ibarra. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

The regenerative potential of fibroblasts in a new diabetes-induced delayed humanised wound healing model

Cutaneous diabetic wounds greatly affect the quality of life of patients, causing a substantial economic impact on the healthcare system. The limited clinical success of conventional treatments is mainly attributed to the lack of knowledge of the pathogenic mechanisms related to chronic ulceration. Therefore, management of diabetic ulcers remains a challenging clinical issue. Within this context, reliable animal models that recapitulate situations of impaired wound healing have become essential. In this study, we established a new in vivo humanised model of delayed wound healing in a diabetic context that reproduces the main features of the human disease. Diabetes was induced by multiple low doses of streptozotocin in bioengineered human-skin-engrafted immunodeficient mice. The significant delay in wound closure exhibited in diabetic wounds was mainly attributed to alterations in the granulation tissue formation and resolution, involving defects in wound bed maturation, vascularisation, inflammatory response and collagen deposition. In the new model, a cell-based wound therapy consisting of the application of plasma-derived fibrin dermal scaffolds containing fibroblasts consistently improved the healing response by triggering granulation tissue maturation and further providing a suitable matrix for migrating keratinocytes during wound re-epithelialisation. The present preclinical wound healing model was able to shed light on the biological processes responsible for the improvement achieved, and these findings can be extended for designing new therapeutic approaches with clinical relevance.This work was supported by grants from the Science and Innovation Ministry of Spain (SAF2010-16976), from the European VI Framework Programme (LSHB-CT-512102), from Comunidad de Madrid (S2010/BMD-2420; CELLCAM) and from Fundacion Ramon Areces (CIVP16A1864)

Establishment of a murine epidermal cell line suitable for in vitro and in vivo skin modelling

<p>Abstract</p> <p>Background</p> <p>Skin diseases are a major health problem. Some of the most severe conditions involve genetic disorders, including cancer. Several of these human diseases have been modelled in genetically modified mice, thus becoming a highly valuable preclinical tool for the treatment of these pathologies. However, development of three-dimensional models of skin using keratinocytes from normal and/or genetically modified mice has been hindered by the difficulty to subculture murine epidermal keratinocytes.</p> <p>Methods</p> <p>We have generated a murine epidermal cell line by serially passaging keratinocytes isolated from the back skin of adult mice. We have termed this cell line COCA. Cell culture is done in fully defined media and does not require feeder cells or any other coating methods.</p> <p>Results</p> <p>COCA retained its capacity to differentiate and stratify in response to increased calcium concentration in the cell culture medium for more than 75 passages. These cells, including late passage, can form epidermis-like structures in three-dimensional <it>in vitro </it>models with a well-preserved pattern of proliferation and differentiation. Furthermore, these cells form epidermis in grafting assays <it>in vivo</it>, and do not develop tumorigenic ability.</p> <p>Conclusions</p> <p>We propose that COCA constitutes a good experimental system for <it>in vitro </it>and <it>in vivo </it>skin modelling. Also, cell lines from genetically modified mice of interest in skin biology could be established using the method we have developed. COCA keratinocytes would be a suitable control, within a similar background, when studying the biological implications of these alterations.</p

Campus virtual y dispositivos con conexión a internet durante las clases: ¿una alternativa a los clickers?

Inicialmente los clickers (una forma de “Personal Response Systems” mediente mandos a distancia) se emplearon en programas de televisión, congresos, reuniones, etc. pero en los últimos años ha pasado a la docencia; en especial a la universitaria. Algunos estudios demuestran su utilidad para fomentar el trabajo, aprendizaje e interactividad entre profesores y alumnos. El problema técnico actual es el software, mandos específicos, ect. A partir de dos hechos que hemos constatado 1º. que en la UEM existe un campus virtual que utiliza moodle y dentro hay una actividad (CONSULTA) en la que se pueden hacer preguntas y mostrar los resultados por porcentajes y 2º que un porcentaje de alumnos asiste a clase con un dispositivo con conexión a la red, hemos planteado una experiencia en el sentido alternativo a los clickers. La experiencia se llevó a cabo en la asignatura de Semiología y Fisiopatología general II de 2º de odontología y se ha empleado el campus virtual de la asignatura y los PC y Smartphones como “mandos a distancia”. Hemos comprobado que si los alumnos tienen en clase Smartphones con conexión 3G, PC o ipad, puede aprovecharse el campus virtual consiguiendo unos efectos parecidos al empleo de los clickers.SIN FINANCIACIÓNNo data 2011UE

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

Histopathology of skin transplants.

<p>Histopathology of skin transplants.</p

Induction of p21 and cyclin A in E7-grafts.

<p>Sections of paraffin-embedded grafts were processed for immunohistochemistry staining with antibodies to cell cycle inhibitor p21 and cell cycle regulator cyclin A. With respect to control vector grafts (<b>A</b> and <b>D</b>), both p21 and cyclin A proteins are induced in suprabasal cells of HPV16 E7 transplants (<b>C</b> and <b>F</b>) and in focal HPV5 E7 areas (<b>B</b> and <b>E</b>). In the inserts, dotted lines indicate the location of the basal membrane.</p

Pocket proteins expression in skin grafts.

<p>(<b>A</b>) Pocket protein and eGFP expression was analyzed by immunoblotting with specific antibodies using graft protein lysates from control vector, 5E7 and 16E7 samples. (<b>B</b>) Pocket protein and eGFP protein expression bands were quantified and relative (pocket protein/eGFP) values plotted. Horizontal green lines represents mean relative values. According to Student’s t-test, significant differences were only detected in pRb/eGFP expression values between vector and 16E7 samples (threshold p-val<0.05). *: p-val<0.05.</p

RNA expression quantification of oncogenic HPV biomarker genes and miRNAs.

<p>mRNA quantification of E2F1, CENPF, MELK and RFC4 genes was conducted by qRT-PCR using RNA purified from PHK cells or grafts, or obtained from reported microarray experiments for CC or VIN. Each dot represents an individual sample. Horizontal lines represent mean values in each sample group. (<b>A</b>) All genes were induced in E7-transduced PHKs, though not significantly (Student’s t-test threshold p-val<0.05). Significant overexpression was also observed for all genes in HPV16 E7-transplants, but only for E2F1 in HPV5 E7. Shown are log₂-based, z-values of expression relative to housekeeping GUSB in PHK cells and transplants (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041743#s2" target="_blank">Materials and Methods</a>). (<b>B</b>) Microarray expression patterns of each gene in CC and VIN infected with HR-HPVs are shown as log₂-based, z-values (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041743#s2" target="_blank">Materials and Methods</a>). All genes display significant overexpression in CC with respect to cervix uteri (N), and in VIN with respect to normal vulva (N). Horizontal lines represent mean values in each sample group. *: p-val <0.05, **: p-val <0.005, ***: p-val <0.0005, ****: p-val <0.00005. <b>V</b>: control vector; <b>5</b>: HPV5 E7; <b>16</b>: HPV16 E7. (<b>C</b>) miRNA was quantified by qRT-PCR for miR-146a, miR-21 and miR-200a, using RNA purified from skin grafts. Shown are log₂-based, z-values of expression relative to housekeeping U6B. Each dot represents an individual sample value. All miRNAs were overexpressed in HPV16 E7-grafts. Only miR-21 displayed significant deregulation in transplants expressing HPV5 E7. Horizontal lines represent mean values in each sample group. *: p-val <0.05, **: p-val <0.005, ***: p-val <0.0005, ****: p-val <0.00005. <b>V</b>: control vector; <b>5</b>: HPV5 E7; <b>16</b>: HPV16 E7.</p