Dermal papilla cells cultured as spheres improve angiogenesis.

Tissue-engineered skin represents a helpful strategy for the treatment of deep skin injuries. Nevertheless, these skin substitutes must promote and encourage proper vascularization for a successful graft take. Previous work showed that dermal papilla cells (DPC) favour an earlier neovascularization process of grafted skin substitute contributing to the rapid maturation of the neovascular network, reducing inflammation and favouring extracellular matrix remodelling in nude mice. Based on these results, we studied the influence of DPC and its culture conditions on the different stages of angiogenesis in in vitro models. Here, we showed that DPC cultured as spheres favour the expression of angiogenic factors such as VEGF, FGF2 and angiogenin compared to their monolayer culture. To study the effects of DPC on the different stages of angiogenesis, an in vitro model has been adapted. DPC cultured as spheres significantly enhanced HUVEC migration and tubule formation, indicating the importance of employing physiological culture systems that provide a closer representation of cell behaviour and interactions occurring in vivo. Overall, these results allow us to speculate that the use of DPC spheres in skin substitutes could promote its grafting, vascularization and vascular network maturation through the secretion of angiogenic factors. This approach has great potential to improve clinical outcomes in regenerative medicine and skin wound repair.

Androgens downregulate BMP2 impairing the inductive role of dermal papilla cells on hair follicle stem cells differentiation.

Hair follicle cyclical regeneration is regulated by epithelial-mesenchymal interactions. During androgenetic alopecia (AGA), hair follicle stem cells (HFSC) differentiation is impaired by deregulation of dermal papilla cells (DPC) secreted factors. We analyzed androgen influence on BMPs expression in DPC and their effect on HFSC differentiation to hair lineage. Androgens downregulated BMP2 and BMP4 in DPC spheroids. Addition of BMP2 restored alkaline phosphatase activity, marker of hair-inductivity in DPC, and DPC-induced HFSC differentiation, both inhibited by androgens. Concomitantly, in differentiating HFSC, an upregulation of BMPRIa and BMPRII receptors and nuclear ß-catenin accumulation, indicative of Wnt/ß-catenin pathway activation, were detected. Our results present BMP2 as an androgen-downregulated paracrine factor that contributes to DPC inductivity and favors DPC-induced HFSC differentiation to hair lineage, possibly through a crosstalk with Wnt/ß-catenin pathway. A comprehensive understanding of androgen-deregulated DPC factors and their effects on differentiating HFSC would help to improve treatments for AGA.

Androgens and androgen receptor action in skin and hair follicles.

Beyond sexual functions, androgens exert their action in skin physiology and pathophysiology. Skin cells are able to synthesize most active androgens from gonadal or adrenal precursors and the enzymes involved in skin steroidogenesis are implicated both in normal or pathological processes. Even when the role of androgens and androgen receptor (AR) in skin pathologies has been studied for decades, their molecular mechanisms in skin disorders remain largely unknown. Here, we analyze recent studies of androgens and AR roles in several skin-related disorders, focusing in the current understanding of their molecular mechanisms in androgenetic alopecia (AGA). We review the molecular pathophysiology of type 2 5α-reductase, AR coactivators, the paracrine factors deregulated in dermal papillae (such as TGF-ß, IGF 1, WNTs and DKK-1) and the crosstalk between AR and Wnt signaling in order to shed some light on new promising treatments.

Androgens modify Wnt agonists/antagonists expression balance in dermal papilla cells preventing hair follicle stem cell differentiation in androgenetic alopecia.

In androgenetic alopecia, androgens impair dermal papilla-induced hair follicle stem cell (HFSC) differentiation inhibiting Wnt signaling. Wnt agonists/antagonists balance was analyzed after dihydrotestosterone (DHT) stimulation in androgen-sensitive dermal papilla cells (DPC) cultured as spheroids or monolayer. In both culture conditions, DHT stimulation downregulated Wnt5a and Wnt10b mRNA while the Wnt antagonist Dkk-1 was upregulated. Notably, tissue architecture of DPC-spheroids lowers Dkk-1 and enhances Wnt agonists' basal expression; probably contributing to DPC inductivity. The role of Wnt agonists/antagonists as mediators of androgen inhibition of DPC-induced HFSC differentiation was evaluated. Inductive DPC-conditioned medium supplemented with DKK-1 impaired HFSC differentiation mimicking androgens' action. This effect was associated with inactivation of Wnt/ß-catenin pathway in differentiating HFSC by both DPC-conditioned media. Moreover, addition of WNT10b to DPC-medium conditioned with DHT, overcame androgen inhibition of HFSC differentiation. Our results identify DKK1 and WNT10b as paracrine factors which modulate the HFSC differentiation inhibition involved in androgen-driven balding.

Epidermal stem cells and skin tissue engineering in hair follicle regeneration.

The reconstitution of a fully organized and functional hair follicle from dissociated cells propagated under defined tissue culture conditions is a challenge still pending in tissue engineering. The loss of hair follicles caused by injuries or pathologies such as alopecia not only affects the patients' psychological well-being, but also endangers certain inherent functions of the skin. It is then of great interest to find different strategies aiming to regenerate or neogenerate the hair follicle under conditions proper of an adult individual. Based upon current knowledge on the epithelial and dermal cells and their interactions during the embryonic hair generation and adult hair cycling, many researchers have tried to obtain mature hair follicles using different strategies and approaches depending on the causes of hair loss. This review summarizes current advances in the different experimental strategies to regenerate or neogenerate hair follicles, with emphasis on those involving neogenesis of hair follicles in adult individuals using isolated cells and tissue engineering. Most of these experiments were performed using rodent cells, particularly from embryonic or newborn origin. However, no successful strategy to generate human hair follicles from adult cells has yet been reported. This review identifies several issues that should be considered to achieve this objective. Perhaps the most important challenge is to provide three-dimensional culture conditions mimicking the structure of living tissue. Improving culture conditions that allow the expansion of specific cells while protecting their inductive properties, as well as methods for selecting populations of epithelial stem cells, should give us the necessary tools to overcome the difficulties that constrain human hair follicle neogenesis. An analysis of patent trends shows that the number of patent applications aimed at hair follicle regeneration and neogenesis has been increasing during the last decade. This field is attractive not only to academic researchers but also to the companies that own almost half of the patents in this field.

Dermal papilla cells improve the wound healing process and generate hair bud-like structures in grafted skin substitutes using hair follicle stem cells.

Tissue-engineered skin represents a useful strategy for the treatment of deep skin injuries and might contribute to the understanding of skin regeneration. The use of dermal papilla cells (DPCs) as a dermal component in a permanent composite skin with human hair follicle stem cells (HFSCs) was evaluated by studying the tissue-engineered skin architecture, stem cell persistence, hair regeneration, and graft-take in nude mice. A porcine acellular dermal matrix was seeded with HFSCs alone and with HFSCs plus human DPCs or dermal fibroblasts (DFs). In vitro, the presence of DPCs induced a more regular and multilayered stratified epidermis with more basal p63-positive cells and invaginations. The DPC-containing constructs more accurately mimicked the skin architecture by properly stratifying the differentiating HFSCs and developing a well-ordered epithelia that contributed to more closely recapitulate an artificial human skin. This acellular dermal matrix previously repopulated in vitro with HFSCs and DFs or DPCs as the dermal component was grafted in nude mice. The presence of DPCs in the composite substitute not only favored early neovascularization, good assimilation and remodeling after grafting but also contributed to the neovascular network maturation, which might reduce the inflammation process, resulting in a better healing process, with less scarring and wound contraction. Interestingly, only DPC-containing constructs showed embryonic hair bud-like structures with cells of human origin, presence of precursor epithelial cells, and expression of a hair differentiation marker. Although preliminary, these findings have demonstrated the importance of the presence of DPCs for proper skin repair.

Prevalencia de papilomavirus humano (HPV) tipo 6, 11, 16 y 18 en la ciudad de Buenos Aires: implicancias en la implementación de vacunas profilácticas / Prevalence of human papillomavirus (HPV-6-11, -16 and -18) in the city of Buenos Aires: epidemiology and vaccination

Introducción: el cáncer cervical es uno de los cáncer más comunes que afectan mujeres en países en desarrollo. Es provocado por la infección persistente del Papilomavirus Humano (HPV). En los últimos años se ha lanzado al mercado una vacuna cuadrivalente contra los HPV de bajo y alto riesgo (HPV-6, 11, 16 y 18), otra vacuna, bivalente para HPV-16 y 18, también ha sido introducida al mercado. En el presente trabajo se evaluó si los tipos de HPV cubiertos por las vacunas profilácticas son los más prevalentes en nuestro país, así como la utilidad de los métodos de detección viral por ADN como herramienta para el diagnóstico en mujeres asintomáticas. Materiales y métodos: éste protocolo se encuentra aprobado por el Comité de Docencia e Investigación (CODEI) del Hospital Gral. de Agudos "C.G. Durand" y por el consejo de Etica de la USAL. Todos los participantes dieron consentimiento escrito voluntario antes de participar del mismo. Se analizaron biopsias cervicales obtenidas en el Servicio de Tocoginecología de dicho Hospital, las cuales fueron agrupadas en 4 categorías: Grupo Control (ctrl.) n= 27, Grupo de lesiones de bajo grado (CIN I) n = 22, Grupo de lesiones de mediano y alto grado (CIN II/III) n = 30, Grupo de carcinomas cervicales (CC) n = 35. El ADN fue extraído por métodos estándar. La determinación de presencia de HPV se realizó mediante PCR específica con cebadores MY09/MY11. La tipificación de ADN específico de HPV-6, -11, -16 y -18 se realizó con PCR multiplex específica. Resultados y conclusiones: hemos observado una alta incidencia de la infección con HPV-16 en todos los grupos analizados (52% p< 0,0001), y varias muestras mostraron coinfección entre HPVs de alto riesgo, y entre HPVs de bajo y alto riesgo (6,85%, p< 0,001 vs. HPV16). Estos resultados, sumados al hecho que el 17,8 % de las muestras no mostró infección con los citados tipos de HPV, sugieren que la prevalencia de los tipos virales cubiertos por las vacunas es menor a la esperada...

Introductions: Cervical cancer is one of the most frequent cancers that affect women in developing countries. It is caused by persistent infection with Human Papillomavirus (HPV). In the last years a cuadrivalent vaccine against High-risk HPVs (HPV-6, -11, -16 and -18) has been launched into the market; another vaccine, bivalent for HPV-16 and -18, has also been introduced intro the market. In the present work we evaluated if HPV types covered by these vaccines are actually the most prevalent in our country, as well as the utility of DNA-based viral detection essays as a diagnosis tool in asymptomatic women. Materials and Methods: this protocol has been approved by the Hospital Durand's Teaching and Ethics committee (CODEI) and by the Ethics committee of USAL. All pariticipants gave voluntary informed consent before being included in the study. we analyzed cervical biopsies obtained in Gynecology Service of Hospital Durand, which were grouped into 4 categories: Control group (ctrl.) n = 27, low grade cervical lesions group (CIN I) n = 22, high grade cervical lesions group (CIN II/III) n = 30, cervical carcinomas (CC) n = 35. DNA was extracted by standard methods. Viral HPV presence was evaluated by specific PCR with MY09/MY11 primers. Viral-specific DNA determination of HPV-6, -11, -16 and -18 was evaluated by specific multiplex PCR. Results and conclusions: We observed a high incidence of and several samples showd co-infection between High-risk HPVs, and between high and low-risk HPVs (6.85%, p< 0.001 vs. HPV16). These results, plus the fact that almost 17.8% of the samples shown no expression of HPV types covered by the vaccines, suggest that the prevalence of HPV-6,-11, -16 and -18 is lower than expected in Buenos Aires city.

Expresión de oncogenes y estado de integración del Papilomavirus Humano (HPV) como posibles marcadores de progresión maligna en pacientes con lesiones cervicales: estudio preliminar en la ciudad de Buenos Aires / Viral oncogene expression and integration status of the human papillomavirus as possible markers of malignant progression in patients with cervical lesions

Introducción: el Papilomavirus Humano (HPV) ha sido identificado como agente causal del cáncer cervical, induciendo lesiones cervicales de bajo grado que, eventualmente, se malignizan debido a distintos factores. La expresión de oncogenes virales y el estado de integración viral son necesarios para el desarrollo de neoplasias, por lo que podrían utilizarse como marcadores de progresión maligna en éste tipo de lesiones. Objetivo: evaluar la expresión de los oncogenes virales E6/E7 de HPV16 y 18 y el estado del genoma viral como posibles marcadores de progresión maligna. Materiales y métodos: se evaluaron muestras de lesiones cervicales (n = 27 controles (Ctrl), n = 18 CIN I, n= 24 CIN II/III y n = 32 Carcinomas invasivos (CC) derivadas del Hospital Gral. de Agudos C. Durand, CABA. Se evaluó la presencia de genoma consenso HPV y específico HPV16 y 18 mediante dPCR. Se determinó la expresión de oncogenes virales E6/E7 de HPV16 y HPV 18 y el estado de integración viral se analizó mediante PCR-APOT. Resultados: se observó un aumento significativo de presencia de genoma viral en correlación con el grado de lesión analizada CIN I: 77,8 %, CIN II/III: 83,3 % y CC: 100 % en comparación con el grupo control (25,9 %, p<0,001). La infección con HPV16 fue significativamente mayor en todos los grupos (vs. HPV18, p<0,001), encontrándose coinfección en varios casos. La expresión de los oncogenes virales de HPV16 fue significativamente mayor en comparación con HPV18 (p<0,0001). El estado físico de los genomas virales mostró una tendencia a la forma integrada en correlación con el grado de lesión analizada, encontrándose mayor presencia de genomas integrados en CC (vs. Episomal y/o episomal e integrado p<0,02) de HPV16. Para HPV18, sólo pudimos observar genomas integrados en CIN II/III (100 %) y CC (50 %). Sólo el 12,5 % de los genomas HPV 18 fueron detectados episomal e integrado. Conclusiones: observamos altas frecuencias de infección con HPV16 en comparación con HPV 18...

Introduction: Human Papillomavirus (HPV) has been identified as causal agent of cervical cancer, inducing cervical low grade lesions that, eventually, maligniza due to different factors. Viral oncogene expression and integration status are necessary for neoplasic development, and they could be used as malignant progression markers in these types of lesions. Aim: Evaluate HPV16 and 18 (E6/E7) viral oncogene expression and genome integration status as possible malignant progression markers. Materials and methods: Cervical samples derived from Hospital GA C. Durand (CABA) were evaluated (n = 27 healthy controls -Ctrl-, n = 18 CINI, n = 24 CIN II/III abd b = 32 Invasive Carcinomas (CC). HPV consensus and HPV16 and 18 specific genomes were evaluated by PCR. Viral oncogene expression (E6/E7) of HPV16 and HPV18 was determined, as well as viral integration status was determined by means of PCR-APOT. Results: A significant increase in viral genome presence was observed in correlation with the degree of the lesion analyzed CIN I: 77,9%, CIN II/III: 83,3% y CC: 100% in comparison to control Group (25,9% p<0,001). The infection with HPV16 was significantly greater in all the groups in comparison with HPV18 (p<0,001); co-infection was detected in several cases. HPV 16 oncogene expression was greater than the one showed by HPV-18 (p<0,0001). The physcal state of the viral genomes showed a tendency to the integrated form in correlation with the degree of analyzed lesion, detecting most of integrated HPV16 genomes in CC group (vs. episomal and/or episomal and integrated, p<0,02), FORM II/III (100%) and CC (50%). Only a minor fraction of HPV18 genomes in CC where found to be, both, episomal and integrated (12,5%). Conclusions: We have detected higher HPV16 infection frequencies in comparison with HPV18 in all analyzed groups. On the other hand, we observed an increase in HPV-16 onocogene expression in comparison to HPV18 ones. HPV16 was found predominantly integrated...