RESUMO
OBJECTIVES: Limbal stem cells (LSC) are self-renewing, highly proliferative cells in vitro, which express a set of specific markers and in vivo have the capacity to reconstruct the entire corneal epithelium in cases of ocular surface injury. Currently, LSC transplantation is a commonly used procedure in patients with either uni- or bilateral total limbal stem cells deficiency (TLSCD). Although LSC transplantation holds great promise for patients, several problems need to be overcome. In order to find an alternative source of cells that can partially substitute LSC in cornea epithelium reconstruction, we aimed at investigating whether human immature dental pulp stem cells (hIDPSC) would present similar key characteristics as LSC and whether they could be used for corneal surface reconstruction in a rabbit TLSCD model. MATERIALS: We used hIDPSC, which co-express mesenchymal and embryonic stem cell markers and present the capacity to differentiate into derivative cells of the three germinal layers. TLSCD was induced by chemical burn in one eye of rabbits. After 30 days, the opaque tissue formed was removed by superficial keratectomy. Experimental group received undifferentiated hIDPSC, while control group only received amniotic membrane (AM). Both groups were sacrificed after 3 months. RESULTS AND CONCLUSIONS: We have demonstrated, using immunohistochemistry and reverse transcription-polymerase chain reaction, that hIDPSCs express markers in common with LSC, such as ABCG2, integrin beta1, vimentin, p63, connexin 43 and cytokeratins 3/12. They were also capable of reconstructing the eye surface after induction of unilateral TLSCD in rabbits, as shown by morphological and immunohistochemical analysis using human-specific antibodies against limbal and corneal epithelium. Our data suggest that hIDPSCs share similar characteristics with LSC and might be used as a potential alternative source of cells for corneal reconstruction.
Assuntos
Queimaduras Químicas/terapia , Polpa Dentária/citologia , Epitélio Corneano/citologia , Queimaduras Oculares/terapia , Transplante de Células-Tronco/métodos , Células-Tronco/citologia , Animais , Biomarcadores , Queimaduras Químicas/patologia , Diferenciação Celular/fisiologia , Células Cultivadas , Córnea/citologia , Córnea/fisiologia , Modelos Animais de Doenças , Queimaduras Oculares/patologia , Humanos , Masculino , Coelhos , Regeneração/fisiologiaRESUMO
OBJECTIVES: In this study, we aimed at determining whether human immature dental pulp stem cells (hIDPSC) would be able to contribute to different cell types in mouse blastocysts without damaging them. Also, we analysed whether these blastocysts would progress further into embryogenesis when implanted to the uterus of foster mice, and develop human/mouse chimaera with retention of hIDPSC derivates and their differentiation. MATERIALS AND METHODS: hIDPSC and mouse blastocysts were used in this study. Fluorescence staining of hIDPSC and injection into mouse blastocysts, was performed. Histology, immunohistochemistry, fluorescence in situ hybridization and confocal microscopy were carried out. RESULTS AND CONCLUSION: hIDPSC showed biological compatibility with the mouse host environment and could survive, proliferate and contribute to the inner cell mass as well as to the trophoblast cell layer after introduction into early mouse embryos (n = 28), which achieved the hatching stage following 24 and 48 h in culture. When transferred to foster mice (n = 5), these blastocysts with hIDPSC (n = 57) yielded embryos (n = 3) and foetuses (n = 6); demonstrating presence of human cells in various organs, such as brain, liver, intestine and hearts, of the human/mouse chimaeras. We verified whether hIDPSC would also be able to differentiate into specific cell types in the mouse environment. Contribution of hIDPSC in at least two types of tissues (muscles and epithelial), was confirmed. We showed that hIDPSC survived, proliferated and differentiated in mouse developing blastocysts and were capable of producing human/mouse chimaeras.
Assuntos
Células-Tronco Adultas/citologia , Polpa Dentária/citologia , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário/fisiologia , Feto/citologia , Quimeras de Transplante/embriologia , Células-Tronco Adultas/transplante , Estruturas Animais/citologia , Estruturas Animais/embriologia , Estruturas Animais/metabolismo , Animais , Blastocisto/citologia , Diferenciação Celular/fisiologia , Cromossomos Humanos Y/química , Transferência Embrionária , Embrião de Mamíferos/embriologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Epitélio/embriologia , Epitélio/metabolismo , Feminino , Feto/embriologia , Feto/metabolismo , Humanos , Hibridização in Situ Fluorescente , Masculino , Camundongos , Camundongos Endogâmicos , Células Musculares/citologia , Células Musculares/metabolismo , Músculos/citologia , Músculos/embriologia , Músculos/metabolismo , Quimeras de Transplante/metabolismoRESUMO
Proper morphology is essential for the ability of Candida albicans to switch between yeast and hyphae and thereby sustain its virulence. Here we identified, by differential screening, a novel C. albicans AAA ATPase encoding gene, CaYLL34 (RIX7), with enhanced expression in hyphae. Phylogenetic analysis suggests that CaYLL34 belongs to a "VCP-like" subgroup of AAA ATPases essential for yeast viability and contains a bipartite nuclear localization signal. Inactivation of one copy of CaYLL34, by the URA-Blaster method, generated the heterozygous mutant strain M61. This strain has severe phenotypic alterations, such as a highly increased vacuole, abnormal cell shape and reduced growth in different conditions. Also, major pathogenicity factors are affected in M61, for instance, a significant decrease of hypha formation (>90%), surface biofilm adhesion (86%) and secreted aspartyl proteinase activity (76.5%). Our results show that the partial impairment of CaYll34p cellular levels is sufficient to affect the proper cellular morphology and pathogenicity factors and suggest that this protein is required for biogenesis of ribosomal subunits. Accordingly, we propose that the product of CaYLL34 could be tested as a novel target for antifungal drugs.
Assuntos
Adenosina Trifosfatases/genética , Ácido Aspártico Endopeptidases/metabolismo , Biofilmes/crescimento & desenvolvimento , Candida albicans/genética , Proteínas de Saccharomyces cerevisiae/genética , Sequência de Bases , Candida albicans/enzimologia , Candida albicans/crescimento & desenvolvimento , Hifas/enzimologia , Hifas/genética , Hifas/crescimento & desenvolvimento , Dados de Sequência Molecular , Mutação , Proteínas Nucleares , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
Proper morphology is essential for the ability of Candida albicans to switch between yeast and hyphae and thereby sustain its virulence. Here we identified, by differential screening, a novel C. albicans AAA ATPase encoding gene, CaYLL34 (RIX7), with enhanced expression in hyphae. Phylogenetic analysis suggests that CaYLL34 belongs to a [quot ]VCP-like[quot ] subgroup of AAA ATPases essential for yeast viability and contains a bipartite nuclear localization signal. Inactivation of one copy of CaYLL34, by the URA-Blaster method, generated the heterozygous mutant strain M61. This strain has severe phenotypic alterations, such as a highly increased vacuole, abnormal cell shape and reduced growth in different conditions. Also, major pathogenicity factors are affected in M61, for instance, a significant decrease of hypha formation (>90%), surface biofilm adhesion (86%) and secreted aspartyl proteinase activity (76.5%). Our results show that the partial impairment of CaYll34p cellular levels is sufficient to affect the proper cellular morphology and pathogenicity factors and suggest that this protein is required for biogenesis of ribosomal subunits. Accordingly, we propose that the product of CaYLL34 could be tested as a novel target for antifungal drugs.
Assuntos
Adenosina Trifosfatases/genética , Biofilmes/crescimento & desenvolvimento , Candida albicans/genética , Ácido Aspártico Endopeptidases/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Sequência de Bases , Candida albicans/enzimologia , Candida albicans/crescimento & desenvolvimento , Hifas/enzimologia , Hifas/genética , Hifas/crescimento & desenvolvimento , Dados de Sequência Molecular , Mutação , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
Paracoccidioidomycosis (PCM) is a severe disease caused by the dimorphic fungus Paracoccidioides brasiliensis, which is characterized by granulomatous pulmonary and systemic lesions, affecting mainly men between 20 and 60 years of age. Reports of PCM disease in animals are rare, but the disease has been described in armadillos. On the other hand, PCM infection of domestic and wild animals detected by serological or cutaneous tests in the absence of apparent disease has been frequently reported. We present here the case of a female adult Doberman that developed cervical lymphadenomegaly. Histopathological examination of a cervical biopsy specimen revealed active PCM, with an epithelioid, granulomatous inflammation containing numerous yeast-like, multiple budding fungal forms. The diagnosis of PCM was confirmed by immunohistochemistry using a specific antibody anti-gp43 and by nested PCR using primers for the amplification of the gp43 gene region. This is the first report of PCM disease occurring in a dog, an animal that has been shown to play an important role in the natural history of North American blastomycosis.