RESUMO
Mutations in the FOXC1/Foxc1 gene in humans and mice and Bmp4 in mice are associated with congenital anterior segment dysgenesis (ASD) and the development of the aqueous outflow structures throughout the limbus. The aim of this study was to advance our understanding of anterior segment abnormalities in mouse models of ASD using a 3-D imaging approach. Holistic imaging information combined with quantitative measurements were carried out on PECAM-1 stained individual components of the aqueous outflow vessels and corneal vasculature of Foxc1(+/-) on the C57BL/6Jx129 and ICR backgrounds, Bmp4(+/-) ICR mice, and wildtype mice from each background. In both wildtype and heterozygotes, singular, bifurcated and plexus forms of Schlemm's canal were noted. Of note, missing portions of the canal were seen in the heterozygous groups but not in wildtype animals. In general, we found the number of collector channels to be reduced in both heterozygotes. Lastly, we found a significant increase in the complexity of the corneal arcades and their penetration into the cornea in heterozygotes as compared with wild types. In conclusion, our 3-D imaging studies have revealed a more complex arrangement of both the aqueous vessels and corneal arcades in Foxc1(+/-) and Bmp4(+/-) heterozygotes, and further advance our understanding of how such abnormalities could impact on IOP and the aetiology of glaucoma.
Assuntos
Segmento Anterior do Olho/anatomia & histologia , Humor Aquoso/metabolismo , Proteína Morfogenética Óssea 4/fisiologia , Fatores de Transcrição Forkhead/fisiologia , Limbo da Córnea/irrigação sanguínea , Análise de Variância , Animais , Segmento Anterior do Olho/irrigação sanguínea , Vasos Sanguíneos/anatomia & histologia , Proteína Morfogenética Óssea 4/deficiência , Corioide/irrigação sanguínea , Fatores de Transcrição Forkhead/deficiência , Heterozigoto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Microscopia ConfocalRESUMO
During wound healing, melanocytes are required to migrate into the wounded area that is still in the process of re-construction. The role and behaviour of melanocytes during this process is poorly understood, that is, whether melanocyte migration into the wound is keratinocyte-dependent or not. This paper attempts, through the use of both two- and three-dimensional in vitro models, to understand the role and behaviour of melanocytes during the process of wound healing. In addition, it sheds light on whether keratinocytes influence/contribute toward melanocyte migration and ultimately wound healing. Scratch assays were performed to analyse migration and Western blot analyses measured cellular E-cadherin expression. Immunohistochemistry was used to analyse the in vivo 3D wound healing effect. Scratch assays performed on co-cultures of melanocytes and keratinocytes demonstrated that melanocytes actively migrated, with the use of their dendrites, into the scratch ahead of the proliferating keratinocyte sheet. Migration of the melanocyte into the wound bed was accompanied by loss of attachment to keratinocytes at the wound front with concomitant downregulation of E-cadherin expression as observed through immunocytochemistry. This result suggests that, in vitro, melanocyte migration occurs independently of keratinocytes but that the migration is influenced by keratinocyte E-cadherin expression. We now demonstrate that melanocyte migration during re-pigmentation is an active process, and suggest that targeting of mechanisms involved in active melanocyte migration (e.g. the melanocyte dendrite) may enhance the re-pigmentation process.
Assuntos
Caderinas/metabolismo , Queratinócitos/citologia , Melanócitos/citologia , Cicatrização , Técnicas de Cultura de Células , Células Cultivadas , Regulação para Baixo , Humanos , Imuno-Histoquímica , Queratinócitos/metabolismo , Melanócitos/metabolismo , Modelos BiológicosRESUMO
Correct formation of the corneal endothelium is essential for continued development of the anterior segment of the eye. Corneal endothelial development is initiated at E12 when precursor peri-ocular mesenchyme cells migrate into the space between the lens and the presumptive corneal epithelium and begin to respond to signals from the lens, undergoing a mesenchymal to epithelial transition (MET) that is complete by E15.5. To study the initiation of MET, peri-ocular mesenchyme cell lines were derived from E12.5 and E13.5 murine embryos. These cells expressed key transcription factors, Foxc1 and Pitx2, as well as Slug and Tsc22, genes involved in MET. We have shown that all these genes must be down-regulated by E13.5 for differentiation to proceed. Lens-derived signals play a role in this down-regulation with Tgfß2 specifically down-regulating Foxc1 and Pitx2. Over-expression and knock-down of Foxc1 significantly and similarly affected the expression of Pitx2, Tsc22 and Slug while Foxc1 was shown to play a role in mediating the lens effects on Slug. Thus, for the progression of initial corneal endothelial development, the key transcription factors, Foxc1 and Pitx2, as well as genes involved in MET, Slug and Tsc-22, must be down-regulated, a process driven by the lens and Foxc1.
Assuntos
Diferenciação Celular , Endotélio Corneano/citologia , Endotélio Corneano/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Cristalino/citologia , Cristalino/metabolismo , Animais , Western Blotting , Células Cultivadas , Galinhas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Transição Epitelial-Mesenquimal , Fatores de Transcrição Forkhead/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta2/genética , Fator de Crescimento Transformador beta2/metabolismo , Proteína Homeobox PITX2RESUMO
Studies on mammalian species with naturally reduced eyes can provide valuable insights into the evolutionary developmental mechanisms underlying the reduction of the eye structures. Because few naturally microphthalmic animals have been studied and eye reduction must have evolved independently in many of the modern groups, novel evolutionary developmental models for eye research have to be sought. Here, we present a first report on embryonic eye development in the Cape dune mole rat, Bathyergus suillus. The eyes of these animals contain all the internal structures characteristic of the normal eye but exhibit abnormalities in the anterior chamber structures. The lens is small but develops normally and exhibits a normal expression of α- and γ-crystallins. One of the interesting features of these animals is an extremely enlarged and highly pigmented ciliary body. In order to understand the molecular basis of this unusual feature, the expression pattern of an early marker of the ciliary zone, Ptmb4, was investigated in this animal. Surprisingly, in situ hybridization results revealed that Ptmb4 expression was absent from the ciliary body zone of the developing Bathyergus eye.
Assuntos
Desenvolvimento Embrionário , Olho/embriologia , Ratos-Toupeira/embriologia , Envelhecimento , Animais , Western Blotting , Corpo Ciliar/metabolismo , Clonagem Molecular , Cristalinas/genética , Cristalinas/metabolismo , Eletroforese em Gel de Poliacrilamida , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Olho/anatomia & histologia , Olho/citologia , Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Cristalino/embriologia , Cristalino/metabolismo , Ratos-Toupeira/genética , Tamanho do Órgão , África do Sul , Timosina/metabolismoRESUMO
The mouse eye has been used as a model for studies on the microanatomy of the outflow pathways but most of what is known comes from histological sections. These studies have focused mainly on the morphological features of the trabecular meshwork, Schlemm's canal and aqueous channels that link to the superficial episcleral vasculature. However, the anatomical architecture of the aqueous outflow vessels and their relationship to each other and to the general vascular circulation is not well understood. The aim of this study was to provide a detailed description of the microarchitecture of the aqueous outflow vessels and their relationship to the superficial limbal/episcleral vasculature throughout the entire limbus. The aqueous outflow vessels and blood and lymphatic vessels were imaged in PECAM-1 and LYVE-1 immunostained whole anterior segments of adult mice and three-dimensional (3-D) reconstructions of the optical sections were generated to reveal the aqueous, blood and lymphatic architecture. The arterial supply, venous drainage, organisation of perilimbal vasculature, collector channels/aqueous veins and the morphology of Schlemm's canal were revealed in their entirety and the relationships between these structures is described. Schlemm's canal was PECAM-1 positive but there was no affinity for the lymphatic marker LYVE-1. We show that Schlemm's canal is a continuous circular structure and more often seen as a single, broad, varicose vessel with short regions appearing as a plexus. Aqueous veins link Schlemm's canal to the superficial vasculature and there were no direct links seen between the canal and the lymphatic vessels.
Assuntos
Segmento Anterior do Olho/anatomia & histologia , Humor Aquoso/metabolismo , Limbo da Córnea/irrigação sanguínea , Animais , Segmento Anterior do Olho/irrigação sanguínea , Camundongos Endogâmicos C57BL , Microscopia Confocal , Modelos AnimaisRESUMO
Spinocerebellar ataxia type 7 (SCA7) is an inherited neurodegenerative disease caused by a polyglutamine repeat expansion in the ATXN7 gene. Patients with this disease suffer from a degeneration of their cerebellar Purkinje neurons and retinal photoreceptors that result in a progressive ataxia and loss of vision. As with many neurodegenerative diseases, studies of pathogenesis have been hindered by a lack of disease-relevant models. To this end, we have generated induced pluripotent stem cells (iPSCs) from a cohort of SCA7 patients in South Africa. First, we differentiated the SCA7 affected iPSCs into neurons which showed evidence of a transcriptional phenotype affecting components of STAGA (ATXN7 and KAT2A) and the heat shock protein pathway (DNAJA1 and HSP70). We then performed electrophysiology on the SCA7 iPSC-derived neurons and found that these cells show features of functional aberrations. Lastly, we were able to differentiate the SCA7 iPSCs into retinal photoreceptors that also showed similar transcriptional aberrations to the SCA7 neurons. Our findings give technical insights on how iPSC-derived neurons and photoreceptors can be derived from SCA7 patients and demonstrate that these cells express molecular and electrophysiological differences that may be indicative of impaired neuronal health. We hope that these findings will contribute towards the ongoing efforts to establish the cell-derived models of neurodegenerative diseases that are needed to develop patient-specific treatments.
Assuntos
Marcadores Genéticos , Células-Tronco Pluripotentes Induzidas/citologia , Neurônios/fisiologia , Retina/fisiologia , Ataxias Espinocerebelares/fisiopatologia , Ataxina-7/genética , Diferenciação Celular , Células Cultivadas , Reprogramação Celular , Fenômenos Eletrofisiológicos , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP70/genética , Histona Acetiltransferases/genética , Humanos , Células-Tronco Pluripotentes Induzidas/química , Modelos Biológicos , Neurônios/química , Neurônios/citologia , Cultura Primária de Células , Retina/química , Retina/citologia , África do Sul , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/patologiaRESUMO
Photodynamic therapy (PDT) as a regime for melanoma is of limited success due to factors such as the efficacy of the photosensitizer used, penetration depth and the presence of pigment. We characterised a pigmented and an unpigmented melanoma cell line with respect to their phenotypes. Cell viability was assessed after exposure to hypericin, a UVA-activated photosensitizer. Exposure to 3 microM activated hypericin induced a cytoprotective (autophagic) response from both cell lines. However, the pigmented cells accumulated a large amount of glycogen in their cytoplasm. We hypothesise that the treatment induces an initial cytoprotective response through autophagy, but with increased stress results in a different mode of cell death in pigmented melanoma cells from unpigmented cells. These results indicate that hypericin-PDT could be an adjuvant therapy for melanoma.
Assuntos
Autofagia/fisiologia , Resistencia a Medicamentos Antineoplásicos , Melanoma/metabolismo , Perileno/análogos & derivados , Radiossensibilizantes/metabolismo , Neoplasias Cutâneas/metabolismo , Antracenos , Autofagia/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Humanos , Melaninas/análise , Melanoma/tratamento farmacológico , Melanoma/ultraestrutura , Monofenol Mono-Oxigenase/metabolismo , Perileno/metabolismo , Perileno/farmacologia , Perileno/uso terapêutico , Fotoquimioterapia , Tolerância a Radiação , Radiossensibilizantes/farmacologia , Radiossensibilizantes/uso terapêutico , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/ultraestrutura , Raios UltravioletaRESUMO
Hypericin, the major component of St. John's Wort, absorbs light in the UV and visible ranges whereupon it becomes phototoxic through the production of reactive oxygen species. Although photodynamic mechanisms (i.e. through endogenous photosensitizers) play a role in UVA phototherapy for the treatment of skin disorders such as eczema and psoriasis, photodynamic therapy employing exogenous photosensitizers are currently being used only for the treatment of certain forms of non-melanoma skin cancers and actinic keratoses. There are few reports however on its use in treating melanomas. This in vitro study analyses the phototoxic effect of UVA (400-315 nm) - activated hypericin in human pigmented and unpigmented melanomas and immortalised keratinocytes and melanocytes. We show that neither hypericin exposure nor UV irradiation alone reduces cell viability. We show that an exposure to 1 microM UVA-activated hypericin does not bring about cell death, while 3 microM activated hypericin induces a necrotic mode of cell death in pigmented melanoma cells and melanocytes and an apoptotic mode of cell death in non-pigmented melanoma cells and keratinocytes. We hypothesis that the necrotic mode of cell death in the pigmented cells is possibly related to the presence of melanin-containing melanosomes in these cells and that the hypericin-induced increase in reactive oxygen species leads to an increase in permeability of melanosomes. This would result in toxic melanin precursors (of an indolic and phenolic nature) leaking into the cytoplasm which in turn leads to cell death. Hypericin localisation in the endoplasmic reticulum in these cells shown by fluorescent microscopy, further support a disruption in cellular processing and induction of cell death. In contrast, this study shows that cells that do not contain melanosomes (non-pigmented melanoma cells and keratinocytes) die by apoptosis. Further, using a mitochondrial-specific fluorescent dye, we show that intracellular accumulation of hypericin induces a mitochondrial-associated caspase-dependent apoptotic mode of cell death. This work suggests that UVA is effective in activating hypericin and that this phototoxicity may be considered as treatment option in some cases of lentigo maligna or lentigo maligna melanoma that are too large for surgical resection.
Assuntos
Queratinócitos/efeitos dos fármacos , Melanócitos/efeitos dos fármacos , Melanoma/patologia , Perileno/análogos & derivados , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/toxicidade , Pele/citologia , Antracenos , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Espaço Intracelular/metabolismo , Queratinócitos/citologia , Melanócitos/citologia , Melanoma/tratamento farmacológico , Necrose , Perileno/metabolismo , Perileno/toxicidade , Fármacos Fotossensibilizantes/metabolismo , Pele/patologiaRESUMO
The naked mole rat (Heterocephalus glaber) is a subterranean rodent whose eyes are thought to be visually nonfunctional and as such is an ideal animal with which to pursue questions in evolutionary developmental biology. This report is the first in-depth study on the development and morphology of the naked mole rat eye. Using standard histological analysis and scanning and transmission electron microscopy, we describe the structural features of the eye. We further report on the morphological changes that accompany the development of this eye from neonate to adult and compare them with those that occur during mouse eye development. We observed numerous abnormalities in the shape and cellular arrangement of the structures of the anterior chamber, with notable malformations of the lens. Cell proliferation and cell death assays were conducted to investigate the possible causes of lens malformation. We found that neither of these processes appeared abnormal, indicating that they were not responsible for the lens phenotype of the mole rat. In order to investigate the process of lens differentiation, we analyzed the expression of gamma-crystallins using Western blots and immunocytochemistry. At birth, levels of gamma-crystallin appear normal, but soon thereafter, the gamma-crystallin expression is terminated. Absence of detectable gamma-crystallins in adults suggests that there is a gradual degradation and loss of these proteins. The evolutionary factors that could be responsible for the eye morphology of the naked mole rat are discussed. A model for abnormal lens differentiation and the role it plays in the morphogenesis of the rest of the eye in the naked mole rats is proposed.
Assuntos
Olho/crescimento & desenvolvimento , Olho/ultraestrutura , Cristalino/crescimento & desenvolvimento , Ratos-Toupeira/anatomia & histologia , Ratos-Toupeira/crescimento & desenvolvimento , Animais , Diferenciação Celular/fisiologia , Cristalino/ultraestrutura , Camundongos , Ratos-Toupeira/fisiologia , Morfogênese , Corpo Vítreo/anatomia & histologia , gama-Cristalinas/metabolismoRESUMO
Lymphatic mapping and sentinel lymphadenectomy provide a minimally invasive means of directly determining the status of the regional lymph nodes in all patients who have a primary melanoma >1 mm thick but no clinical evidence of nodal involvement. Since the histological status of the sentinel node (SN) has been shown to be the most important prognostic factor in primary melanoma patients, the World Health Organization has recently recommended that sentinel lymphadenectomy should become the new standard of care for primary melanoma patients. This paper reviews the literature with regards to developments in and the current status of SN evaluation. Developments in the histopathological versus molecular detection of melanoma nodal metastases are reviewed, with specific emphasis on the strengths, limitations and clinical significance of these techniques. Molecular evaluation of the SN offers several advantages over standard histopathological analysis. These include an improved sensitivity, the cost-effective use of multiple markers for the improvement of detection rate and prognosis, as well as being less labour-intensive and costly. Moreover, molecular analysis has the potential to allow estimation of tumour burden. We review the potential causes of technical false-negative and false-positive reverse transcription-polymerase chain reaction (RT-PCR) results and how these could be eliminated by a systematic approach consisting of (i) careful and systematic assay design, which would include efficient tissue homogenization, choice of reagents and molecular markers, primer design and the use of one-stage versus two-stage PCR; (ii) careful optimization of the RT-PCR parameters (in particular the PCR cycle number) through the use of appropriate control tissues; and (iii) aiming for high assay reproducibility and lastly by applying the necessary positive and negative controls with each batch of samples. We also review the significant improvement in patient prognosis and management that has been made possible by the development of sentinel lymphadenectomy and histopathological evaluation of the SN, and compare the clinical (predictive) value of histopathological analysis with that of RT-PCR. Although RT-PCR is able to detect additional, clinically significant SN metastases that are missed by routine histopathology, its current limitation is that it overestimates the number of patients who have clinically significant melanoma metastases. Therefore, we suggest and discuss appropriate steps that need to be taken in order to minimize these false-positives and make this molecular tool more acceptable for routine clinical use.
Assuntos
Melanoma/patologia , Melanoma/secundário , Estadiamento de Neoplasias/métodos , Biópsia de Linfonodo Sentinela/métodos , Neoplasias Cutâneas/patologia , Biomarcadores Tumorais/análise , Humanos , Linfonodos/patologia , Metástase Linfática , Proteínas de Neoplasias/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Critically ill patients frequently display unexplained or incompletely explained features of gastrointestinal (GI) dysfunction, including gastric stasis, ileus, and diarrhea. This makes nutrition delivery challenging, and may contribute to poor outcomes. The typical bowel dysfunction seen in severely ill patients includes retarded gastric emptying, unsynchronized intestinal motility, and intestinal hyperpermeability. These functional changes appear similar to the corticotropin-releasing factor (CRF)-mediated bowel dysfunctions associated with stress of various types and some GI disorders and diseases. CRF has been shown to be present within the GI tract and its action on CRF receptors within the gut have been shown to reduce gastric emptying, alter intestinal motility, and increase intestinal permeability. However, the precise role of CRF in the GI dysfunction in critical illness remains unclear. In this short review, we provide an update on GI dysfunction during stress and review the possible role of CRF in the aetiology of gut dysfunction. We suggest that activation of CRF signaling pathways in critical illness might be key to understanding the mechanisms underlying the gut dysfunction that impairs enteral feeding in the intensive care unit.
Assuntos
Hormônio Liberador da Corticotropina/metabolismo , Estado Terminal , Gastroenteropatias/fisiopatologia , Trato Gastrointestinal/fisiopatologia , Nutrição Enteral , Esvaziamento Gástrico/fisiologia , Trato Gastrointestinal/metabolismo , Humanos , Transdução de Sinais , Estresse FisiológicoRESUMO
OBJECTIVE: Corticotropin-releasing factor (CRF) is implicated in stress-related gastrointestinal dysfunction, possibly causing gut dysfunction following trauma and surgery. We investigated plasma and intestinal tissue CRF levels and gut function in patients with traumatic shock or those undergoing elective abdominal surgery. RESEARCH METHODS AND PROCEDURES: In a prospective, parallel, observational study in a university hospital surgical intensive care unit (ICU), 8 shocked patients (systolic blood pressure <90 mmHg and/or acidosis and/or urine output <1 mL/kg/hr and/or requiring >2 L of intravenous fluid resuscitation) undergoing small bowel resection during emergency laparotomy following abdominal injury and 17 stable patients undergoing elective hepatobiliary surgery were included if they required postoperative ICU management. Serial plasma and intestinal CRF was measured and postoperative gastric emptying and intestinal permeability were evaluated. RESULTS: Plasma CRF was significantly increased in the shocked patients compared with the elective surgery patients at all times. CRF peptide was quantified in intestinal tissue at similar levels in both groups. Intestinal permeability was increased and associated with shock and resuscitation fluid volume. Gastric emptying was retarded and correlated significantly with shock but not with plasma CRF. Delayed gastric emptying in shocked patients was associated with longer ICU stay. CONCLUSIONS: The novel finding is the presence of CRF in the small bowel of both elective and emergency gastrointestinal surgery patients with concomitant gastrointestinal dysfunction. Circulating CRF is associated with poor gastric emptying, which prolongs ICU stay, whereas shock significantly impairs gastric emptying and gut permeability.
Assuntos
Abdome/cirurgia , Traumatismos Abdominais/cirurgia , Hormônio Liberador da Corticotropina/metabolismo , Gastroenteropatias/etiologia , Mucosa Intestinal/metabolismo , Complicações Pós-Operatórias/fisiopatologia , Choque Traumático/fisiopatologia , Traumatismos Abdominais/fisiopatologia , Adulto , Hormônio Liberador da Corticotropina/sangue , Serviços Médicos de Emergência , Feminino , Hidratação/efeitos adversos , Esvaziamento Gástrico , Gastroenteropatias/sangue , Gastroenteropatias/metabolismo , Gastroenteropatias/fisiopatologia , Motilidade Gastrointestinal , Humanos , Absorção Intestinal , Intestinos/fisiopatologia , Tempo de Internação , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/sangue , Complicações Pós-Operatórias/metabolismo , Estudos Prospectivos , Choque Traumático/etiologia , Choque Traumático/metabolismo , Choque Traumático/terapia , Adulto JovemRESUMO
Immunolabeling of tissue sections requires careful optimization of protocols in order to achieve accurate and consistent data. Multiple immunolabeling is desirable when determining the exact location and phenotype of cell populations in the same cellular compartment. 5-bromodeoxyuridine (BrdU)-immunolabeling is commonly used to assess cellular proliferation in vitro. However, the technical limitations of standard methods preclude multiple antigen immunolabeling. The aim was therefore to develop a robust protocol for simultaneous labeling using anti-BrdU and a melanocyte-specific marker in formalin-fixed paraffin-embedded (FFPE) skin samples. Human skin samples were obtained from patients undergoing elective plastic surgery. The tissue was incubated with BrdU, and a standard sample procedure for FFPE tissue was used. Heat-induced antigen retrieval was performed in a conventional pressure cooker, followed by immunolabeling with anti-BrdU and anti-Melan A/MART-1 antibodies. Fluorescent-conjugated secondary antibodies were used for signal detection. We have demonstrated both proliferating cells (BrdU-immunopositive) and melanocytes (Melan A/MART-1-immunopositive) in the basal compartment of the epidermis in our skin samples. Successful double labeling requires heat-induced epitope retrieval to replace the harsh pretreatment protocols of standard BrdU immunolabeling methods. We have optimized a robust protocol for the double labeling of proliferating cells and cells bearing melanocyte-specific antigens (melanocytes and/or melanoblasts) in FFPE human skin samples.
Assuntos
Anticorpos Monoclonais/imunologia , Biomarcadores/metabolismo , Bromodesoxiuridina , Imunofluorescência/métodos , Melanócitos/metabolismo , Pele/metabolismo , Humanos , Inclusão em ParafinaRESUMO
Immunocytochemical identification of skin cells are difficult due to numerous endogenous autofluorescent components within the cell and the environment. This is particularly evident in hair follicles. This paper reports on a serendipitous modification to an existing method which results in a drastically reduced background fluorescence. Immediately after antigen retrieval, sections exposed to 0.3% hydrogen peroxide in methanol for 30 min at room temperature exhibited low background fluorescence, increased antigenicity and revealed quantifiable numbers of melanocytes. This method is applicable to both human and mouse melanocytes particularly in the hair follicle.
Assuntos
Folículo Piloso/citologia , Técnicas Imunoenzimáticas/métodos , Melanócitos/citologia , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Feminino , Técnica Direta de Fluorescência para Anticorpo , Técnica Indireta de Fluorescência para Anticorpo , Folículo Piloso/metabolismo , Melanócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , GravidezRESUMO
The lacrimal gland produces secretions that lubricate and protect the cornea of the eye. Foxc1 encodes a forkhead/winged helix transcription factor required for the development of many embryonic organs. Autosomal dominant mutations in human FOXC1 cause eye disorders such as Axenfeld-Rieger Syndrome and glaucoma iris hypoplasia, resulting from malformation of the anterior segment of the eye. We show here that lacrimal gland development is severely impaired in homozygous null Foxc1 mouse mutants, with reduced outgrowth and branching. Foxc1 is expressed in both the epithelium of the lacrimal gland and the surrounding mesenchyme. FGF10 stimulates the growth and branching morphogenesis in cultures of wild type and Foxc1 mutant gland epithelial buds. However, using micromass culture of lacrimal gland mesenchyme, we show that Bmp7 induces wild type mesenchyme cells to aggregate, but Foxc1 mutant cells do not respond. This study demonstrates that Foxc1 mediates the BMP signaling required for lacrimal gland development.
Assuntos
Fatores de Transcrição Forkhead/genética , Aparelho Lacrimal/embriologia , Morfogênese , Animais , Proteína Morfogenética Óssea 7 , Proteínas Morfogenéticas Ósseas/metabolismo , Células Cultivadas , Células Epiteliais , Fatores de Transcrição Forkhead/fisiologia , Imuno-Histoquímica , Hibridização In Situ , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Camundongos Knockout , Mutação , Técnicas de Cultura de Órgãos , Fator de Crescimento Transformador beta/metabolismo , beta-Galactosidase/metabolismoRESUMO
Craniofacial development is severely affected by null mutations in Foxc1, indicating a multifunctional role for Foxc1 in ocular, maxilla and mandible, skull and facial gland development. To delineate signaling pathways in which Foxc1 is involved we compared the transcriptomes of whole heads of Foxc1+/+ and Foxc1-/- embryos using a candidate cDNA array comprising genes expressed in the head mesenchyme and ocular region, and a 7K oligo array. Absence of Foxc1 led to downregulation of Stat1 and Galnt4, and upregulation of Tgf beta1i4 at embryonic day 13.5 in the developing head mesenchyme. Comparative analyses revealed differences in the expression pattern of Tgf beta1i4 in the head mesenchyme of Foxc1-/- and Foxc1+/+ embryos. In the ocular regions of Foxc1-/- embryos, Tgf beta1i4 was expressed in higher levels in the conjunctival epithelium and in the condensing mesenchyme on the nasal aspect of the developing eye while in wild-type embryos more intense expression was seen in the mesenchyme on the temporal aspect of the eye. Such data indicate that Foxc1 regulation of Tgf beta1i4 is complex and may be cell-type dependent. Analysis of the regulation of Tgf beta1i4 by Foxc1 in a more homogenous cell population, mesenchymal cells isolated from the periocular region revealed that, in these cells, Foxc1 negatively regulated Tgf beta1i4 expression, presumably via secreted factors such as TGF-beta1. Since Foxc1 expression is essential for normal craniofacial development, it is possible that its downstream targets play a role in the development of the phenotypes associated with null mutations in Foxc1.
Assuntos
Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica no Desenvolvimento , Cabeça/embriologia , Mesoderma , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Animais , Linhagem Celular , Regulação para Baixo , Olho/citologia , Olho/embriologia , Feminino , Fatores de Transcrição Forkhead/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Organismos Livres de Patógenos EspecíficosRESUMO
Plasminogen activator inhibitor type 1 (PAI1) is considered to be the main regulator of fibrinolytic activity in blood and has been identified as a key-enzyme in the metastasis and vascularization of solid tumors. The aim of this study was to determine whether high or low plasma levels and/or activity of PAI1 correlate with the presence of metastatic disease for patients with melanoma. We hypothesized that the presence of metastases could result in a disturbance of the fibrinolytic balance of the blood. To test our hypothesis, we have developed a unique enzyme-linked immunosorbent assay (ELISA) that can measure both the total amount as well as the active fraction of PAI1 in the plasma. We then used this novel assay to analyze the plasmatic PAI1 levels and activity of patients with advanced melanoma (AM, n = 18) and primary melanoma (PM, n = 21) and compare it to a control population (n = 38). We found no statistically significant difference in the total plasmatic PAI1 levels between the controls and patients with PM or AM (P = 0.6199). In contrast, there was a significant difference in the active fraction of PAI1 between the controls and patients with PM or AM (P = 0.0076). The difference between the control and AM groups was highly significant (P = 0.0042). A value of less than 44% active PAI1 was shown to be clinically meaningful by linear discriminant analysis. Surprisingly, the difference between the control and PM groups was also significant--although borderline (P = 0.0488). Of the patients with PM, 19% had PAI1 activity values less than 44%, which strongly supports further investigations to determine whether plasmatic PAI1 activity might be a biological marker of increased metastatic risk.