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While skin is readily available for sampling and direct studies of its constituents, an important intermediate step is to design in vitro and/or in vivo models to address scientific or medical questions in dermatology and skin biology. Pioneered more than 30 years ago, human skin equivalents (HSEs) have been refined with better cell culture techniques and media, together with sophisticated cell biology tools including genetic engineering and cell reprogramming. HSEs mimic key elements of human skin biology and have been instrumental in demonstrating the importance of cell-cell interactions in skin homeostasis and the role of a complex cellular microenvironment to coordinate epidermal proliferation, differentiation and pigmentation. HSEs have a wide field of applications from cell biology to dermocosmetics, modelling diseases, drug development, skin ageing, pathophysiology and regenerative medicine. In this article we critically review the major current approaches used to reconstruct organotypic skin models and their application with a particular emphasis on skin biology and pathophysiology of skin disorders.
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Modelos Biológicos , Dermatopatias/patologia , Pele/crescimento & desenvolvimento , Animais , Queimaduras/patologia , Queimaduras/fisiopatologia , Comunicação Celular/fisiologia , Técnicas de Cultura de Células , Homeostase/fisiologia , Humanos , Queratinócitos/fisiologia , Melanócitos/fisiologia , Camundongos , Transtornos de Fotossensibilidade/patologia , Transtornos de Fotossensibilidade/fisiopatologia , Pele/citologia , Dermatopatias/fisiopatologia , Células-Tronco/fisiologia , Cicatrização/fisiologiaRESUMO
Current international guidelines lack definite conclusions regarding repeat stool sampling for the detection of toxigenic Clostridium difficile. We assessed the value of repeat sampling and compared the diagnostic yield in an epidemic to a non-epidemic setting. Consecutive fecal samples obtained during two time frames were analyzed using direct stool immunoassay toxin testing (enzyme immunoassay [EIA]), direct stool real-time PCR toxin gene testing, and toxigenic culture. Samples collected within 7 days of the initial sample were considered repeat tests. In the epidemic setting 989 patients were analyzed, and in the non-epidemic setting 1,015. In the epidemic setting 204 patients had two or more specimens included for analysis and in the non-epidemic setting 287 patients. In the epidemic setting 136 samples yielded a positive results, either by EIA or toxigenic culture; of these, 108 were positive according to EIA and 123 according to toxigenic culture. In the first test round 98 (90.7%, 95% CI 85.3 to 96.2), 114 (92.7%, 88.1 to 97.3), and 126 (92.6%, 88.3 to 97.0) positives were detected. Subsequent test rounds yielded 10 (9.3%, 3.8 to 14.7), 9 (7.3%, 2.7 to 11.9), and 10 (7.4%, 3.0 to 11.7) extra positives. In the non-epidemic setting EIA, toxigenic culture and PCR detected 33, 66, and 83 positives. The three tests combined 93 detected positives. In the first test round 30 (90.9%, 81.1 to 100.7), 63 (95.5%, 90.4 to 110.5), 76 (91.6%, 85.6 to 97.5), and 87 (93.5%, 88.6 to 98.5) positives were detected. Subsequent test rounds yielded 3 (9.1%, -0.7 to 18.9), 3 (4.5%, -0.5 to 9.6), 7 (8.4%, 2.5 to 14.4), and 6 (6.5%, 1.5 to 11.4) extra positives. In conclusion, repeat testing resulted in 4.5% to 9.3% extra positives. No significant difference between the settings studied could be demonstrated. Repeat sampling and multimodality testing may be chosen in an outbreak situation to detect all cases, effectively controlling nosocomial spread.
Assuntos
Clostridioides difficile/isolamento & purificação , Infecções por Clostridium/diagnóstico , Diarreia/diagnóstico , Manejo de Espécimes/métodos , Técnicas de Cultura de Células/métodos , Infecções por Clostridium/epidemiologia , Infecções por Clostridium/microbiologia , Diarreia/epidemiologia , Diarreia/microbiologia , Fezes/química , Fezes/microbiologia , Humanos , Imunoensaio/métodos , Estudos Prospectivos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Estudos RetrospectivosRESUMO
BACKGROUND: Dimethyl fumarate (DMF) is an oral immunomodulatory drug used in the treatment of autoimmune diseases. Here, we sought to study whether the effect of DMF can be detected using positron emission tomography (PET) targeting the 18-kDa translocator protein (TSPO) in the focal delayed-type hypersensitivity rat model of multiple sclerosis (fDTH-EAE). The rats were treated orally twice daily from lesion activation (day 0) with either vehicle (tap water with 0.08% Methocel, 200 µL; control group n = 4 (3 after week four)) or 15 mg/kg DMF (n = 4) in 0.08% aqueous Methocel (200 µL) for 8 weeks. The animals were imaged by PET using the TSPO tracer [18F]GE-180 in weeks 0, 1, 2, 4, 8, and 18 following lesion activation, and the non-displaceable binding potential (BPND) was calculated. Immunohistochemical staining for Iba1, CD4, and CD8 was performed in week 18, and in separate cohorts of animals, following 2 or 4 weeks of treatment. RESULTS: Using the fDTH-EAE model, DMF reduced the [18F]GE-180 BPND in the DMF-treated animals compared to control animals after 1 week of treatment (two-tailed unpaired t test, p = 0.031), but not in weeks 2, 4, 8, or 18 when imaged in vivo by PET. Immunostaining for Iba1 showed that DMF had no effect on the perilesional volume or the core lesion volume after 2 or 4 weeks of treatment, or at 18 weeks. However, the optical density (OD) measurements of CD4+ staining showed reduced OD in the lesions of the treated rats. CONCLUSIONS: DMF reduced the microglial activation in the fDTH-EAE model after 1 week of treatment, as detected by PET imaging of the TSPO ligand [18F]GE-180. However, over an extended time course, reduced microglial activation was not observed using [18F]GE-180 or by immunohistochemistry for Iba1+ microglia/macrophages. Additionally, DMF did affect the infiltration of CD4+ and CD8+ T-lymphocytes at the fDTH-EAE lesion.
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In the present work, we report the imaging of Au nanostars nanoparticles (AuNSt) and their multifunctional applications in biomedical research and theranostics applications. Their optical and spectroscopic properties are considered for the multimodal imaging purpose. The AuNSt are prepared by the seed-meditated method and characterized for use as an agent for bio-imaging. To demonstrate imaging with AuNSt, penetration and localization in different biological models such as cancer cell culture (A549 lung carcinoma cell), 3D tissue model (multicellular tumor spheroid on the base of human oral squamous carcinoma cell, SAS) and murine skin tissue are studied. AuNSt were visualized using fluorescence lifetime imaging (FLIM) at two-photon excitation with a pulse duration 140 fs, repetition rate 80 MHz and 780 nm wavelength femtosecond laser. Strong emission of AuNSt at two-photon excitation in the near infrared range and fluorescence lifetime less than 0.5 ns were observed. It allows using AuNSt as a fluorescent marker at two-photon fluorescence microscopy and lifetime imaging (FLIM). It was shown that AuNSt can be observed inside a thick sample (tissue and its model). This is the first demonstration using AuNSt as an imaging agent for FLIM at two-photon excitation in biosystems. Increased scattering of near-infrared light upon excitation of AuNSt surface plasmon oscillation was also observed and rendered using a possible contrast agent for optical coherence tomography (OCT). AuNSt detection in a biological system using FLIM is compared with OCT on the model of AuNSt penetrating into animal skin. The AuNSt application for multimodal imaging is discussed.
RESUMO
Morphogenesis of embryonic organs is regulated by epithelial-mesenchymal interactions associating with changes in the extracellular matrix (ECM). The response of the cells to the changes in the ECM must involve integral cell surface molecules that recognize their matrix ligand and initiate transmission of signal intracellularly. We have studied the expression of the cell surface proteoglycan, syndecan, which is a matrix receptor for epithelial cells (Saunders, S., M. Jalkanen, S. O'Farrell, and M. Bernfield. J. Cell Biol. In press.), and the matrix glycoprotein, tenascin, which has been proposed to be involved in epithelial-mesenchymal interactions (Chiquet-Ehrismann, R., E. J. Mackie, C. A. Pearson, and T. Sakakura. 1986. Cell. 47:131-139) in experimental tissue recombinations of dental epithelium and mesenchyme. Our earlier studies have shown that in mouse embryos both syndecan and tenascin are intensely expressed in the condensing dental mesenchyme surrounding the epithelial bud (Thesleff, I., M. Jalkanen, S. Vainio, and M. Bernfield. 1988. Dev. Biol. 129:565-572; Thesleff, I., E. Mackie, S. Vainio, and R. Chiquet-Ehrismann. 1987. Development. 101:289-296). Analysis of rat-mouse tissue recombinants by a monoclonal antibody against the murine syndecan showed that the presumptive dental epithelium induces the expression of syndecan in the underlying mesenchyme. The expression of tenascin was induced in the dental mesenchyme in the same area as syndecan. The syndecan and tenascin positive areas increased with time of epithelial-mesenchymal contact. Other ECM molecules, laminin, type III collagen, and fibronectin, did not show a staining pattern similar to that of syndecan and tenascin. Oral epithelium from older embryos had lost its ability to induce syndecan expression but the presumptive dental epithelium induced syndecan expression even in oral mesenchyme of older embryos. Our results indicate that the expression of syndecan and tenascin in the tooth mesenchyme is regulated by epithelial-mesenchymal interactions. Because of their early appearance, syndecan and tenascin may be used to study the molecular regulation of this interaction. The similar distribution patterns of syndecan and tenascin in vivo and in vitro and their early appearance as a result of epithelial-mesenchymal interaction suggest that these molecules may be involved in the condensation and differentiation of dental mesenchymal cells.
Assuntos
Glicoproteínas de Membrana/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Biossíntese de Proteínas , Proteoglicanas , Germe de Dente/metabolismo , Animais , Células Cultivadas , Desenvolvimento Embrionário e Fetal , Epitélio/fisiologia , Imunofluorescência , Mandíbula/embriologia , Mandíbula/metabolismo , Camundongos , Camundongos Endogâmicos , Morfogênese , Sulfatos/metabolismo , Radioisótopos de Enxofre , Sindecanas , TenascinaRESUMO
We have studied the expression of an integral cell surface proteoglycan, syndecan, during the healing of cutaneous wounds, using immunohistochemical and in situ hybridization methods. In normal mouse skin, both syndecan antigen and mRNA were found to be expressed exclusively by epidermal and hair follicle cells. After incision and subsequent suturing, remarkably increased amounts of syndecan on the cell surfaces of migrating and proliferating epidermal cells and on hair follicle cells adjacent to wound margins were noted. This increased syndecan expression was shown to be a consequence of greater amounts of syndecan mRNA. Induction was observed already 1 d after wounding, was most significant at the time of intense cell proliferation, and was still observable 14 d after incision. The migrating cells of the leading edge of the epithelium also showed enhanced syndecan expression, although clearly less than that seen in the proliferating epithelium. The merging epithelial cells at the site of incision showed little or no syndecan expression; increased syndecan expression, however, was detected during later epithelial stratification. When wounds were left unsutured, in situ hybridization experiments also revealed scattered syndecan-positive signals in the granulation tissue near the migrating epidermal sheet. By immunohistochemical analysis, positive staining in granulation tissue was observed around vascular endothelial cells in a subpopulation of growing capillaries. Induction of syndecan in granulation tissue both at the protein and mRNA levels was temporally and spatially highly restricted. Granulation tissue, which formed in viscose cellulose sponge cylinders placed under the skin of rats, was also found to produce 3.4 and 2.6 kb mRNA species of syndecan similar to that observed in the normal murine mammary epithelial cell line, NMuMG. These results suggest that syndecan may have a unique and important role as a cell adhesion and a growth factor-binding molecule not only during embryogenesis but also during tissue regeneration in mature tissues.
Assuntos
Glicoproteínas de Membrana/biossíntese , Proteoglicanas/biossíntese , Cicatrização , Animais , Diferenciação Celular , Linhagem Celular , Movimento Celular , Endotélio Vascular/metabolismo , Tecido de Granulação/metabolismo , Imuno-Histoquímica , Queratinócitos/citologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Hibridização de Ácido Nucleico , Pele/citologia , Pele/lesões , Pele/metabolismo , SindecanasRESUMO
BACKGROUND: Positron emission tomography (PET) can be used for in vivo evaluation of the pathology associated with multiple sclerosis. We investigated the use of longitudinal PET imaging and the 18-kDa translocator protein (TSPO) binding radioligand [18F]GE-180 to detect changes in a chronic multiple sclerosis-like focal delayed-type hypersensitivity experimental autoimmune encephalomyelitis (fDTH-EAE) rat model during and after anti-VLA-4 monoclonal antibody (mAb) treatment. Thirty days after lesion activation, fDTH-EAE rats were treated with the anti-VLA-4 mAb (n = 4) or a control mAb (n = 4; 5 mg/kg, every third day, subcutaneously) for 31 days. Animals were imaged with [18F]GE-180 on days 30, 44, 65, 86 and 142. Another group of animals (n = 4) was used for visualisation the microglia with Iba-1 at day 44 after a 2-week treatment period. RESULTS: After a 2-week treatment period on day 44, there was a declining trend (p = 0.067) in [18F]GE-180-binding in the anti-VLA-4 mAb-treated animals versus controls. However, cessation of treatment for 4 days after a 31-day treatment period increased [18F]GE-180 binding in animals treated with anti-VLA-4 mAb compared to the control group (p = 0.0003). There was no difference between the groups in TSPO binding by day 142. CONCLUSIONS: These results demonstrated that cessation of anti-VLA-4 mAb treatment for 4 days caused a transient rebound increase in neuroinflammation. This highlights the usefulness of serial TSPO imaging in the fDTH-EAE model to better understand the rebound phenomenon.
RESUMO
OBJECTIVES: The aim of this verification study was to compare the QuantiFERON®-TB Gold Plus (QFT-Plus) to the QuantiFERON®-TB Gold In Tube (QFT-GIT). The new QFT-Plus test contains an extra antigen tube which, according to the manufacturer additionally elicits a CD8+ T-cell response above the CD4+ T-cell response. We assessed the value of this tube in detecting recent latent tuberculosis infections. METHODS: Between May 2015 and December 2016, 1031 subjects underwent QFT-Plus and QFT-GIT test. Overall agreement between both tests and performance for different test indications and/or immune states was assessed. A difference of >0.6 IU/mL interferon-γ release between the two antigen tubes of the QFT-Plus assay was considered a true difference and used as estimation for CD8+ T-cell response. RESULTS: Analysis of the QuantiFERON tests resulted in an overall agreement between assays of 95%. Subjects considered to be recently exposed to tuberculosis had significantly more often a true difference in interferon-γ release compared to all other subjects (p = 0.029). CONCLUSION: Results of QFT-Plus are highly comparable to QFT-GIT. Although there is an indication that a true difference in interferon-γ release between the antigen tubes is associated with recent latent tuberculosis infection, the QFT-Plus could not be used to exclude recent exposure.
Assuntos
Antígenos de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Testes de Liberação de Interferon-gama , Interferon gama/imunologia , Tuberculose Latente/diagnóstico , Mycobacterium tuberculosis/imunologia , Adulto , Bélgica , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/microbiologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/microbiologia , Feminino , Interações Hospedeiro-Patógeno , Humanos , Interferon gama/metabolismo , Tuberculose Latente/imunologia , Tuberculose Latente/microbiologia , Masculino , Pessoa de Meia-Idade , Países Baixos , Valor Preditivo dos Testes , Reprodutibilidade dos TestesRESUMO
We have analyzed the expression of early growth response gene (Egr-1) by mRNA in situ hybridization during mouse embryonic tooth development and in experimental recombinations of dental epithelium and mesenchyme. Egr-1 was transiently and recurrently expressed both in epithelial and mesenchymal cells starting from day 13 of gestation and up to 4 days after birth. The expression correlated with developmental transition points of dental mesenchymal and epithelial cells suggesting a role for Egr-1 in sequential determination and differentiation of cells. In recombination cultures of early dental epithelium and mesenchyme Egr-1 RNA was localized at the epithelial-mesenchymal interface in mesenchymal cells, and in two cases also in epithelial cells. These data indicate that Egr-1 expression may be regulated by epithelial-mesenchymal interactions when they are specific enough to initiate differentiation. We have also analyzed by in situ hybridization whether Wilms' tumour-1 gene (wt-1) is expressed in the developing tooth as it was proposed on the bases of in vitro studies that it may inhibit Egr-1 expression. No wt-1 expression was detected at any stage of tooth development showing that wt-1 is not obligatory for regulation of Egr-1 expression.
Assuntos
Regulação da Expressão Gênica , Odontogênese/genética , Animais , Células Epiteliais , Genes do Tumor de Wilms , Hibridização In Situ , Mesoderma/citologia , Camundongos/embriologia , Camundongos/genética , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Dente Molar/embriologia , Morfogênese , RNA Mensageiro/análiseRESUMO
Development of an organ is directed by cell and tissue interactions and these also occur during the formation of functional kidney. During vertebrate development inductive signalling between mesenchyme and epithelium controls the organogenesis of all three kinds of kidneys: pronephros, mesonephros and metanephros. In higher animals the metanephros differentiates into the permanent kidney and in this review we will mainly concentrate on its development. Molecular interactions currently known to function during nephrogenesis have primarily been based on the use of knockout techniques. These studies have highlighted the role for transcription factors, signalling molecules, growth factors and their receptors and also for extracellular matrix components in kidney development. Finally in this review we will represent our own model for kidney development according to the knowledge of the genes involved in the development of the functional excretory organ, kidney.
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Rim/embriologia , Animais , Apoptose , Aves/embriologia , Adesão Celular , Comunicação Celular , Divisão Celular , Linhagem da Célula , Movimento Celular , Genes Homeobox/fisiologia , Mamíferos/embriologia , Mesoderma/fisiologia , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Morfogênese , Mutação , Neovascularização Fisiológica , Transdução de SinaisRESUMO
We demonstrate that Sprouty genes 1, 2 and 4 are expressed in several developing organs of the craniofacial area and trunk, including the brain, cochlea, nasal organs, teeth, salivary gland, lungs, digestive tract, kidneys and limb buds. In organs such as the semicircular canal, Rathke's pouch, nasal organs, the follicle of vibrissae and teeth, Sprouty1 and Sprouty2 are expressed in the epithelium and Sprouty4 in the mesenchyme or neuronal tissue, while in the lung Sprouties1, 2 and 4 are all expressed mainly in the epithelial tissue. In the kidney, Sprouty1 is prominent in the ureteric bud whereas Sprouty2 and 4 are expressed in both the ureteric bud and the kidney mesenchyme and glomeruli deriving from it. The expression profiles suggest roles for these Sprouties in the epithelial-mesenchymal interactions that govern organogenesis.
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Embrião de Mamíferos/metabolismo , Proteínas de Membrana/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Fosfoproteínas/biossíntese , Proteínas Adaptadoras de Transdução de Sinal , Animais , Sistema Digestório/embriologia , Etiquetas de Sequências Expressas , Cabeça/embriologia , Hibridização In Situ , Pulmão/embriologia , Camundongos , Glândulas Salivares/embriologia , Fatores de Tempo , Distribuição TecidualRESUMO
A chain of reciprocal interactions between the epithelial and mesenchymal tissues regulates both morphogenesis and cell differentiation in the developing tooth. The very early interactions lead to budding of the oral epithelium and to the characteristic condensation of the neural crest-derived mesenchymal cells around the epithelial bud. During the bell stage of morphogenesis, the mesenchymal cells which are in contact with the dental epithelium differentiate into odontoblasts. In this reveiw article we summarize the results of our descriptive and experimental studies, which indicate that differentiation of the dental mesenchymal cells into odontoblasts, as well the condensation of dental mesenchymal cells at the bud stage, are regulated by interactions between the cell surface and the extracellular matrix. Transfilter studies where the dental epithelium and mesenchyme were cultured on opposite sides of Nuclepore filters, led to the hypothesis that the differentiation of dental mesenchymal cells into odontoblasts is triggered by interactions between the cell surface and the epithelial basement membrane matrix. Immunohistochemical localization of various matrix molecules showed that the matrix glycoproteins fibronectin and tenascin are accumulated in the dental basement membrane at the time of odontoblast differentiation. Fibronectin and tenascin are known to interact with each other, with other matrix molecules as well as with the cell surface, and also to influence cell shape. We suggest that fibronectin and tenascin are involved in the cell-matrix interaction which leads to the polarization and differentiation of odontoblasts.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Matriz Extracelular/fisiologia , Odontogênese , Dente/embriologia , Animais , Epitélio/fisiologia , Glicoproteínas/biossíntese , Mesoderma/metabolismo , Morfogênese , Germe de Dente/fisiologiaRESUMO
Since the discovery that inductive tissue interactions regulate nephrogenesis, one of the aims has been to identify the molecules that mediate this induction. The small size of embryonic tissue has limited the possibilities to identify the inducers biochemically, even though such efforts were directed to study, e.g. neural induction (for a comprehensive review, Saxén and Toivonen, Primary embryonic induction, Academic Press, London, 1962). The rapid progress in molecular biology made it possible to identify genes from minute amounts of tissue and provided techniques to generate recombinant proteins to assay their action in classic experimental systems. This led to the identification of some signals that are involved in primary and secondary inductive interactions during embryogenesis. Here, we will review evidence suggesting that secreted signaling molecules from the Wnt gene family mediate kidney tubule induction.
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Indução Embrionária , Túbulos Renais/embriologia , Proteínas Proto-Oncogênicas/metabolismo , Animais , Túbulos Renais/metabolismo , Mesoderma/metabolismo , Néfrons/metabolismo , Proteoglicanas/fisiologia , Transdução de Sinais/fisiologia , Proteínas Wnt , Proteína Wnt4RESUMO
The final step in the biosynthesis of testosterone is reduction of androstenedione by the enzyme 17beta-hydroxysteroid dehydrogenase/ 17-ketosteroid reductase (17betaHSD/17KSR). In this study, we have examined expression of the four known reductive isoforms of 17betaHSD/ 17KSR (types 1, 3, 5, and 7) in the developing mouse testis and have determined changes in the localization of isoform expression and testosterone secretion during development. Using RT-PCR isoforms 1, 3, and 7 were shown to be expressed in the seminiferous tubules of neonatal testis, whereas isoforms 3 and 7 were expressed in the interstitial tissue of the adult testis. The type 7 isoform is unlikely to be involved in androgen synthesis and further study concentrated on the type 3 isoform. Developmentally, isoform type 3 was expressed in the seminiferous tubules up to day 10, showed little or no expression on day 20 and from day 30 was confined to the interstitial tissue. In situ hybridization confirmed that the type 3 isoform was expressed only in the seminiferous tubules in fetal testes and in the interstitial tissue in adult testes. In accordance with the localization of enzyme messenger RNA expression 17-ketosteroid reductase enzyme activity was very low in isolated interstitial tissue from neonatal testes while interstitial tissue from adult testes showed high activity. Seminiferous tubules from both neonatal and adult testes showed high levels of enzyme activity. The major androgen secreted by the interstitial tissue of prepubertal animals was androstenedione up to day 20 while 5alpha-androstanediol and/or testosterone were the major androgens secreted from day 30 onwards. These results show that fetal Leydig cells do not express significant levels of a reductive isoform of 17betaHSD/ 17KSR and that androstenedione is the major androgen secreted by these cells. Production of testosterone up until puberty is dependent upon 17betaHSD/17KSR activity in the seminiferous tubules--a "two cell" requirement for testosterone synthesis. Expression of the 17betaHSD/17KSR type 3 isoform (the main reductive isoform in the testis) declines in the seminiferous tubules before puberty but then reappears in the developing adult Leydig cell population.
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17-Hidroxiesteroide Desidrogenases/metabolismo , Envelhecimento/metabolismo , Animais Recém-Nascidos/metabolismo , Feto/metabolismo , Isoenzimas/metabolismo , Testículo/enzimologia , Androgênios/biossíntese , Androstenodiona/metabolismo , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Desenvolvimento Embrionário e Fetal , Hibridização In Situ , Técnicas In Vitro , Células Intersticiais do Testículo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C3H , Testículo/citologia , Testículo/embriologia , Testículo/crescimento & desenvolvimento , Distribuição TecidualRESUMO
UNLABELLED: Polymorphism Ala54Thr of the intestinal fatty acid-binding protein 2 (FABP2) has been reported to have an effect on the protein's affinity for long chain fatty acids and to be associated with serum lipid and insulin levels in fasting and especially postprandial states. We wanted to test whether this genetic variation is associated with fasting and postprandial glucose, insulin or lipid levels in 666 male university students participating in the second European Atherosclerosis Study (EARS II). We also studied whether the subgroup of 330 students with paternal history of myocardial infarction (MI) before the age of 55 have different genotype distribution than 336 matched controls. RESULTS: No difference in genotype distribution was observed between offspring with and without paternal history of MI or between populations from 11 European countries. The frequency of the threonine encoding allele was 0.276 in cases and 0.266 in controls. There were no differences in fasting or postprandial serum lipid, glucose or insulin levels between subjects having different genotypes. CONCLUSIONS: In this study FABP2 Ala54Thr polymorphism was not associated with lipid or glucose metabolism. In addition to environmental and genetic factors, selection of study population also may explain the difference between this and earlier studies.
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Proteínas de Transporte/genética , Gorduras na Dieta/administração & dosagem , Ácidos Graxos/genética , Teste de Tolerância a Glucose , Mucosa Intestinal/metabolismo , Proteínas de Neoplasias , Polimorfismo Genético , Proteínas Supressoras de Tumor , Adolescente , Adulto , Arteriosclerose/genética , Arteriosclerose/metabolismo , Glicemia/análise , Proteínas de Transporte/metabolismo , Códon , Jejum , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Ácidos Graxos/metabolismo , Genótipo , Humanos , Insulina/sangue , Lipídeos/sangue , Masculino , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Período Pós-PrandialRESUMO
17beta-Hydroxysteroid dehydrogenase type 2 (17HSD type 2) catalyzes the inactivation of estradiol, testosterone and dihydrotestosterone into biologically less active 17-keto forms. Our recent Northern analysis indicated that the enzyme is expressed both in mouse placenta and fetus. The present data indicate that in the placenta the distribution of enzyme expression changes during pregnancy. In the choriovitelline placenta (day 8) 17HSD type 2 was expressed both in mural and polar giant cells. Later, on days 9-12.5, the mRNA was also detected in the junctional zone, and in late gestation (days 14.5-17.5), 17HSD type 2 mRNA was predominantly expressed only at the labyrinth region. In the fetus, 17HSD type 2 expression appears in the liver on day 11. At day 12 the expression was strongly increased in the liver, and at the same time moderate mRNA expression was also detected in the esophagus and intestine. In these tissues, high constitutive expression of 17HSD type 2 was then maintained throughout pregnancy. At later stages of development (days 15-16) the mRNA was, furthermore, detected in epithelial cells of the stomach, tongue, oropharynx and nasopharynx as well as in the kidney. We conclude that the expression pattern of 17HSD type 2 in the developing placenta and fetus suggests a role for the enzyme in maintaining a barrier to the transfer of active 17-hydroxy forms of sex steroids between the fetus and maternal circulation.
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17-Hidroxiesteroide Desidrogenases/genética , Feto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Placenta/metabolismo , RNA Mensageiro/metabolismo , Animais , Células Epiteliais/metabolismo , Feminino , Camundongos , Especificidade de Órgãos , Placenta/enzimologia , Gravidez , RNA Mensageiro/genéticaRESUMO
The expression of the gene COL1A2, coding for the pro-alpha 2 chain of type I pro-collagen, was analyzed in fully developed human permanent teeth. The teeth were fixed with formalin, demineralized with EDTA for about ten weeks, and embedded in paraffin. Pro-alpha 2(I) mRNA was localized in the sections by in situ hybridization, with use of [35S)]-labeled single-stranded RNA probes. The amount of mRNA for pro-alpha 2(I) collagen chain, as indicated by the relative densities of silver grains and the grain counts per cell in autoradiography, was high in odontoblasts, whereas in pulpal fibroblasts it was low. High levels of pro-alpha 2(I)mRNA expression were also present in those odontoblasts which had elaborated new dentin matrix in response to dental caries. Expression in the periodontal ligament, including the cementoblast layer, was slightly stronger than that in odontoblasts. The intense expression of pro-alpha 2(I) mRNA in odontoblasts of adult teeth suggests that even after the completion of primary dentin formation, they continue to synthesize heterotrimeric type I collagen molecules. Cell type-specific differences in the expression of pro-alpha 2(I) mRNA imply that type I collagen probably plays a major role in the regulation of the structure and function of dental tissues. Finally, in situ hybridization enabled pro-alpha 2(I) collagen mRNA to be detected in tissue sections even after prolonged demineralization, and thus it proved to be a valuable technique for analysis of gene expression in adult dental tissues, as shown here for COL1A2.
Assuntos
Colágeno/genética , Polpa Dentária/química , Odontoblastos/química , Ligamento Periodontal/química , RNA Mensageiro/análise , Adulto , Colágeno/biossíntese , Dentina/química , Expressão Gênica , Humanos , Dente Molar/metabolismo , Hibridização de Ácido Nucleico , Sondas RNARESUMO
The molecular specificity of the dental papilla of a bell-stage tooth was studied by production of dental-papilla-reactive monoclonal antibodies (Mabs). One of the Mabs, designated 7C5, recognized an epitope present in glycosaminoglycan. Several lines of evidence suggested that the 7C5-epitope consists of chondroitin 6-sulfate. The Mab did not react with mouse dental epithelium, but reacted uniformly with mesenchymal tissue in the mandibular process and accumulated in the dental sac and in the papilla of bell-stage tooth germs. The 7C5-staining was lost from the differentiating odontoblasts, while the staining in the molar tooth papilla was accumulated in the subodontoblastic layer. In the developing mouse incisor, the 7C5-epitope was restricted to the lingual-posterior area. The 7C5-epitope was also present in pulpal tissue and predentin of different types of teeth of various mammalian species, including man, sheep, swine, and rat. Collagenase pre-treatment of tissue sections abolished the bulk of the 7C5-reactivity in peridental mesenchyme during embryonic stages while leaving the staining of the dental papilla intact. In newborn and adult teeth, collagenase also impaired the reactivity in the pulp except for the subodontoblastic layer. This suggests the existence of different subpopulations of the 7C5-epitope containing proteoglycans in dental papilla and pulp. A high-molecular-weight proteoglycan, sensitive to chondroitinase ABC but not to heparinase or heparitinase, was immunoprecipitated by 7C5 from extracts of bell-stage mouse tooth germs. We suggest that the evolutionary conservation of chondroitin 6-sulfate in the dental pulp reflects its properties as non-terminally differentiated tissue and perhaps the retention of a potential to differentiate to odontoblasts.
Assuntos
Sulfatos de Condroitina/análise , Papila Dentária/química , Polpa Dentária/química , Epitopos/análise , Animais , Anticorpos Monoclonais , Bovinos , Membrana Celular/química , Membrana Celular/imunologia , Membrana Celular/ultraestrutura , Sulfatos de Condroitina/genética , Sulfatos de Condroitina/imunologia , Papila Dentária/imunologia , Papila Dentária/patologia , Polpa Dentária/imunologia , Polpa Dentária/patologia , Saco Dentário/química , Saco Dentário/imunologia , Saco Dentário/patologia , Epitopos/genética , Matriz Extracelular/química , Matriz Extracelular/imunologia , Matriz Extracelular/ultraestrutura , Expressão Gênica , Humanos , Hibridomas , Mesoderma/química , Mesoderma/imunologia , Mesoderma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Odontogênese/genética , Odontogênese/imunologia , Periodonto/química , Periodonto/imunologia , Periodonto/patologia , Ratos , Ovinos , SuínosRESUMO
OBJECTIVE: Acute alcohol exposure induces malformation and malfunction of placenta-yolk sac tissues in rodents, reducing the labyrinth zone in the placenta and altering the permeability and fluidity of the cell membrane. During normal mouse placentation the cells line up in an optimal way to form a hemotrichorial placenta where layers II and III are connected through gap junctions. These act as molecular sieves that limit the passage of large molecules. PlGF is a developmentally regulated protein that controls the passage of molecules in the vasculosyncytial membranes and media of large blood vessels in the placental villi. In addition to the chorioallontoic placenta, rodents also have another type of placenta that consists of Reichert's membrane within the trophoblast cell layer on the maternal side and the parietal endodermal cells on the embryonic site. This forms a separate materno-fetal transport system. We study here whether alcohol affects these two placental barriers, leading to placental malfunction that in turn diminishes the nutrient supply to the embryo. STUDY DESIGN: CD-1 mice received two intraperitoneal injections of 3 g/kg ethanol at 4 h intervals at 8.75 days post coitum (dpc). The placentas were collected on 9.5, 11.5 and 14.5 dpc and used for histopathological protein studies. Hemotrichorial cell layer structure interactions through connective tissue and gap junction were analyzed by electron microscopy. The permeability of the feto-maternal barrier was visualized with Evans Blue. RESULTS: VEGF, a permeability inducer, was found to be up-regulated in the mouse placenta after acute alcohol exposure, and permeability was also affected by altered structures in the barriers that separate the feto-maternal blood circulation which destroyed the gap junctions in the hemotrichorial cell layer, reduced the thickness of Reichert's membrane and interfered with with Reichert's trophoblast/Reichert's parietal interaction. These defects together could have caused the permeability malfunction of the placenta-yolk sac tissues as visualized and quantified here by Evans Blue leakage. CONCLUSIONS: An altered PlGF/VEGF ratio together with barrier malformation may contribute to placental malfunction by altering the permeability of the feto-maternal barriers. Further studies are needed in order to show whether premature permeability is involved in the intrauterine growth restriction observed in human FAS embryos.