RESUMEN
BACKGROUND: Increased abdominal fat and chronic inflammation in the expanded adipose tissue of obesity contribute to the development of insulin resistance and type 2 diabetes mellitus (T2D). The emerging immunoregulatory and anti-inflammatory properties of Sertoli cells have prompted their application to experimental models of autoimmune/inflammatory disorders, including diabetes. The main goal of this work was to verify whether transplantation of microencapsulated prepubertal porcine Sertoli cells (MC-SC) in the subcutaneous abdominal fat depot of spontaneously diabetic and obese db/db mice (homozygous for the diabetes spontaneous mutation [Leprdb ]) would: (i) improve glucose homeostasis and (ii) modulate local and systemic immune response and adipokines profiles. METHODS: Porcine prepubertal Sertoli cells were isolated, according to previously established methods and enveloped in Barium alginate microcapsules by a mono air-jet device. MC-SC were then injected in the subcutaneous abdominal fat depot of db/db mice. RESULTS: We have preliminarily shown that graft of MC-SC restored glucose homeostasis, with normalization of glycated hemoglobin values with improvement of the intraperitoneal glucose tolerance test in 60% of the treated animals. These results were associated with consistent increase, in the adipose tissue, of uncoupling protein 1 expression, regulatory B cells, anti-inflammatory macrophages and a concomitant decrease of proinflammatory macrophages. Furthermore, the treated animals showed a reduction in inducible NOS and proinflammatory molecules and a significant increase in an anti-inflammatory cytokine such as IL-10 along with concomitant rise of circulating adiponectin levels. The anti-hyperglycemic graft effects also emerged from an increased expression of GLUT-4, in conjunction with downregulation of GLUT-2, in skeletal muscle and liver, respectively. CONCLUSIONS: Preliminarily, xenograft of MC-SC holds promises for an effective cell therapy approach for treatment of experimental T2D.
Asunto(s)
Glucemia/metabolismo , Diabetes Mellitus Tipo 2/inmunología , Xenoinjertos/citología , Homeostasis/inmunología , Células de Sertoli/trasplante , Trasplante Heterólogo , Tejido Adiposo/citología , Animales , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2/terapia , Composición de Medicamentos , Prueba de Tolerancia a la Glucosa/métodos , Xenoinjertos/inmunología , Resistencia a la Insulina/fisiología , Masculino , Ratones Transgénicos , Porcinos , Trasplante Heterólogo/métodosRESUMEN
Occupational and environmental exposure to the heavy metal cadmium (Cd) and its inhalation from cigarette smoke are associated with emphysema. Many growth factors and extracellular matrix (ECM) cell signaling molecules are directly involved in the epithelial bronchial cell pathway. This study investigated the direct effects of Cd on the production of several ECM components in human bronchial epithelial cells (BEAS-2B) that were exposed in vitro for 48 h to sub-toxic and toxic concentrations of Cd. Gene expression of collagens, metalloproteases (MMPs), integrins, tenascin and vitronectin were quantified by RT-PCR. To study apoptosis cascade, annexin assay and cellular cytotoxicity by MTT assay were performed. We also investigated whether an imbalance in the TGFß/TGFß receptor (TGFßR) expression mediated Cd effects. The results showed the sub-toxic Cd dose significantly increased tenascin, vitronectin, ß1 and ß5 integrin gene expression. The toxic Cd dose decreased type IV and V collagen, α1, α2 and ß3 integrins. Both Cd doses down-regulated type I collagen and up-regulated metalloproteases. Each Cd dose caused a different imbalance in the complex pattern of TGFß and its receptors. No alteration in classic apoptotic marker protein expression was observed in presence of the sub-toxic dose of Cd, suggesting this metal alters ECM production without apoptotic activation. In conclusion, all these data show even sub-toxic Cd dose exposure alters the specific gene expression of several ECM components that are crucially implicated in the mechanical properties of lung parenchyma supporting the hypothesis that the mechanism underlying Cd-induced lung disease may involve downstream changes in TGFß/TGFßR signaling.
Asunto(s)
Bronquios/citología , Cadmio/toxicidad , Células Epiteliales/efectos de los fármacos , Proteínas de la Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular , Colágeno/genética , Regulación hacia Abajo , Expresión Génica , Humanos , Integrinas/genética , Metaloproteasas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal , Tenascina/genética , Factor de Crecimiento Transformador beta/metabolismo , Regulación hacia Arriba , Vitronectina/genéticaRESUMEN
BACKGROUND: Porcine Sertoli cells (pSCs) have been employed for cell therapy in pre-clinical studies for several chronic/immune diseases as they deliver molecules associated with trophic and anti-inflammatory effects. To be employed for human xenografts, pSCs products need to comply with safety and stability. To fulfill such requirements, we employed a microencapsulation technology to increase pre-transplant storage stability of specific pathogen-free pSCs (SPF-pSCs) and evaluated the in vivo long-term viability and safety of grafts. METHODS: Specific pathogen free neonatal pigs underwent testis excision under sterility. pSCs were isolated, characterized by immunofluorescence (IF) and cytofluorimetric analysis (CA) and examined in terms of viability and function [namely, production of anti-müllerian hormone (AMH), inhibin B, and transforming growth factor beta-1 (TFGß-1)]. After microencapsulation in barium alginate microcapsules (Ba-MC), long-term SPF-pSCs (Ba-MCpSCs) viability and barium concentrations were evaluated at 1, 24 throughout 40 h to establish pre-transplant storage conditions. RESULTS: The purity of isolated pSCs was about 95% with negligible contaminating cells. Cultured pSCs monolayers, both prior to and after microencapsulation, maintained high function and full viability up to 24 h of storage. At 40 h post-encapsulation, pSCs viability decreased to 80%. Barium concentration in Ba-MCpSCs lagged below the normal maximum daily allowance and was stable for 4 months in mice with no evident side effects. CONCLUSIONS: Such results suggest that this protocol for the isolation and microencapsulation of pSCs is compatible with long-haul transportation and that Ba-MCpSCs could be potentially employable for xenotransplantation.
Asunto(s)
Células de Sertoli/trasplante , Trasplante Heterólogo/métodos , Alginatos , Animales , Animales Recién Nacidos , Separación Celular , Trasplante de Células/métodos , Células Cultivadas , Ácido Glucurónico , Ácidos Hexurónicos , Humanos , Masculino , Ratones , Células de Sertoli/citología , Células de Sertoli/fisiología , Organismos Libres de Patógenos Específicos , PorcinosRESUMEN
Exposure to nicotine and other compounds contained in cigarette smoking affects human health. This study examined the effects of exposure to a single or multiple sub-toxic nicotine concentrations on human osteoblasts. Cell growth and expression of genes involved in bone differentiation, extracellular matrix (ECM) metabolism, and growth factor signaling pathways were investigated in nicotine-treated cells compared to untreated cells. Depending on osteoblast concentration and maturation stages, nicotine differently regulated cell growth. Real-time PCR showed regulated expressions of genes expressed by nicotine-treated osteoblasts compared to untreated cells. Among ECM genes, type I collagen was down-regulated and osteonectin was up-regulated in nicotine-treated osteoblasts; similarly, fibroblast growth factor-1 (FGF1) and fibroblast growth factor-2 (FGF2), two members of FGF signaling system, were discordantly modulated; genes involved in osteoblast maturation and differentiation such as alkaline phosphatase (ALP), runt-related transcription factor-2 (RUNX2), and bone sialoprotein (BSP) were over-expressed after drug treatment. Our results show a positive association between nicotine exposure and osteoblast phenotype and illustrate for the first time a mechanism whereby acute or chronic exposure to sub-toxic nicotine concentrations may affect bone formation through the impairment of growth factor signaling system and ECM metabolism.
Asunto(s)
Matriz Extracelular/genética , Factor 1 de Crecimiento de Fibroblastos/biosíntesis , Factor 2 de Crecimiento de Fibroblastos/biosíntesis , Nicotina/toxicidad , Osteoblastos/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Proliferación Celular/efectos de los fármacos , Colágeno Tipo I/biosíntesis , Matriz Extracelular/efectos de los fármacos , Factor 1 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/genética , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Osteopontina/biosíntesis , Transducción de SeñalRESUMEN
Nonsyndromic cleft lip with or without cleft palate (CLP) is a frequent craniofacial malformation caused by both genetic and environmental factors. Maternal smoking during pregnancy is a known risk factor, due to the teratogenic role of nicotine. To assess and compare the impact of CLP and nicotine, we studied the quantitative expression of genes involved in signaling pathways and extracellular matrix (ECM) metabolism in human normal nicotine-treated (NicN) and CLP fibroblasts compared to normal control (CTRL) cells. Palatal fibroblast cultures from seven CLP children and seven age-matched CTRL subjects were established and subconfluent cells incubated for 24 h without (CTRL and CLP fibroblasts) or with (NicN fibroblasts) 0.6 mM nicotine. Gene expressions were analyzed by real-time quantitative PCR. For the first time, a regulated cholinergic signaling in our human fibroblasts in vitro was demonstrated. Members of TGF-beta, retinoic acid (RA), and GABA-ergic signaling systems were also differently regulated. Among the ECM genes, fibronectin, syndecan, integrin alpha2, and MMP13 genes were concordantly modulated, while integrin beta5, and decorin genes were discordantly modulated. Interestingly, nicotine treatment regulated gene expressions of CD44 and CLPTM1, two candidate genes for CLP. Our findings show a positive association between nicotine treatment and CLP phenotype. Results suggest that nicotine deranges normal palate development, which might contribute to the development of a CLP malformative phenotype, through the impairment of some important signaling systems and ECM composition.
Asunto(s)
Labio Leporino/genética , Fisura del Paladar/genética , Matriz Extracelular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Transducción de Señal/efectos de los fármacos , Estudios de Casos y Controles , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Preescolar , Labio Leporino/inducido químicamente , Labio Leporino/metabolismo , Labio Leporino/patología , Fisura del Paladar/inducido químicamente , Fisura del Paladar/metabolismo , Fisura del Paladar/patología , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Genotipo , Humanos , Masculino , Nicotina/toxicidad , Agonistas Nicotínicos/toxicidad , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genéticaRESUMEN
Prenatal exposure to diazepam, a prototype sedative drug that belongs to Benzodiazepines, can lead to orofacial clefting in human newborns. By using real-time PCR, in the present study we investigated whether diazepam elicits gene expression alterations in extracellular matrix (ECM) components, growth factors and gamma-aminobutyric acid receptor (GABRB3), implicated in the coordinate regulation of palate development. Palate fibroblasts were treated with diazepam (Dz-N fibroblasts) and compared to cleft lip-palate (CLP) fibroblasts obtained from patients with no known exposure to diazepam or other teratogens. Untreated fibroblasts from non-CLP patients were used as control. The results showed significant convergences in gene expression pattern of collagens, fibromodulin, vitronectin, tenascin C, integrins and metalloprotease MMP13 between Dz-N and CLP fibroblasts. Among the growth factors, constitutive Fibroblast Growth Factor 2 (FGF2) was greatly enhanced in Dz-N and CLP fibroblasts and associated with a higher reduction of FGF receptor. Transforming Growth Factor beta 3 (TGFbeta(3)) resulted up-regulated in CLP fibroblasts and decreased in Dz-N fibroblasts. We found phenotypic differences exhibited by Dz-N and CLP fibroblasts in GABRB3 gene regulation, so further studies are necessary to determine whether GABAergic system could be involved in the development of diazepam mediated CLP phenotype. Taken together the results elucidate the molecular mechanisms underlying possible toxicology effects induced by diazepam. Counselling of women on the safety of diazepam exposure is clinically important, also for the forensic consequences.
Asunto(s)
Ansiolíticos/toxicidad , Labio Leporino/inducido químicamente , Fisura del Paladar/inducido químicamente , Diazepam/toxicidad , Proteínas de la Matriz Extracelular/genética , Fibroblastos/efectos de los fármacos , Paladar Duro/efectos de los fármacos , Estudios de Casos y Controles , Proliferación Celular , Forma de la Célula/efectos de los fármacos , Células Cultivadas , Niño , Labio Leporino/genética , Labio Leporino/patología , Fisura del Paladar/genética , Fisura del Paladar/patología , Proteínas de la Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Paladar Duro/crecimiento & desarrollo , Paladar Duro/metabolismo , ARN Mensajero/metabolismo , Receptores de GABA-A/genéticaRESUMEN
The study evaluated the effects of 4â¯wt% nanohydroxyapatite (HA), 6â¯wt% zinc l-carnosine (MDA) and 1.5â¯wt% Ciprofloxacin (AB) on the mechanical, thermal and biological properties of glass ionomer cements (GIC). Filler and additive concentrations were selected after a previous study had tested single components and different percentages. Specimens included five silicon molds of each GIC cement for all tests. They were stored at room temperature for 24â¯h from specimen collection to analysis. Mechanical tests, calorimetric analysis, morphological investigation, antibacterial and cell viability assays were conducted. One-way analysis of variance (ANOVA) was used for data analysis with significance set at pâ¯<â¯0.05. Adding HA, MDA and AB to GICs modified their thermal, mechanical and microbiological properties. Polymerization increased. A slight decrease in the compressive strength of modified GICs was observed in dry condition (pâ¯<â¯0.05). Cement extracts affected cell viability in relation to extract dilution. Mechanical behavior improved in modified glass ionomer cements, especially with the powder formulated antibiotic. Overall cytotoxicity was reduced. Therefore adding nanohydroxyapatite, antibiotic and a mucosal defensive agent to conventional glass ionomer cement in special need patients could improve the clinical, preventive and therapeutic performance of the cements, without altering their mechanical properties.
Asunto(s)
Carnosina/análogos & derivados , Ciprofloxacina/farmacología , Durapatita/química , Cementos de Ionómero Vítreo/química , Nanopartículas/química , Compuestos Organometálicos/farmacología , Temperatura , Rastreo Diferencial de Calorimetría , Carnosina/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Fuerza Compresiva , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Humanos , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Ensayo de Materiales , Pruebas de Sensibilidad Microbiana , Nanopartículas/ultraestructura , Streptococcus mutans/efectos de los fármacos , Estrés Mecánico , Compuestos de Zinc/farmacologíaRESUMEN
Universal adhesives are the most important innovation in restorative dentistry. They are composed of different monomers, solvents and fillers. The potential cytotoxic effect of these materials is an important scientific aspect in recent literature. The aim of this study was to determine, using different in vitro techniques, the cytotoxicity evaluation of seven universal enamel-dental adhesives on human gingival fibroblasts. For this purpose, seven universal dental enamel adhesives have been evaluated by in vitro cytotoxicity tests using direct contact tests (an unpolymerized and a polymerized method) and an indirect contact test: preparation of extracts. The polymerized method showed a cytotoxicity range from 36% (G-PremioBond, GPB) to 79% (FuturaBond M+, FB). With the unpolymerized direct methods the range was from 4% (Prime&Bond Active, PBA) to 40% (Ibond Universal, IB) for undiluted adhesives; generally passing to the major dilutions the test showed a strong inhibitory activity by all the adhesives. Whereas with the indirect method by diluting the extracts of all dental adhesives the cell viability increased. The data obtained from the work has shown a lower cytotoxic effect of Optibond Solo Plus (OB) and Adhesive Universal (AU) with more reliable results with the extracts technique. The choice of reliable in vitro cytotoxic technique could represent, in dental practice, an important aid for clinical procedures in the use of adhesive systems.
Asunto(s)
Cementos Dentales/toxicidad , Fibroblastos/efectos de los fármacos , Encía/citología , Metacrilatos/toxicidad , Línea Celular , Supervivencia Celular/efectos de los fármacos , HumanosRESUMEN
Nicotine contained in cigarette smoke contributes to the onset of several diseases, including osteoporosis, whose emerging pathogenic mechanism is associated with osteoblasts apoptosis. Scanty information is available on the molecular mechanisms of nicotine on osteoblasts apoptosis and, consequently, on an important aspect of the pathogenesis of smokers-related osteoporosis. Glyoxalase 1 (Glo1) is the detoxification enzyme of methylglyoxal (MG), a major precursor of advanced glycation end products (AGEs), potent pro-apoptotic agents. Hydroimidazolone (MG-H1) is the major AGE derived from the spontaneous MG adduction of arginine residues. The aim of this study was to investigate whether, and by means of which mechanism, the antiglycation defence Glo1 was involved in the apoptosis induced by 0.1 and 1µM nicotine in human primary osteoblasts chronically exposed for 11 and 21 days. By using gene overexpression/silencing and scavenging/inhibitory agents, we demonstrated that nicotine induces a significant intracellular accumulation of hydrogen peroxide (H2O2) that, by inhibiting Glo1, drives MG-H1 accumulation/release. MG-H1, in turn, triggers H2O2 overproduction via receptor for AGEs (RAGE) and, in parallel, an apoptotic mitochondrial pathway by inducing Transglutaminase 2 (TG2) downregulation-dependent NF-kB desensitization. Measurements of H2O2, Glo1 and MG-H1 circulating levels in smokers compared with non-smokers or in smokers with osteoporosis compared with those without this bone-related disease supported the results obtained in vitro. Our findings newly pose the antiglycation enzymatic defense Glo1 and MG-H1 among the molecular events involved in nicotine-induced reactive oxygen species-mediated osteoblasts apoptosis, a crucial event in smoker-related osteoporosis, and suggest novel exposure markers in health surveillance programmes related to smokers-associated osteoporosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Fumar Cigarrillos/efectos adversos , Nicotina/efectos adversos , Osteoblastos/efectos de los fármacos , Osteoporosis/etiología , Proteínas de Unión al GTP/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Imidazoles/metabolismo , Lactoilglutatión Liasa/metabolismo , FN-kappa B/metabolismo , Nicotina/toxicidad , Ornitina/análogos & derivados , Ornitina/metabolismo , Osteoblastos/metabolismo , Osteoporosis/metabolismo , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transducción de Señal/fisiología , Transglutaminasas/metabolismoRESUMEN
Environmental pollution is one of the main factors responsible for reducing fertility in males. Lead is one of the major heavy metal contaminants that impairs several organs; it preferentially accumulates in male reproductive organs and alters sperm quality both in vivo and in vitro. However, the underlying mechanisms remain unclear. Sertoli cells (SCs) provide structural and physiological support to spermatogenic cells within seminiferous tubules. Therefore, changes in SCs affect the developing germ cells and alter spermatogenesis. This study aimed to assess whether exposure to subtoxic doses of adversely affects SC functioning in higher mammals. Purified and functional porcine neonatal SCs were exposed to lead acetate at three different concentrations. Lead exposure decreased the mRNA expression and protein levels of inhibin B and anti-Mullerian hormone (AMH) compared to control, indicating loss of FSH-r integrity in terms of 17-ß-oestradiol production under FSH stimulation. In addition, we observed an increase in the mRNA levels of Akt and mTOR, and the phosphorylation of p38 and Akt in SCs exposed to lead at all concentrations compared to unexposed control SCs. In conclusion, lead is toxic to SCs, even at low concentrations, and is expected to alter spermatogenesis.
Asunto(s)
Compuestos Organometálicos/toxicidad , Células de Sertoli/efectos de los fármacos , Animales , Animales Recién Nacidos , Hormona Antimülleriana/metabolismo , Recuento de Células , Supervivencia Celular/efectos de los fármacos , Células Germinativas/efectos de los fármacos , Inhibinas/metabolismo , Masculino , ARN Mensajero/biosíntesis , ARN Mensajero/efectos de los fármacos , Receptores de HFE/efectos de los fármacos , Túbulos Seminíferos/efectos de los fármacos , Espermatogénesis/efectos de los fármacos , PorcinosRESUMEN
Spermatogenesis is a highly complicated biological process that occurs in the epithelium of the seminiferous tubules. It is regulated by a complex network of endocrine and paracrine factors and by juxtacrine testicular cross-talk. Sertoli cells (SC) play a key role in spermatogenesis due to their production of trophic, differentiation and immune-modulating factors, but many of the molecular pathways of SC action remain controversial and unclear. Over the last two decades, research has focused on extracellular vesicles as an important mechanism of intercellular communication. The aim of this study was to investigate the presence of extracellular vesicles (EVs) in SC and the modulation of their content in SC after FSH and testosterone stimulation. Highly purified porcine pre-pubertal Sertoli cells were isolated according to previously established methods. After 48 h of culture with FSH or FSH + testosterone stimulation, we identified sertolian EVs containing specific mRNAs. Proteomic analysis of EVs content identified 29 proteins under non-stimulatory conditions, most of which were related to receptor binding activity. FSH stimulation induced increases in inhibin-alpha, inhibin-beta, plakoglobin, haptoglobin, D-3-phosphoglycerate dehydrogenase and sodium/potassium-transporting ATPase in sertolian EVs. Testosterone stimulation enhanced the abundance of inhibin-alpha, inhibin-beta, tissue-type plasminogen activator, epidermal growth factor-like protein 8, elongating factor 1-gamma and D-3-phosphoglycerate dehydrogenase. These results are likely to help determine the unknown molecular secretion of Sertoli cells.
Asunto(s)
Hormona Folículo Estimulante/farmacología , Proteómica/métodos , Células de Sertoli/metabolismo , Testosterona/farmacología , Animales , Separación Celular , Vesículas Extracelulares/efectos de los fármacos , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/ultraestructura , Masculino , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células de Sertoli/efectos de los fármacos , PorcinosRESUMEN
OBJECTIVES: Preoperative chemotherapy may play a role in postoperative respiratory complications due to subclinical parenchymal damage. We investigated the gene expression of lung tissue components after neoadjuvant chemotherapy of alveolar-capillary membrane, extracellular matrix and membrane proteins. METHODS: The study group included 14 patients submitted to pulmonary resection for lung cancer after 3 cycles of gemcitabine-cisplatin, while the control group included 14 naive-treatment patients. RNA was extracted from frozen tissue obtained by healthy lung specimens using EZ1 RNA Universal Tissue kit and automatically purified by BioRobot EZ1 instrument. Three hundred nanograms of total RNA was reverse transcribed to complementary DNA and used to evaluate the gene expression of type I and III collagen, elastin, syndecan, metalloproteinase 13 and aquaporins (AQPs) in real-time polymerase chain reaction. Results were expressed as the mean ± standard deviation of 3 independent experiments. Analysis of variance followed by Sheffe's F-test was performed. RESULTS: Among the alveolar-capillary membrane and extracellular matrix genes, type I-III collagens and syndecan were significantly up-regulated (+645%, +327% and +261%, respectively), while elastin and metalloproteinase 13 were down-regulated in the study group versus control group (-46% and -77%, respectively). Furthermore, chemotherapy was associated with a significant up-regulation of AQP expressions (AQP1:+51% and AQP5:+36%). CONCLUSIONS: We observed, in the treated group, increases in the mean values of gene expressions for macromolecules involved in the remodelling of both the alveolar septa and parenchyma scaffold, thereby supporting the hypothesis that induction chemotherapy may foster a fibrosing effect on the pulmonary parenchyma and lead to altering the alveolar-capillary membrane.
Asunto(s)
Cisplatino/uso terapéutico , Desoxicitidina/análogos & derivados , Proteínas de la Matriz Extracelular/genética , Regulación Neoplásica de la Expresión Génica , Quimioterapia de Inducción/métodos , Neoplasias Pulmonares/genética , Proteínas de la Membrana/genética , Antineoplásicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Desoxicitidina/uso terapéutico , Quimioterapia Combinada , Proteínas de la Matriz Extracelular/biosíntesis , Estudios de Seguimiento , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/terapia , Proteínas de la Membrana/biosíntesis , Neumonectomía , Cuidados Preoperatorios , Pronóstico , Estudios Prospectivos , ARN Neoplásico/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , GemcitabinaRESUMEN
INTRODUCTION: Immune dysfunction, promoted by pro-inflammatory cytokines, plays a pivotal role in neurodegeneration associated with Huntington's disease. AIMS: The aim of this study was to investigate the emerging immunoregulatory and antiinflammatory properties of Sertoli cells in Huntington's disease. METHODS: The experimental R6/2 mouse model of Huntington's disease was treated by a single intraperitoneal injection of microencapsulated prepubertal porcine Sertoli cells and lifespan, motor performance and striatal inflammatory pattern have been evaluated. RESULTS: The results of this study demonstrated that a single intraperitoneal injection of microencapsulated prepubertal porcine Sertoli cells uniquely improved performances and extended the life expectancy of R6/2 Huntington's disease mice, by immune dysfunction modulation in brain. CONCLUSIONS: This study highlights the immunomodulatory and trophic role of Sertoli cells that could be of help in the treatment of neurodegenerative disorders.
Asunto(s)
Composición de Medicamentos/métodos , Enfermedad de Huntington/cirugía , Células de Sertoli/fisiología , Células de Sertoli/trasplante , Animales , Animales Recién Nacidos , Apoptosis/genética , Apoptosis/fisiología , Cuerpo Estriado/citología , Ciclooxigenasa 2/metabolismo , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica/fisiología , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/genética , Enfermedad de Huntington/mortalidad , Enfermedad de Huntington/fisiopatología , Masculino , Ratones , Ratones Transgénicos , Actividad Motora/fisiología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Análisis de Supervivencia , Porcinos , Repeticiones de Trinucleótidos/genéticaRESUMEN
Alginate represents one of the most appealing biopolymers for pharmaceutical and biomedical applications. Alginate as a biomaterial for clinical use has been established, although not free from issues. Here we provide a critical review on some of the main recent advances in alginate research in drug delivery and its prominent role in cell microencapsulation for the treatment of diseases, such as type 1 diabetes mellitus. A brief description of the basic properties of the polymer will be provided as well. Based on our experience and contributions, as well as wide research in the field, the correlation between physicochemical and biological properties of alginate systems and clinical outcomes will be investigated and discussed to address the actual future clinical impact of alginatebased delivery strategies.
Asunto(s)
Alginatos/química , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos , Animales , Biopolímeros/química , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diseño de Fármacos , HumanosRESUMEN
The Crouzon syndrome, which is associated with fibroblast growth factor receptor (FGFR2) mutations, is characterized by premature fusion of cranial sutures. We used an in vitro model of cultured periosteal fibroblasts from normal subjects and from Crouzon patients with FGFR2 mutation. We analyzed the matrix turnover rate and the effects of adding FGF2 by evaluating fibronectin synthesis and the activity of some proteolytic enzymes. To assess the role of some FGF signaling molecules involved in FGFR2 regulation, we studied Grb2 tyrosine phosphorylation and the phosphotyrosine proteins associated with Grb2. The iodinate FGF binding assay was performed to quantify FGFR expression. Compared with normal fibroblasts, fibronectin synthesis was decreased in Crouzon fibroblasts, and protease activities in cells and medium were enhanced, suggesting that excess fibronectin catabolism is present. Differences were more marked when FGF2 was added. Very few phosphoproteins were visible in anti-Grb2 immunoprecipitations from Crouzon fibroblasts, which showed a significant increase in the number of high-affinity and low-affinity FGF2 receptors. These results suggest that the abnormal genotype and the Crouzon cellular phenotype are related. To compensate the low levels of tyrosine phosphorylation, Crouzon cells might increase the numbers of FGFR2, thus increasing the cell surface binding sites for FGF2.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Disostosis Craneofacial/genética , Matriz Extracelular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Fibroblastos/química , Periostio/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/genética , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal , Adolescente , Catepsina B/análisis , Disostosis Craneofacial/metabolismo , Disostosis Craneofacial/patología , Endopeptidasas/análisis , Matriz Extracelular/efectos de los fármacos , Fibronectinas/biosíntesis , Fibronectinas/efectos de los fármacos , Proteína Adaptadora GRB2 , Humanos , Calicreínas/análisis , Periostio/patología , Fosforilación , Activadores Plasminogénicos/análisis , Mutación Puntual , Proteínas/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/efectos de los fármacos , Tirosina/metabolismoRESUMEN
BACKGROUND: Silicosis is mediated by macrophages, their soluble mediators, and extracellular matrix molecules. In this study, we investigated the effects of silica and/or hyaluronate (HA) on several alveolar macrophage responses. METHODS: We evaluated glycosaminoglycan (GAG) production by radiolabeled precursors, nitric oxide (NO) release by its oxidation product, phagocytic activity by Candida albicans internalization, and the secretion of two fibrogenic cytokines, tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta, by specific assays. RESULTS: Silica significantly reduced GAG secretion, particularly HA secretion. Alone, it decreased Candida uptake; associated with HA, it enhanced the reduction. Silica and Candida reduced NO release, which was not significantly affected when silica- or Candida-exposed cells were also treated with HA. TNF-alpha and TGF-beta activities were stimulated by silica but reduced by HA. CONCLUSIONS: The results suggest that silica and HA modify alveolar macrophage functional differentiation. Silica- and HA-induced modifications of the microenvironment could determine whether the response proceeds toward healing and repair or toward lung chronic pathology.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Ácido Hialurónico/farmacología , Macrófagos Alveolares/efectos de los fármacos , Dióxido de Silicio/farmacología , Animales , División Celular/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , Combinación de Medicamentos , Glicosaminoglicanos/metabolismo , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patología , Óxido Nítrico/metabolismo , Fagocitosis/efectos de los fármacos , Ratas , Factor de Crecimiento Transformador beta/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: Transforming growth factor-beta (TGF-beta) interference with interleukin 6 (IL-6) activity and the role of the latter in early human embryonic development prompted us to examine the effects IL-6 on matrix synthesis and the effects of TGF-beta3 on IL-6 expression human cleft lip and palate (CLP) fibroblasts. METHODS: Collagen and glycosaminoglycan (GAG) synthesis were determined by radiolabeled precursors and biglycan expression by Northern blotting before and after adding IL-6. The effects of TGF-beta3 on IL-6 production were assayed by evaluating IL-6 transcript by Northern blotting and IL-6 protein secretion by enzyme-linked immunosorbent assay. RESULTS: The results showed that IL-6 elicited an inhibitory effect on collagen and GAG levels in CLP fibroblasts by lowering hyaluronan and dermatan sulfate secretion. IL-6 up-regulated biglycan expression, but less strongly than TGF-beta3. TGF-beta3 significantly down-regulated IL-6 transcript and secretion in CLP fibroblasts. CONCLUSIONS: These data suggest the increase in matrix components that characterize the CLP fibroblast phenotype might be due to a concerted TGF-beta3-IL-6 action. We hypothesize changes in cross-talk between TGF-beta3 and IL-6 signal transduction pathways are involved in the induction of cleft palate.
Asunto(s)
Fisura del Paladar/metabolismo , Matriz Extracelular/metabolismo , Interleucina-6/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Análisis de Varianza , Células Cultivadas , Preescolar , Labio Leporino/metabolismo , Fisura del Paladar/etiología , Colágeno/biosíntesis , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Glicosaminoglicanos/biosíntesis , Humanos , Interleucina-6/farmacología , Proteoglicanos/biosíntesis , Receptor Cross-Talk , Transducción de Señal/efectos de los fármacos , Estadísticas no Paramétricas , Factor de Crecimiento Transformador beta/farmacología , Factor de Crecimiento Transformador beta3RESUMEN
Recombinant human IGF-1 currently represents the only available treatment option for the Laron Syndrome, a rare human disorder caused by defects in the gene encoding growth hormone receptor, resulting in irreversibly retarded growth. Unfortunately, this treatment therapy, poorly impacts longitudinal growth (13% in females and 19% in males), while burdening the patients with severe side effects, including hypoglycemia, in association with the unfair chore of taking multiple daily injections that cause local intense pain. In this study, we have demonstrated that a single intraperitoneal graft of microencapsulated pig Sertoli cells, producing pig insulin-like growth factor-1, successfully promoted significant proportional growth in the Laron mouse, a unique animal model of the human Laron Syndrome. These findings indicate a novel, simply, safe and successful method for the cell therapy-based cure of the Laron Syndrome, potentially applicable to humans.
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Factor I del Crecimiento Similar a la Insulina/metabolismo , Síndrome de Laron/terapia , Células de Sertoli/trasplante , Trasplante Heterólogo/métodos , Alginatos/química , Animales , Peso Corporal , Desarrollo Óseo , Modelos Animales de Enfermedad , Composición de Medicamentos , Femenino , Ácido Glucurónico/química , Ácidos Hexurónicos/química , Masculino , Ratones , Ratones Transgénicos , Receptores de Somatotropina/genética , PorcinosRESUMEN
Skin rejection remains a major hurdle in skin reconstructive transplantation surgery. In fact, 85% of the grafted patients experience at least one episode of acute skin rejection in the first year. It has been observed that Sertoli cells (SC), when co-transplanted with allo- or xenogeneic cell/tissues, can induce graft acceptance in the absence of systemic immunosuppression. A method aimed at significantly prolonging skin allografts in rats transplanted with barium alginate-based microencapsulated xenogeneic porcine SC (SC-MCs) is described. Results demonstrated that intraperitoneal (IP) transplantation of SC-MCs with high cellular viability and function can significantly prolong allogeneic skin grafts when compared to transplantation controls receiving only empty alginate capsules (E-MCs). Lymphocytic infiltration at the skin graft site was not observed in 80% of the SC-MCs transplanted rats and these recipient animals showed a significant increased expression of T regulatory (Tregs) cells when compared to E-MCs transplantation controls. The findings of this report further substantiate the positive therapeutic effects of SC on transplantation technology mediated by Sertoli cell-induced alterations of the host's immune system and indicate new perspectives and new strategies for successful skin tissue allografts.
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Composición de Medicamentos/métodos , Supervivencia de Injerto/inmunología , Células de Sertoli/trasplante , Trasplante de Piel/inmunología , Animales , Animales Recién Nacidos , Cápsulas , Separación Celular , Células Cultivadas , Citometría de Flujo , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Estimación de Kaplan-Meier , Masculino , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Long-Evans , Ratas Wistar , Células de Sertoli/citología , Piel/patología , Sus scrofa , Trasplante HeterólogoRESUMEN
IMPORTANCE OF THE FIELD: This review analyses international studies investigating the combined genetic and environmental causes of cleft lip with or without cleft palate (CL/P) and describes successes and limitations in identifying underlying genetic and environmental factors. CL/P, the most common congenital facial malformation, is a major public health burden in terms of medical costs and emotional stress to patients and families. Because genetic and environmental factors determine risk of occurrence, CL/P has a complex, multifactor aetiology. AREAS COVERED IN THIS REVIEW: English language reports from 1980 to 2010 were searched for in Medline, PubMed, Science Citation Index, textbooks and review articles on drugs and pregnancy. Key words were diazepam or benzodiazepine(s) combined with cleft lip, cleft palate, oral malformations, prenatal exposure, GABA, gene expression and extracellular matrix. WHAT THE READER WILL GAIN: This review presents an updated assessment of the mutagenic and genotoxic effects of diazepam (DZ), one of the most commonly used benzodiazepines, on CL/P occurrence. TAKE HOME MESSAGE: Data are divergent; more studies are needed for an in-depth picture of the effects of DZ during gestation on the child's development, particularly on orofacial clefts.