ANA LUCIA GARIPPO
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conferenceObject Confocal Microscopy Image Analysis of Pancreatic beta-Cells K-ATP Channel Proteins in Congenital Hyperinsulinism (CHI)(2012) LOVISOLO, S. M.; GARIPPO, A. L.; GUEDES, F.; ZERBINI, M. C.Background: CHI is a life-threatening disorder of glucose metabolism of neonates characterized by serum insulin levels unresponsive to blood glucose concentrations. Pancreatic ß-cells regulates insulin secretion through ATP sensitive potassium channels (KATP channel) formed by sulfonylurea receptor 1 (SUR1) and potassium inward rectifying 6.2 (Kir 6.2) proteins. The ß-cell K ATP channel dependent CHI is classically associated with loss-of-function mutations of these subunits genes. In a previous study [1], no mutations in the Kir6.2 gene and in the 33-37 exons hot spot region of the SUR1 gene were identified in these patients. The aim of this study is to investigate if these subunits were present in the ß-cells of CHI patients. Design: Pancreatic surgical paraffin tissue from 7 neonates (3F/4M, 4-13 mo) with CHI and 8 autopsy pancreatic control tissue (5F/3M, 4-11 mo) were included in the study. Confocal microscopy double-staining immunofluorescence (insulin/SUR1 and insulin/Kir6.2) was performed in order to detect each protein specifically in the ß-cells All sections were analyzed under a Zeiss 510 Meta confocal laser scanning microscope (63x). At least 10 different endocrine islets were captured to evaluate the expression of either Kir6.2 or SUR1 (green-488nm) and insulin (red-633nm). Co-expression of insulin/SUR1 and insulin/Kir6.2 was analysed visually (green x red→yellow) and through correlation Pearson’s coefficient(PC) that estimates the goodness of rate association of the two fluorochromes. PC was compared among cases and controls using a nonparametric method (Mann-Whitney). Results: Visual observation clearly revealed the presence of Kir6.2 and SUR1 in a granular cytoplasmic pattern in the ß-cells of cases and controls. Nevertheless, overlap of insulin/Kir6.2 and insulin/SUR1 seemed to be more constant and uniform in controls than in CHI cases, confirmed by the analysis through PC showing a statistically significant decrease in Kir6.2 (P= 0.0084) and SUR1 (P= 0.041) in the ß-cells of CHI patients. Conclusions: This is the first demonstration of K ATP channels subunits Kir6.2 and SUR1 in the pancreatic ß-cells of CHI patients using an in situ method. Our results show that both subunits were present in the ß-cells, but are under-expressed in CHI patients compared to controls, adding a new information to this rare condition with a complex pathogenesis.- Enriched inorganic compounds in diesel exhaust particles induce mitogen-activated protein kinase activation, cytoskeleton instability, and cytotoxicity in human bronchial epithelial cells(2015) SERIANI, Robson; JUNQUEIRA, Mara S.; CARVALHO-SOUSA, Claudia E.; ARRUDA, Alessandra Ct.; MARTINEZ, Diana; ALENCAR, Adriano M.; GARIPPO, Ana L.; BRITO, Jose Mara; MARTINS, Milton A.; SALDIVA, Paulo H. N.; NEGRI, Elnara M.; MAUAD, Thais; MACCHIONE, MariangelaThis study assessed the effects of the diesel exhaust particles on ERR and JNK MAPKs activation, cell rheology (viscoelasticity), and cytotoxicity in bronchial epithelial airway cells (BEAS-2B). Crude DEP and DEP after extraction with hexane (DEP/HEX) were utilized. The partial reduction of some DEP/HEX organics increased the biodisponibility of many metallic elements. JNK and ERR were activated simultaneously by crude DEP with no alterations in viscoelasticity of the cells. Mitochondrial activity, however, revealed a decrease through the MIT assay. DEP/HEX treatment increased viscoelasticity and cytotoxicity (membrane damage), and also activated JNK. Our data suggest that the greater bioavailability of metals could be involved in JNK activation and, consequently, in the reduction of fiber coherence and increase in the viscoelasticity and cytotoxicity of BEAS cells. The adverse findings detected after exposure to crude DEP and to DEP/HEX reflect the toxic potential of diesel compounds. Considering the fact that the cells of the respiratory epithelium are the first line of defense between the body and the environment, our data contribute to a better understanding of the pathways leading to respiratory cell injury and provide evidence for the onset of or worsening of respiratory diseases caused by inorganic compounds present in DEP.
conferenceObject Confocal Microscopy Image Analysis of Pancreatic beta-Cells K-ATP Channel Proteins in Congenital Hyperinsulinism (CHI)(2012) LOVISOLO, S. M.; GARIPPO, A. L.; GUEDES, F.; ZERBINI, M. C.Background: CHI is a life-threatening disorder of glucose metabolism of neonates characterized by serum insulin levels unresponsive to blood glucose concentrations. Pancreatic ß-cells regulates insulin secretion through ATP sensitive potassium channels (K ATP channel) formed by sulfonylurea receptor 1 (SUR1) and potassium inward rectifying 6.2 (Kir 6.2) proteins. The ß-cell K ATP channel dependent CHI is classically associated with loss-of-function mutations of these subunits genes. In a previous study [1], no mutations in the Kir6.2 gene and in the 33-37 exons hot spot region of the SUR1 gene were identified in these patients. The aim of this study is to investigate if these subunits were present in the ß-cells of CHI patients. Design: Pancreatic surgical paraffin tissue from 7 neonates (3F/4M, 4-13 mo) with CHI and 8 autopsy pancreatic control tissue (5F/3M, 4-11 mo) were included in the study. Confocal microscopy double-staining immuno fluorescence (insulin/SUR1 and insulin/Kir6.2) was performed in order to detect each protein specifically in the ß-cells. All sections were analyzed under a Zeiss 510 Meta confocal laser scanning microscope (63x). At least 10 different endocrine islets were captured to evaluate the expression of either Kir6.2 or SUR1 (green-488nm) and insulin (red-633nm). Co-expression of insulin/SUR1 and insulin/Kir6.2 was analysed visually (green x red→yellow) and through correlation Pearson’s coefficient(PC) that estimates the goodness of rate association of the two fluorochromes. PC was compared among cases and controls using a nonparametric method (Mann-Whitney). Results: Visual observation clearly revealed the presence of Kir6.2 and SUR1 in a granular cytoplasmic pattern in the ß-cells of cases and controls. Nevertheless, overlap of insulin/Kir6.2 and insulin/SUR1 seemed to be more constant and uniformin controls than in CHI cases, confirmed by the analysis through PC showing a statistically significant decrease in Kir6.2 ( P = 0.0084) and SUR1 ( P = 0.041) in the ß-cells of CHI patients. Conclusions: This is the first demonstration of K ATP channels subunits Kir6.2 and SUR1 in the pancreatic ß-cells of CHI patients using an in situ method. Our results show that both subunits were present in the ß-cells, but are under-expressed in CHI patients compared to controls, adding a new information to this rare condition with a complex pathogenesis.- Unusual remodeling of the hyalinization band in vulval lichen sclerosus by type V collagen and ECM 1 protein(2015) GODOY, Charles A.P.; TEODORO, Walcy R.; VELOSA, Ana Paula P.; GARIPPO, Ana Lucia; EHER, Esmeralda Miristeni; PARRA, Edwin Roger; SOTTO, Mirian N.; CAPELOZZI, Vera L.OBJECTIVES: The vulva is the primary site affected in lichen sclerosus, a chronic dermatosis in women that is histologically characterized by a zone of collagen remodeling in the superior dermis. The normal physiological properties of the vulva depend on the assembly of collagen types I (COLI), III (COLIII) and V (COLV), which form heterotypic fibers, and extracellular matrix protein interactions. COLV regulates the heterotypic fiber diameter, and the preservation of its properties is important for maintaining normal tissue architecture and function. In the current work, we analyzed the expression of COLV and its relationship with COLI, COLIII, elastic fibers and extracellular matrix protein 1 in vulvar biopsies from patients with lichen sclerosus. METHODS: Skin biopsies from 21 patients with lichen sclerosus, classified according to Hewitt histological criteria, were studied and compared to clinically normal vulvar tissue (N=21). Morphology, immunohistochemistry, immunofluorescence, 3D reconstruction and morphometric analysis of COLI, COLIII, COLV deposition, elastic fibers and extracellular matrix 1 expression in a zone of collagen remodeling in the superior dermis were performed. RESULTS: A significant decrease of elastic fibers and extracellular matrix 1 protein was present in the hyalinization zone of lichen sclerosus compared to healthy controls. The non-homogeneous distribution of collagen fibers visualized under immunofluorescence in the hyalinization zone of lichen sclerosus and control skin was confirmed by histomorphometry. Lichen sclerosus dermis shows a significant increase of COLI, COLIII and COLV expression compared to the healthy controls. Significant inverse associations were found between elastic fibers and COLV and between COLV and extracellular matrix 1 expression. A direct association was found between elastic fiber content and extracellular matrix 1 expression. Tridimensional reconstruction of the heterotypic fibers of the lichen sclerosus zone of collagen remodeling confirmed the presence of densely clustered COLV. CONCLUSIONS: Increased deposition of abnormal COLV and its correlation with extracellular matrix 1 and elastic fibers suggest that COLV may be a trigger in the pathogenesis of lichen sclerosus.
article 25 Citação(ões) na Scopus A comparative study of extracellular matrix remodeling in two murine models of emphysema(2013) LOPES, F. D. T. Q. S.; TOLEDO, A. C.; OLIVO, C. R.; PRADO, C. M.; LEICK, E. A.; MEDEIROS, M. C.; SANTOS, A. B. G.; GARIPPO, A.; MARTINS, M. A.; MAUAD, T.A single instillation of porcine pancreatic elastase (PPE) results in significant airspace enlargement on the 28th day after instillation, whereas cigarette smoke (CS) exposure requires 6 months to produce mild emphysema in rodents. Considering that there are differences in the pathogenesis of parenchymal destruction in these different experimental models, it is likely that there may be different patterns of extracellular matrix (ECM) remodeling. To evaluate ECM remodeling, C57BL/6 mice were submitted to either a nasal drop of PPE (PPE 28 Days) or exposed for 6 months to cigarette smoke (CS 6 months). Control groups received either an intranasal instillation of saline solution (Saline 28 Days) or remained without any smoke inhalation for six months (Control 6 months). We measured the mean linear intercept and the volume proportion of collagen type I, collagen type III, elastin and fibrillin. We used emission-scanning confocal microscopy to verify the fiber distribution. Both models induced increased mean linear intercept in relation to the respective controls, being larger in the elastase model in relation to the CS model. In the CS model, emphysema was associated with an increase in the volume proportion of fibrillin, whereas in the PPE model there was an increase in the parenchymal elastin content. In both models, there was an increase in collagen type III, which was higher in the CS-exposed mice. We concluded that ECM remodeling is different in the two most used experimental models of emphysema.