A difficult airway approach in a merosin-deficient congenital muscular dystrophy patient: a case report

Abstract Merosin-deficient muscular dystrophy is caused by an autosomal recessive mutation on laminin-α2 gene characterized by severe progressive muscle weakness associated with neuromuscular scoliosis and restrictive lung disease. In this case report, we describe an alternative airway approach performed in a child with anticipated difficult airway and merosin-deficient muscular dystrophy. Significant anesthetic implications may increase the perioperative risk, requiring accurate knowledge to anticipate an adequate management and provide patient-safety strategies.

Bioactivity effects of extracellular matrix proteins on apical papilla cells

Abstract Potent signaling agents stimulate and guide pulp tissue regeneration, especially in endodontic treatment of teeth with incomplete root formation. Objective This study evaluated the bioactive properties of low concentrations of extracellular matrix proteins on human apical papilla cells (hAPCs). Methodology Different concentrations (1, 5, and 10 µg/mL) of fibronectin (FN), laminin (LM), and type I collagen (COL) were applied to the bottom of non-treated wells of sterilized 96-well plates. Non-treated and pre-treated wells were used as negative (NC) and positive (PC) controls. After seeding the hAPCs (5×103 cells/well) on the different substrates, we assessed the following parameters: adhesion, proliferation, spreading, total collagen/type I collagen synthesis and gene expression (ITGA5, ITGAV, COL1A1, COL3A1) (ANOVA/Tukey; α=0.05). Results We observed greater attachment potential for cells on the FN substrate, with the effect depending on concentration. Concentrations of 5 and 10 µg/mL of FN yielded the highest cell proliferation, spreading and collagen synthesis values with 10 µg/mL concentration increasing the ITGA5, ITGAV, and COL1A1 expression compared with PC. LM (5 and 10 µg/mL) showed higher bioactivity values than NC, but those were lower than PC, and COL showed no bioactivity at all. Conclusion We conclude that FN at 10 µg/mL concentration exerted the most intense bioactive effects on hAPCs.

Effects of ultrasound and stretching on skeletal muscle contusion in rats: immunohistochemistry analysis / Efectos de ultrasonido y estiramiento en la contusión muscular esqueletal en ratas: análisis inmunohistoquímico

SUMMARY: The aim of this study was to evaluate the effects of stretching and therapeutic ultrasound (TUS) on desmin and laminin contents of rat muscle after contusion. Male Wistar rats (n = 35, 8-9 weeks of age, 271 ± 14g body weight) were divided into five groups: Control group (CG) (n= 03); Injured group (IG) (n= 8); Injured + ultrasound group (IUSG) (n= 8); Injured+stretching group (ISG) (n= 8); Injured +ultrasound + stretching group (IUSSG) (n= 8). The application of ultrasound started 72 hours after the contusion, using the 50 % pulsed mode, 0.5 W/cm2, 5 min, once a day, for five consecutive days. Passive manual stretching was started on the tenth day after injury, with four repetitions of 30 s each and 30 s rest between repetitions, once a day, five times per week, for a total of ten applications. After 22 days, the rats were euthanazied and the gastrocnemius of both limbs removed for desmin and laminin immunohistochemistry morphometric measurement. Analysis was conducted using ANOVA one way post-hoc Tukey to parametric data and Kruskall-Wallis for non-parametric data. The IUSSG animals showed a larger area of desmin than ISG (p<0.05). It was found a decrease in laminin comparing IUSG to IG. However, laminin area was higher in ISG than all groups (p<0.05). UST isolated or in combination with stretching influenced gastrocnemius regeneration in different manners. While stretching applied isolated enhanced gastrocnemius regeneration noticed by the increase in laminin area, in combination with TUS strengthened the muscle healing rising desmin area.

RESUMEN: El objetivo de este estudio fue evaluar los efectos del estiramiento y la ecografía en los contenidos de desmina y laminina del músculo de rata después de la lesión. Ratas Wistar macho (n = 35, 8-9 semanas de edad, 271 ± 14 g de peso corporal) se dividieron en cinco grupos: grupo de control (CG) (n = 03); Grupo lesionado (GL) (n = 8); Lesionado + grupo de ultrasonido (LGU) (n= 8); Lesionado + grupo de estiramiento (LGE) (n = 8); Lesionado + ultrasonido + grupo de estiramiento (LUGE) (n = 8). La aplicación de ultrasonido comenzó 72 horas después de la lesión, usando el modo pulsado al 50 %, 0,5W / cm2, 5 min, una vez al día, durante cinco días consecutivos. El estiramiento manual pasivo se inició el décimo día después de la lesión, con cuatro repeticiones de 30 seg cada una y 30 seg de descanso entre repeticiones, una vez al día, cinco veces por semana, para un total de diez aplicaciones. Las ratas fueron sacrificadas después de 22 días, y se extrajo el músculo gastrocnemio de ambos miembros para la medición morfométrica de desmina y laminina a través de inmunohistoquímica. El análisis se realizó utilizando ANOVA unidireccional Tukey post-hoc para datos paramétricos y Kruskall-Wallis para datos no paramétricos. Los animales LUGE mostraron un área mayor de desmina que LGE (p <0,05). Se encontró una disminución en la laminina comparando LGU con GL. Sin embargo, el área de laminina fue mayor en LGE que en todos los grupos (p <0,05). El tratamiento con ultrasonido aislado o en combinación con estiramiento influyó en la regeneración del músculo gastrocnemio de diferentes maneras. Si bien el estiramiento aplicado, en combinación con tratamiento de ultrasonido, fortaleció el área de desmina, la regeneración del músculo gastrocnemio mejoró por el aumento en el área de laminina aumentando la curación muscular.

Ameloblastoma cell lines derived from different subtypes demonstrate distinct developmental patterns in a novel animal experimental model

Abstract Objective Ameloblastoma is a representative odontogenic tumor comprising several characteristic invasive forms, and its pathophysiology has not been sufficiently elucidated. A stable animal experimental model using immortalized cell lines is crucial to explain the factors causing differences among the subtypes of ameloblastoma, but this model has not yet been disclosed. In this study, a novel animal experimental model has been established, using immortalized human ameloblastoma-derived cell lines. Methodology Ameloblastoma cells suspended in Matrigel were subcutaneously transplanted into the heads of immunodeficient mice. Two immortalized human ameloblastoma cell lines were used: AM-1 cells derived from the plexiform type and AM-3 cells derived from the follicular type. The tissues were evaluated histologically 30, 60, and 90 days after transplantation. Results Tumor masses formed in all transplanted mice. In addition, the tumors formed in each group transplanted with different ameloblastoma cells were histologically distinct: the tumors in the group transplanted with AM-1 cells were similar to the plexiform type, and those in the group transplanted with AM-3-cells were similar to the follicular type. Conclusions A novel, stable animal experimental model of ameloblastoma was established using two cell lines derived from different subtypes of the tumor. This model can help clarify its pathophysiology and hasten the development of new ameloblastoma treatment strategies.

Identificação das proteínas interagentes de Yes-Associated protein (YAP), um efetor da via Hippo, em células epiteliais mamárias expostas à matriz extracelular rica em laminina / Identification of interacting proteins of Yes-Associated Protein (Yap) in mammary epithelial cells exposed to laminin-rich extracellular matrix

A sinalização da matriz extracelular (MEC) é essencial para a determinação do destino e comportamento de células epiteliais da glândula mamária. Entretanto, pouco é conhecido sobre os mecanismos moleculares envolvidos nesse processo. A via Hippo, uma cascata de sinalização que participa da regulação de diversos comportamentos celulares, incluindo o tamanho de órgãos, parece ser uma importante candidata a mediadora sinalização da MEC. Resultados preliminares do laboratório indicam que a arquitetura tecidual e a membrana basal, componente da MEC de epitélios e outros tecidos, influenciam a localização, concentração e atividade de YAP, uma proteína efetora da via Hippo, em células epiteliais mamárias. Neste contexto, o objetivo deste trabalho foi identificar as proteínas que interagem com Yap (ortólogo de YAP em camundongo) nas células epiteliais da glândula mamária em resposta à membrana basal. Foram utilizadas células EpH4, uma linhagem mamária não-tumoral murina, como modelo de diferenciação funcional e formação de ácinos em um ensaio de cultura tridimensional (3D). O tratamento de estruturas multicelulares 3D pré-formadas em placas nãoadesiva com uma matriz rica em laminina (lrECM) alterou a localização e o padrão subcelular de Yap, assim como a expressão gênica de membros da via Hippo e dos alvos de Yap, mas não alterou a expressão das proteínas da via em nível de proteína. O ensaio de co-imunoprecipitação (CoIP) seguida de análise por espectrometria de massas identificou um conjunto diferencial de proteínas que interagem com Yap na fração citoplasmática de células EpH4 cultivadas na ausência ou na presença de lrECM em um modelo de ECM-overlay. Uma análise realizada junto à database KEGG Pathways revelou que os possíveis interagentes Yap nas células cultivadas não-tratadas com lrECM participam de processos relacionados à proteólise mediada por ubiquitina, enquanto nas células expostas à lrECM os possíveis interagentes estão associados a processos metabólicos e são especialmente proteínas-chave do metabolismo de lipídios. A busca na plataforma de redes de interação STRING não identificou trabalhos que destaquem a interação de Yap com estas proteínas. A plataforma Vizit indica a participação de Yap em processos relacionados à síntese e atividade de lipídios e hormônios, o que reforça as evidências de que está pode ser uma nova função de Yap ainda não explorada em detalhes. A fim de se obter resultados complementares à CoIP, padronizamos o ensaio de identificação por biotinilação dependente de proximidade (BioID) em células embrionárias de rim humano da linhagem 293FT. As proteínas isoladas por pulldown foram identificadas por espectrometria de massas e uma análise junto à database Gene Ontology indicou que os possíveis interagentes de Yap nestas células são em sua maioria proteínas relacionadas à via Hippo, o que reforça a robustez do ensaio. Nós pretendemos transpor este sistema para as células EpH4. A expectativa é que, em conjunto, estes resultados nos orientem em projetos futuros para compreender os mecanismos de sinalização da MEC na morfogênese e diferenciação da glândula mamária

Extracellular matrix (ECM)-signaling is crucial for determination of epithelial cell fate and behavior in the mammary gland. However, little is known about the molecular mechanisms involved in these processes. The Hippo pathway, a signaling cascade involved in the regulation of several cellular processes, including organ size, seems to be an important candidate as a mediator of this signaling. Our preliminary results indicate that the tissue architecture and the basement membrane, an ECM component of epithelia and other tissues, influence the location, level and activity of YAP, an effector of the Hippo pathway. In this context, the goal of this work was to identify the proteins that interact with Yap (ortholog of YAP in mouse) in mammary epithelial cells in response to the basement membrane. We used EpH4 cells, a nontumoral murine mammary cell, in a functional differentiation and acini-forming in tridimensional (3D) culture assay. Treatment of 3D multicellular structures pre-formed on nonadhesive plates with a laminin-rich extracellular matrix (lrECM) altered the subcellular localization and pattern of Yap, as well as gene expression of Hippo pathway proteins and Yap targets, but did not altered the expression of the pathway members at the protein level. Coimmunoprecipitation (CoIP) followed by mass spectrometry analysis identified a differential set of proteins interacting with Yap in cytoplasmic fractions of EpH4 cells in the absence or presence of lrECM in an ECM-overlay culture model. An analysis performed with the KEGG Pathways database revealed that putative Yap interactors in non-treated cells participate in processes related to ubiquitin-mediated proteolysis, whereas in cells exposed to lrECM Yap interactors are associated to metabolic processes and are mainly key-proteins of metabolism of lipids and carbohydrates. A search in interaction networks platform STRING did not identify previous works that showing the interaction of Yap with these proteins. Vizit platform indicated the participation of Yap in processes related to the synthesis and activity of lipids and hormones, which reinforces the evidences that Yap can play a novel poorly explored role. To obtain complementary results to CoIP, we devised the proximity-dependent biotinylation identification (BioID) assay on embryonic renal cells of 293FT cell line. Pulldown-isolated proteins were identified by mass spectrometry and an analysis performed with Gene Ontology database revealed that putative Yap interactors are Hippo pathway-related proteins, which reinforces the robustness of the assay. We intend to transpose this system to the EpH4 cells. We expect that, together, these results will guide us in future projects to understand the signaling mechanisms of ECM in mammary gland morphogenesis and differentiation

TNF-alpha increases angiogenic potential in a co-culture system of dental pulp cells and endothelial cells

Abstract We recently demonstrated that a co-culture system of human umbilical vein endothelial cells (HUVECs) and human dental pulp stem cells (hDPSCs) could enhance angiogenesis ability in vitro. However, whether tumor necrosis factor α (TNF-α) could promote blood vessel formation during pulp regeneration remained unknown. The aim of this study was to investigate the effects of TNF-α on the formation of endothelial tubules and vascular networks in a co-culture system of hDPSCs and HUVECs. hDPSCs were co-cultured with HUVECs at a ratio of 1:5. The Matrigel assay was performed to detect the total tubule branching lengths and numbers of branches, and the Cell-Counting Kit 8 assay was performed to examine the effect of TNF-α on cell proliferation. Real-time polymerase chain reactions and western blot were used to detect vascular endothelial growth factor (VEGF) mRNA and protein expression. The Matrigel assay showed significantly greater total branching lengths and numbers of branches formed in the experimental groups treated with different concentrations of TNF-α compared with the control group. The decomposition times of the tubule structures were also significantly prolonged (P < 0.05). Treatment with 50 ng/ml TNF-α did not significantly change the proliferation of co-cultured cells, but it significantly increased the VEGF mRNA and protein expression levels (p < 0.05). In addition, the migration abilities of HUVECs and hDPSCs increased after co-culture with TNF-α (p < 0.05). TNF-α enhanced angiogenic ability in vitro in the co-culture system of hDPSCs and HUVECs.

Morphological observation of bone marrow mesenchymal stem cells under matrigel three dimensional culture conditions / Observación morfológica de células madre mesenquimales de médula ósea en condiciones de cultivo tridimensional con matrigel

SUMMARY: Matrigel is a basement membrane matrix extracted from the EHS mouse tumor containing extracellular matrix protein, its main components are laminin, type IV collagen, nestin, heparin sulfate, growth factor and matrix metalloproteinase.At room temperature, Matrigel polymerized to form a three dimensional matrix with biological activity. It can simulate the structure, composition, physical properties and functions of the cell basement membrane in vivo, which is beneficial to the culture and differentiation of the cells in vitro, and can be used for the study of cell morphology, biochemical function, migration, infection and gene expression. In this study, Matrigel three-dimensional culture model of bone marrow mesenchymal stem cells(BMSCs) was established, and its morphology, proliferation and survival were observed. BMSCs were isolated and cultured with whole bone marrow adherence method. The Second generation BMSCs with good growth condition were selected and mixed with Matrigel to form cell gel complexes. The morphology and proliferation of mesenchymal stem cells were observed by phase contrast microscope and HE staining,Live/Dead staining was used to evaluate the cell activity.Phase contrast microscopy showed that BMSCs were reticulated in Matrigel and proliferated well, After 7 days, the matrix gel gradually became soft and collapsed, a few cell reticular crosslinking growth was seen at 14 days; HE staining showed that the cytoplasm of the cells was larger on the fourth day and the cells were elongated and cross-linked on the seventh day; Live/dead staining showed that most cells showed green fluorescence with the prolongation of culture time, on the first, 4 and 7 days, the activity of bone marrow mesenchymal stem cells in Matrigel gradually increased, and the percentages were 92.57 %, 95.54 % and 97.37 %, respectively. Matrigel three-dimensional culture system can maintain the morphology, function and proliferation ability of bone marrow mesenchymal stem cells.

RESUMEN: Matrigel es una matriz de membrana basal extraída del tumor de ratón EHS que contiene proteína de matriz extracelular. Los componentes principales son laminina, el colágeno tipo IV, nestina, sulfato de heparina, factor de crecimiento y metaloproteinasa de matriz. A temperatura ambiente, Matrigel se polimerizó para formar una matriz tridimensional. Es posible simular la estructura, la composición, las propiedades físicas y las funciones de la membrana basal celular in vivo, lo que es beneficioso para el cultivo y la diferenciación de las células in vitro, y se puede utilizar para el estudio de la morfología celular, la función bioquímica, la migración, infección y expresión génica. En este estudio, se estableció el modelo de cultivo tridimensional Matrigel de células madre mesenquimales de médula ósea (BMSC), y se observó su morfología, proliferación y supervivencia. Las BMSC fueron aisladas y cultivadas con el método de adherencia de la médula ósea completa. Se seleccionaron las BMSC de segunda generación con buenas condiciones de crecimiento y se mezclaron con Matrigel para formar complejos de gel de células. La morfología y la proliferación de las células madre mesenquimales se observaron con microscopio de contraste de fase y se tiñó con Hematoxilina-Eosina (HE); para evaluar la actividad celular se usó la tinción Live/Dead. La microscopía de contraste mostró que las BMSC se reticularon en Matrigel y proliferaron bien. Después de 7 días, se observó que el gel de matriz gradualmente se volvió blando y colapsó, y se visualizó un cruce transversal de algunas células reticulares a los 14 días. La tinción mostró que la mayoría de las células mostraron una fluorescencia verde con la prolongación del tiempo de cultivo; en los primeros 4 y 7 días, la actividad de las células madre mesenquimales de la médula ósea en Matrigel aumentó gradualmente y los porcentajes fueron de 92,57 %, 95,54 % y 97,37 %, respectivamente. El sistema de cultivo tridimensional de Matrigel puede mantener la morfología, la función y la capacidad de proliferación de las células madre mesenquimales de la médula ósea.

Terapias alternativas para o diabetes mellitus tipo 1: caracterização funcional do gene Txnip na diferenciação ß-pancreática e desenvolvimento de biomaterial inovador para microencapsulamento celular / Alternative therapies for type 1 diabetes mellitus: functional characterization of Txnip gene during -pancreatic differentiation and generation of an innovative biomaterial for cell microencapsulation

O diabetes mellitus do tipo 1 (DM1) é uma doença causada pela destruição autoimune das células-ß produtoras de insulina do pâncreas. O transplante de ilhotas pancreáticas é um procedimento tecnicamente simples sendo uma alternativa terapêutica interessante para o DM1. Entretanto, a oferta limitada de pâncreas de doadores falecidos e a necessidade de imunossupressão crônica são fatores que limitam a aplicabilidade dessa modalidade de transplante. Neste trabalho foram estudadas duas estratégias que visam oferecer soluções aos fatores limitantes do transplante de ilhotas pancreáticas. Na primeira parte do trabalho, o mecanismo molecular que dirige o processo de diferenciação de células-tronco embrionárias murinas (murine embryonic stem cells, mESCs) em células produtoras de insulina (insulin producing cells, IPCs) foi analisado visando otimizar o processo de diferenciação. Nós selecionamos o gene Thioredoxin interacting protein (Txnip), diferencialmente expresso ao longo da diferenciação ß-pancreática, para realizar um estudo funcional através da modificação genética de mESCs. Os resultados obtidos permitiram verificar que a inibição de Txnip na diferenciação ß-pancreática pode induzir a diferenciação de IPCs com maior expressão de marcadores de células- e mais responsivas ao estímulo de glicose. Além disso, o modelo de zebrafish permitiu elucidar in vivo o papel de Txnip durante a organogênese pancreática, revelando que a inibição desse gene é capaz de aumentar a massa de células-ß através do estimulo de células presentes no ducto extra-pancreático. Dessa forma, a inibição de Txnip pode aprimorar os protocolos para obtenção de IPCs a partir de células-tronco pluripotentes. A exposição crônica a agentes imunossupressores diabetogênicos e a perda de componentes de matriz extracelular durante o isolamento de ilhotas pancreáticas são causas para a perda de funcionalidade do enxerto. Dessa forma, na segunda parte do trabalho, um biomaterial inovador foi desenvolvido, contendo um polímero de laminina (polilaminina, PLn) para o encapsulamento e a imunoproteção de ilhotas pancreáticas. As cápsulas produzidas com o biomaterial desenvolvido, Bioprotect-Pln, são térmica- e mecanicamente estáveis, além de serem biocompatíveis e capazes de imunoproteger ilhotas pancreáticas humanas in vitro. O encapsulamento com Bioprotect-Pln preserva a funcionalidade de ilhotas pancreáticas. Além disso, quando cápsulas vazias de Bioprotect-Pln foram implantadas em camundongos imunocompetentes, houve atenuação da resposta inflamatória ao implante, uma das principais causas para perda de funcionalidade de enxertos encapsulados. Os resultados obtidos indicam que a presença de polilaminina na malha capsular induz uma resposta anti-inflamatória que pode beneficiar a preservação do enxerto de ilhotas pancreáticas encapsuladas. Atualmente, o transplante de ilhotas pancreáticas é visto como a terapia celular mais promissora para atingir a independência de insulina em pacientes de DM1, porém, a aplicabilidade desse transplante ainda é limitada. Este trabalho contribuiu para a elucidação dos mecanismos moleculares que podem aprimorar o processo de diferenciação de célulastronco pluripotentes em IPCs, estabelecendo uma fonte alternativa de células para a terapiade reposição, e, também, estabeleceu um biomaterial inovador, capaz de diminuir a resposta inflamatória ao implante de microcápsulas e de imunoproteger células microencapsuladas. Desta forma, este trabalho contribui para o estabelecimento da terapia de reposição celular para pacientes de DM1

Type 1 diabetes mellitus (DM1) is a disease caused by the autoimmune destruction of insulin-producing pancreatic ß-cells. Pancreatic islet transplantation is a technically simple procedure and an interesting alternative therapy for DM1, however, the limited supply of cadaveric donated pancreas and the need of life-long immunosuppression are factors which limit its applicability. In the present work, two strategies were employed aiming at establishing viable solutions for the factors limiting pancreatic islet transplantation. In the first part of this study, the molecular mechanism which drives differentiation of murine embryonic stem cells (mESCs) into insulin producing cells (IPCs) was analyzed in order to optimize the differentiation process. The Thioredoxin interacting protein (Txnip) gene, which is differentially expressed along -pancreatic differentiation, was selected to undergo a functional analysis by genetically modifying mESCs. The results allowed us to verify that Txnip inhibition during the ß-pancreatic differentiation process can induce differentiation of IPCs displaying higher expression of ß-cell markers and being more responsive to glucose stimuli. In addition, the zebrafish model allowed us to elucidate in vivo the role of Txnip during pancreatic organogenesis, revealing that its inhibition is able to increase the mass of ß-cells through stimulation of extra-pancreatic ductal cells. Therefore, Txnip inhibition may turbinate IPCs differentiation from pluripotent stem cells. The chronic exposure to diabetogenic immunosuppressive agents and the loss of extracellular matrix components during isolation of pancreatic islets are probable causes for the loss of pancreatic islet graft functionality. Therefore, in the second part of this study, an innovative biomaterial was developed by incorporating a laminin polymer (polylaminin, PLn) for the encapsulation and immunoprotection of pancreatic islets. The capsules produced with the novel biomaterial, Bioprotect-Pln, are biocompatible, thermally and mechanically stable and are able to immunoprotect human pancreatic islets in vitro. Encapsulation with Bioprotect-Pln preserves the functionality of pancreatic islets. In addition, when empty Bioprotect-Pln capsules were implanted into immunocompetent mice, an attenuation of the inflammatory response to the implant occurred, this being one of the main causes of encapsulated graft loss. The results indicate that polylaminin addition to the capsular mesh induces an anti-inflammatory response which may favor preservation of the engrafted encapsulated pancreatic islets. Pancreatic islet transplantation is currently seen as the most promising cell therapy to achieve insulin independence in DM1 patients, however, the applicability of this transplant is still limited. This work contributed to the elucidation of the molecular mechanisms which can turbinate the differentiation of pluripotent stem cells into IPCs, establishing an alternative source of cells for the replacement therapy, and, also, established an innovative biomaterial which is able to decrease the inflammatory response to the graft, thereby immunoprotecting the microencapsulated cells. Therefore, this work contributes to the establishment of the cell replacement therapy for DM1 patients

Decellularization of placentas: establishing a protocol

Biological biomaterials for tissue engineering purposes can be produced through tissue and/or organ decellularization. The remaining extracellular matrix (ECM) must be acellular and preserve its proteins and physical features. Placentas are organs of great interest because they are discarded after birth and present large amounts of ECM. Protocols for decellularization are tissue-specific and have not been established for canine placentas yet. This study aimed at analyzing a favorable method for decellularization of maternal and fetal portions of canine placentas. Canine placentas were subjected to ten preliminary tests to analyze the efficacy of parameters such as the type of detergents, freezing temperatures and perfusion. Two protocols were chosen for further analyses using histology, scanning electron microscopy, immunofluorescence and DNA quantification. Sodium dodecyl sulfate (SDS) was the most effective detergent for cell removal. Freezing placentas before decellularization required longer periods of incubation in different detergents. Both perfusion and immersion methods were capable of removing cells. Placentas decellularized using Protocol I (1% SDS, 5 mM EDTA, 50 mM TRIS, and 0.5% antibiotic) preserved the ECM structure better, but Protocol I was less efficient to remove cells and DNA content from the ECM than Protocol II (1% SDS, 5 mM EDTA, 0.05% trypsin, and 0.5% antibiotic).

Extracellular matrix and fibroblast injection produces pterygium-like lesion in rabbits

BACKGROUND: Translational research to develop pharmaceutical and surgical treatments for pterygium requires a reliable and easy to produce animal model. Extracellular matrix and fibroblast are important components of pterygium. The aim of this study was to analyze the effect of the subconjunctival injection of fibroblast cells (NIH3T3 cell line) and exogenous extracellular matrix in rabbits in producing a pterygium-like lesion. METHODS: Six 3-month-old white New Zealand rabbits were injected with 20,000 NIH3T3 cells and 5 µL of Matrigel in the right conjunctiva, and with only 5 µL of Matrigel in the left conjunctiva. The eyes were photographed under a magnification of 16× using a 12-megapixel digital camera attached to the microscope on day 1,3 and 7. Conjunctival vascularization was measured by analyzing images to measure red pixel saturation. Area of corneal and conjunctival fibrovascular tissue formation on the site of injection was assessed by analyzing the images on day 3 and 7 using area measurement software. Histopathologic characteristics were determined in the rabbit tissues and compared with a human primary pterygium. RESULTS: The two treatments promoted growth of conjunctival fibrovascular tissue at day 7. The red pixel saturation and area of fibrovascular tissue developed was significantly higher in right eyes (p < 0.05). Tissues from both treatments showed neovascularization in lesser extent to that observed in human pterygium. Acanthosis, stromal inflammation, and edema were found in tissues of both treatments. No elastosis was found in either treatment. CONCLUSIONS: Matrigel alone or in combination with NIH3T3 cells injected into the rabbits' conjunctiva can promote tissue growth with characteristics of human pterygium, including neovascularization, acanthosis, stromal inflammation, and edema. The combination of Matrigel with NIH3T3 cells seems to have an additive effect on the size and redness of the pterygium-like tissue developed.

MatrigelBD, a biocompatible scaffold that improves gingival mesenchymal stem cells proliferation / MatrigelBD, un andamiaje biocompatible que estimula la proliferación de celulas troncales mesenquimales derivadas del tejido gingival

MatrigelBD is a hydrogel scaffold with three-dimensional intercrossed networks of hydrophilic polymers with high water content. Human gingival tissue might represent a better source of MSCs, allowing these cells to be easily obtained in a relatively non-invasive way. The objective of this study was to evaluate the biocompatibility of MatrigelBD with GMSCs in vitro. Gingival connective tissue samples were obtained from healthy donors. Fresh tissue was minced and cultured during two weeks, after which cells at passage fourth were analyzed for their immune phenotype by flow cytometry. Differentiation into osteogenic, chondrogenic, and adipogenic lineages was induced and evaluated by culture staining. The "construct" was made of MatrigelBD with GMSC. To assess the biocompatibility, an MTT cellular proliferation assay was performed. The differentiation potential of the cells toward the osteogenic, adipogenic, and chondrogenic lineages was analyzed after 21 days of growth in MatrigelBD with induction differentiation media. The MTT analysis showed that MatrigelBD stimulated cell proliferation; the GMSCs maintained the expression of MSC markers. Importantly, the growth of GMSCs within the MatrigelBD did not interfere with the cell differentiation potential. These findings indicate that MatrigelBD is biocompatible with GMSCs, and this matrix improves cell proliferation in vitro.

MatrigelBD es un andamiaje de hidrogel con redes tridimensionales entrecruzadas de polímeros hidrófilos con un alto contenido de agua. El tejido gingival humano podría representar una mejor fuente de MSCs, estas células pueden obtenerse fácilmente de una manera relativamente no invasiva. El objetivo de este estudio fue evaluar la biocompatibilidad de MatrigelBD con GMSCs in vitro. Muestras gingivales de tejido conectivo se obtuvieron de donantes sanos. El tejido se trituró y se cultivó durante dos semanas, y cuando las células se encontraban en el cuarto pasaje se les analizó su fenotipo inmunológico utilizando citometría de flujo. Se indujo la diferenciación hacia los linajes osteogénico, condrogénico y adipogénico, evaluandose con tinciones. El "constructo" se hizo de MatrigelBD con GMSC. Para evaluar la biocompatibilidad, se realizó un ensayo de proliferación celular: MTT. Se analizó el potencial de diferenciación de las células hacia los linajes osteogénico, adipogénico y condrogénico después de 21 días de cultivo en MatrigelBD con medio de diferenciación de inducción. El análisis de MTT mostró que MatrigelBD estimula la proliferación celular; GMSCs mantiene la expresión de marcadores de MSC. Es importante destacar que el crecimiento de GMSCs en MatrigelBD no interfirió con el potencial de diferenciación celular. Estos hallazgos indican que MatrigelBD es biocompatible con GMSCs, y esta matriz mejora la proliferación celular in vitro.

Growth hormone in the presence of laminin modulates interaction of human thymic epithelial cells and thymocytes in vitro

BACKGROUND: Several evidences indicate that hormones and neuropeptides function as immunomodulators. Among these, growth hormone (GH) is known to act on the thymic microenvironment, supporting its role in thymocyte differentiation. The aim of this study was to evaluate the effect of GH on human thymocytes and thymic epithelial cells (TEC) in the presence of laminin. RESULTS: GH increased thymocyte adhesion on BSA-coated and further on laminin-coated surfaces. The number of migrating cells in laminin-coated membrane was higher in GH-treated thymocyte group. In both results, VLA-6 expression on thymocytes was constant. Also, treatment with GH enhanced laminin production by TEC after 24 h in culture. However, VLA-6 integrin expression on TEC remained unchanged. Finally, TEC/thymocyte co-culture model demonstrated that GH elevated absolute number of double-negative (CD4-CD8-) and single-positive CD4+ and CD8+ thymocytes. A decrease in cell number was noted in double-positive (CD4+CD8+) thymocytes. CONCLUSIONS: The results of this study demonstrate that GH is capable of enhancing the migratory capacity of human thymocytes in the presence of laminin and promotes modulation of thymocyte subsets after co-culture with TEC.

Effects of penicillin and erythromycin on adherence of invasive and noninvasive isolates of Streptococcus pyogenes to laminin

This study investigated the possible relationship between the invasiveness of group A Streptococcus (GAS) strains and their abilities to adhere to laminin and assessed the effects of subinhibitory concentrations of penicillin and erythromycin on the ability of GAS to adhere to laminin. The adherence of noninvasive and highly invasive isolates of GAS to laminin was significantly higher than the adherence displayed by isolates of low invasiveness. Antibiotic treatment caused significant reductions in adherence to laminin in all three groups of strains. Penicillin was more successful in reducing the adherence abilities of the tested GAS strains than erythromycin.

Redução das desigualdades no uso de consultas médicas no Brasil: análise das regiões Nordeste e Sudeste entre 2003 e 2008 / Reduction of inequalities in medical visits in Brazil: analysis of the Northeastern and Southeastern regions between 2003 and 2008

OBJETIVO: Analisar as desigualdades socioeconômicas na utilização de consultas médicas (CM) no último ano no Brasil. MÉTODOS: Dados da Pesquisa Nacional por Amostra de Domicílios (≥ 20 anos de idade) das Regiões Nordeste (2003, n = 75.652 e 2008, n = 79.779) e Sudeste (2003, n = 76.029 e 2008, n = 79.356) foram analisados segundo CM. Compararam-se as prevalências de CM segundo as variáveis exploratórias demográficas e de saúde no primeiro (D1) e último (D10) decil de renda familiar per capita. As análises consideraram o desenho amostral complexo. RESULTADOS: A proporção de pessoas com CM aumentou no período na Região Nordeste (61,2 para 66,9%) e Sudeste (67,9 para 73,5%). A diferença absoluta de CM, segundo D1 e D10 no período, foi de 6,4 pontos percentuais (pp) no Nordeste e 4,2 pp no Sudeste. Houve importante redução das desigualdades entre os homens; naqueles sem doenças crônicas; naqueles que tinham uma percepção positiva da sua saúde e naqueles sem plano de saúde com direito a CM. A Região Sudeste ainda apresentou redução entre aqueles com apenas uma morbidade autorreferida (8 pp) e com percepção negativa da saúde (6 pp). CONCLUSÃO: Houve aumento de CM no Brasil. Observa-se ainda persistente desigualdade entre os mais pobres e os mais ricos, maior no Nordeste do que no Sudeste. Políticas para a redução da desigualdade em saúde mais eficazes e equânimes devem ser adotadas no Brasil. .

OBJECTIVE: To analyze the socioeconomic inequalities in medical visits (MV) in the past year in Brazil. METHODS: Data from adults aged ≥ 20 years old who participated in the Brazilian National Household Surveys and living in the Northeastern (2003; n = 75,652 and 2008, n = 79,779) and Southeastern (2003; n = 76,029 and 2008; n = 79,356) regions were analyzed according to MV. We compared MVs according to demographic and health variables in the first (D1) and last (D10) per capita family income deciles. All analyses considered the complex cluster design. RESULTS: The proportion of people who had MV during this period increased in the Northeastern (from 61.2 to 66.9%) and the Southeastern (from 67.9 to 73.5%) regions. The absolute difference (AD) in the use of MV, according to D1 and D10 in this period, was equal to 6.4 percentage points (pp) in the Northeastern and 4.2 pp in the Southeastern regions. Significant reduction in inequalities was observed among men without chronic diseases, in those who had a positive perception of their health, and among those without health insurance which included MV. The Southeastern region has also showed significant reduction among those with chronic disease (8 pp) and with negative health self-perception (6 pp). CONCLUSION: The increasing number of MVs was found in Brazil. However, persistent inequalities were observed between the poorest and the richest, higher in the Northeastern than in the Southeastern region. More effective and equitable policies to reduce health inequalities should be adopted in Brazil. .

Interações proteína príon e seus ligantes STI1 e laminina: possíveis implicações na doença de Alzheimer / Interactions of the prion protein and its ligands STI1 and laminin: possible implications for Alzheimer’s disease

A Doença de Alzheimer (DA) é uma demência progressiva que tem como principais características a disfunção sináptica e a neurodegeneração em áreas específicas do cérebro, que levam a um quadro grave de perda de memória e outras habilidades cognitivas. Uma das principais características neuropatológicas é a deposiçãode placas amiloides extracelulares, que contêm principalmente o peptídeo beta-amiloide (Aβ), que é formado por um processamento alterado da proteína precursora amiloide (APP). Os oligômeros formados por Aβ (AβO) são considerados os elementos tóxicos mais importantes deste processo, esses se ligam às sinapses e estão estreitamente relacionados com a patogênese da DA. Recentemente, foi descrito que a proteína príon celular (PrPC) é um receptor para AβO, porém, os mecanismos envolvidos nesta interação e de que forma esta pode estar relacionada à DA ainda não foram elucidados. PrPC é uma glicoproteína ancorada à membrana plasmática que interage com diversos ligantes, como a proteína de matriz extracelular laminina e a co-chaperona STI1 (Stress Inducible Protein 1). Estas interações induzem neuroproteção, neuritogênese e modulam a formação de memória. Deste modo, torna-se interessante verificar um possível efeito neuroprotetor dos ligantes de PrPC contra a toxicidade produzida pelos AβO. Utilizando culturas neuronais, observamos que os tratamentos com AβO levam uma diminuição nos níveis da proteína sináptica sinaptofisina (Syp), devido a um aumento da degradação..

Alzheimer's disease (AD) is a progressive dementia mainly characterized by synaptic dysfunction and neurodegeneration in specific areas of the brain, leading to severe memory loss, and others cognitive inabilities. One of the main neuropathological characteristics is the formation of extracellular amyloid plaques, which mainly contain beta-amyloid peptide (Aβ). This peptide is formed by an alteration in the processing of the amyloid precursor protein (APP). The Aβ oligomers (AβO) are considered the major toxic components within this process where they bind to synapses and are closely related to the pathogenesis of the AD. Recently, it was reported that PrPC is a receptor for AβO, however, the mechanisms involved in this interaction and how this may be related to AD have not yet been elucidated. The cellular prion protein (PrPC) is a glycoprotein anchored to the plasma membrane that interacts with several ligands such as the co-chaperone STI1 (Stress inducible protein 1) and the extracellular matrix protein laminin. These interactions induce neuroprotection, neuritogenesis, and modulate memory formation. Therefore, it becomes interesting to verify a possible neuroprotective effect of PrPC ligands against the toxicity induced by AβO. We observed that the treatment of neuronal cultures with AβO lead to a decrease in the levels of synaptic protein synaptophysin (Syp) due to an increase of Syp degradation by the proteasome...

O corpo da nação: posicionamentos governamentais sobre a educação física no Brasil monárquico / The body of the nation: government positions on physical education during tHe Brazilian monarchy

Associadas a projetos de construção da ideia de nação, no Brasil monárquico foram encaminhadas, pelo governo imperial, algumas iniciativas no sentido de materializar propostas de educação física. O objetivo deste artigo é investigar os sentidos e significados atribuídos ao tema na legislação e nos relatórios anuais do Ministério dos Negócios do Império (1831-1889), com especial interesse pelo que se refere ao Rio de Janeiro. A abordagem do assunto nas fontes pesquisadas evidencia que as visões sobre a educação física se deram a partir de uma matriz que articulava concepções de moral, saúde e civilização, tendo que lidar com as condições concretas de um país recém-independente, periférico e com uma burocracia ainda em formação.

In association with its nation building projects, the imperial government in Brazil under monarchic rule took some concrete actions based on proposals for physical education. The aim of this article is to investigate the meanings and significations attributed to this subject in the legislation and the annual reports issued by the Ministry of Business of the Empire (1831-1889), giving special attention to Rio de Janeiro. The approach to the subject in the sources researched demonstrates that the views of physical education took shape through a web of ideas that associated moral, health and civilization conceptions, in a bid to deal with the concrete circumstances of a newly independent peripheral nation with a bureaucratic structure in the process of formation.

Cost of the Cervical Cancer Screening Program at the Mexican Social Security Institute / Costo del Programa de Detección Oportuna de Cáncer Cervical en el Instituto Mexicano del Seguro Social

Objective. To estimate the annual cost of the National Cervical Cancer Screening Program (CCSP) of the Mexican Institute of Social Security (IMSS). Materials and methods. This cost analysis examined regional coverage rates reported by IMSS. We estimated the number of cytology, colposcopy, biopsy and pathology evaluations, as well as the diagnostic test and treatment costs for cervical intraepithelial neoplasia grade II and III (CIN 2/3) and cervical cancer. Diagnostic test costs were estimated using a micro-costing technique. Sensitivity analyses were performed. Results. The cost to perform 2.7 million cytology tests was nearly 38 million dollars, which represents 26.1% of the total program cost (145.4 million). False negatives account for nearly 43% of the program costs. Conclusion. The low sensitivity of the cytology test generates high rates of false negatives, which results in high institutional costs from the treatment of undetected cervical cancer cases.

Objetivo. Estimar el costo anual del Programa Nacional de Detección Oportuna de Cáncer Cervical en el Instituto Mexicano del Seguro Social (IMSS). Material y métodos. Este análisis de costos examinó las distintas coberturas por región reportadas por el IMSS. Se estimó el número de citologías, colposcopías, biopsias y evaluaciones de patología y los costos de pruebas de diagnóstico y de tratamientos por neoplasia cervical intraepitelial de grado II y III (NIC 2/3) y cáncer cervical. Los costos de las pruebas de diagnóstico se estimaron utilizando una técnica de microcosteo. Se llevó a cabo un análisis de sensibilidad. Resultados. El costo de realizar 2.7 millones de citologías fue de 38 millones de dólares, lo que representa 26.1% del costo total del programa (145.4 millones). Los falsos negativos corresponden a casi 43% de los costos del programa. Conclusiones. La baja sensibilidad de la citología genera un alto número de falsos negativos que resultan en costos elevados para la institución por el tratamiento de estos casos no detectados.

Immobilization and therapeutic passive stretching generate thickening and increase the expression of laminin and dystrophin in skeletal muscle

Extracellular matrix and costamere proteins transmit the concentric, isometric, and eccentric forces produced by active muscle contraction. The expression of these proteins after application of passive tension stimuli to muscle remains unknown. This study investigated the expression of laminin and dystrophin in the soleus muscle of rats immobilized with the right ankle in plantar flexion for 10 days and subsequent remobilization, either by isolated free movement in a cage or associated with passive stretching for up to 10 days. The intensity of the macrophage response was also evaluated. One hundred and twenty-eight female Wistar rats were divided into 8 groups: free for 10 days; immobilized for 10 days; immobilized/free for 1, 3, or 10 days; or immobilized/stretched/free for 1, 3, or 10 days. After the experimental procedures, muscle tissue was processed for immunofluorescence (dystrophin/laminin/CD68) and Western blot analysis (dystrophin/laminin). Immobilization increased the expression of dystrophin and laminin but did not alter the number of macrophages in the muscle. In the stretched muscle groups, there was an increase in dystrophin and the number of macrophages after 3 days compared with the other groups; dystrophin showed a discontinuous labeling pattern, and laminin was found in the intracellular space. The amount of laminin was increased in the muscles treated by immobilization followed by free movement for 10 days. In the initial stages of postimmobilization (1 and 3 days), an exacerbated macrophage response and an increase of dystrophin suggested that the therapeutic stretching technique induced additional stress in the muscle fibers and costameres.

Papel da laminina na migração de linfócitos T em modelo murino de transplante cardíaco alogênico / Role of laminina in the migration of T lymphocyte in a murine model of allogeneic cardiac transplantation

Durante o processo de rejeição, linfócitos T do receptor são ativados e migram para o enxerto. A glicoproteína laminina (LM) é importante na migração e posicionamento de linfócitos durante a rejeição, porém dados sobre o papel das diferentes isoformas neste processo são escassos. Usando um modelo de transplante cardíaco alogênico, nosso grupo verificou que o tratamento com o anticorpo monoclonal anti-LM diminuiu o infiltrado inflamatório e a deposição de LM nos enxertos cardíacos. No presente trabalho, caracterizamos os linfócitos T presentes nos linfonodos de drenagem e aloenxertos, a expressão das isoformas de LM nos aloenxertos e seu papel na migração dos linfócitos T. Nos aloenxertos, observamos um aumento de linfócitos T CD4+ e CD8+, e um maior enriquecimento de linfócitos CD8+ ativados, quando comparado aos enxertos isogênicos. Por qPCR, verificamos que somente a cadeia LAMB3, constituinte da LM332, teve sua expressão aumentada nos aloenxertos. Por imunofluorescência, verificamos uma maior deposição de LM332 e da cadeia LAMB3 nos aloenxertosPara verificar o papel da LM332 na migração dos linfócitos T, realizamos ensaios de migração e observamos uma migração reduzida dos linfócitos T frente a LM332, quando comparado aos grupos controle. Esse resultado sugere que a LM332 pode promover uma forte adesão, influenciando a migração dos linfócitos T alorreativos. A análise das isoformas de LM durante a rejeição será importante para o entendimento da migração das células efetoras durante esse processo e poderá ajudar a definir estratégias terapêuticas...

During rejection process, recipient T cells are activated in lymph nodes and migrate to the graft. The glycoprotein laminina (LM) is important in lymphocyte positioningand effector function during alloreactive responses. Using a model of allogeneic heart transplantation, where hearts from neonatal C57BL/6 mice were transplanted in the ear of adult BALB/c recipients, treatment with anti-LM mAb decreased the cellular infiltrate and LM deposition within the grafts. In this work, we characterized T cells inallografts, evaluate LM isoform expression and their role on T lymphocyte migration in the model described above. In allografts, we observed an increase of CD4+and CD8+ lymphocytes, and an enrichment of activated CD8+T cells, when compared tothe isografts. By qPCR, only LAMB3 chain, component of the LM332 isoform, was increased in allografts. Additionally, we observed higher deposition of LM332 in allografts by immunofluorescence. To check the role of LM332 on T cell migration, we performed ex vivo experiments and observed a reduced LM332-driven migratory response of lymphocytes from lymph nodes. Our data suggest that the predominanceof T cells in allografts can be related to an enhanced contact of T cells with LM332, as no haptotactic effect of the intact LM332 was observed. Analysis of the role of LM isoforms during the rejection process will be important to understand the trafficking process of effector cells during graft rejection and might help to define new therapeutical strategies...

Primary tumorectomy promotes angiogenesis and pulmonary metastasis in osteosarcoma-bearing nude mice

PURPOSE: To investigate the effect of primary tumorectomy on angiogenesis and pulmonary metastasis in osteosarcoma-bearing nude mice. METHODS: Osteosarcoma was introduced to nude mice via subcutaneous injection of MG-63 cells. One hundred and eighty osteosarcoma-bearing mice were used equally in 3 parallel experiments. The effect of tumorectomy (TR) on the expression of vascular endothelial growth factor (VEGF) and endostatin was investigated by ELISA. Meanwhile, the effect on angiogenesis was evaluated by Matrigel plug assay, and pulmonary metastasis assessed by calculating the metastatic foci. Sham-operation (SO) and untreated (UT) groups served as controls. RESULTS: The VEGF (TR: 79.55 ± 7.82 pg/mL vs. SO: 110.01 ± 5.69 pg/mL, UT: 123.50 ± 10.41 pg/mL; p < 0.01) and endostatin (TR: 47.09 ± 6.22 ng/mL vs. SO: 117.64 ± 7.39 ng/mL, UT: 126.73 ± 6.55 ng/mL; p<0.01) were down-regulated significantly after tumorectomy, and angiogenesis was significantly promoted simultaneously. The incidence of pulmonary metastatic foci was 80.0% in the TR group, 40.0% in the SO group and 35.0% in the UT group. CONCLUSION: Primary tumorectomy can down-regulate the expression of VEGF and endostatin and promote angiogenesis which leads to the acceleration of pulmonary metastasis. These findings imply that anti-angiogenic treatment can be considered after primary tumorectomy.