RESUMEN

Fibronectin (FN)-loaded nanofiber scaffolds were developed and assessed concerning their bioactive potential on human apical papilla cells (hAPCs). First, random (NR) and aligned (NA) nanofiber scaffolds of polycaprolactone (PCL) were obtained by electrospinning technique and their biological properties were evaluated. The best formulations of NR and NA were loaded with 0, 5, or 10 µg/ml of FN and their bioactivity was assessed. Finally, FN-loaded NR and NA tubular scaffolds were prepared and their chemotactic potential was analyzed using an in vitro model to mimic the pulp regeneration of teeth with incomplete root formation. All scaffolds tested were cytocompatible. However, NR and NA based on 10% PCL promoted the highest hAPCs proliferation, adhesion and spreading. Polygonal and elongated cells were observed on NR and NA, respectively. The higher the concentration of FN added to the scaffolds, greater cell migration, viability, proliferation, adhesion and spreading, as well as collagen synthesis and gene expression (ITGA5, ITGAV, COL1A1, COL3A1). In addition, tubular scaffolds with NA loaded with FN (10 µg/ml) showed the highest chemotactic potential on hAPCs. It was concluded that FN-loaded NA scaffolds may be an interesting biomaterial to promote hAPCs-mediated pulp regeneration of endodontically compromised teeth with incomplete root formation.

Asunto(s)

Materiales Biocompatibles/química , Fibronectinas/química , Nanofibras/química , Poliésteres/química , Andamios del Tejido/química , Adolescente , Adhesión Celular , Proliferación Celular , Colágeno/química , Cadena alfa 1 del Colágeno Tipo I/genética , Cadena alfa 1 del Colágeno Tipo I/metabolismo , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Pulpa Dental , Femenino , Regeneración Tisular Dirigida , Humanos , Integrinas/genética , Integrinas/metabolismo , Masculino , Regeneración , Ingeniería de Tejidos

RESUMEN

Aim: The aim of this study was to analyze the prognostic value of integrin subunit ß1 and laminin γ1 chain in patients with cervical cancer (CC). Materials & methods: The study included 96 samples. Cytological diagnosis, human papillomavirus (HPV) genotyping, HPV integration status and integrin subunit ß1 and laminin γ1 chain expressions were performed or determined using Papanicolaou smear, INNO-LiPA® Genotyping Extra Kit, in situ hybridization, and immunocytochemistry, respectively. The association between variables was calculated using chi-squared and Fisher's exact test; logistic regression analysis was performed to calculate odds ratios and CI at 95%. Results: Our results show that integrin subunit ß1 and laminin γ1 chain expressions increase according to tumor progression. Integrin subunit ß1 and laminin γ1 chain expressions are associated with cytological diagnosis (p < 0.001 and p = 0.001, respectively) and laminin γ1 chain expression with the integration status of HPV (p < 0.001). Moderate/high expressions of integrin subunit ß1 and laminin γ1 chain were correlated with overall survival and increased risk of CC (6.86 and 3.75, respectively), the odds ratio was 12.91 when the moderate/high expression of integrin subunit ß1 and laminin γ1 chain were combined. Conclusion: Our results suggest that integrin subunit ß1 and laminin γ1 chain expressions could be a prognostic biomarker in CC.

Asunto(s)

Integrina beta1/genética , Laminina/genética , Neoplasias del Cuello Uterino/metabolismo , Adulto , Alphapapillomavirus/patogenicidad , Biomarcadores de Tumor/genética , Femenino , Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Inmunohistoquímica , Hibridación in Situ , Integrina beta1/metabolismo , Integrinas/genética , Laminina/metabolismo , México , Persona de Mediana Edad , Infecciones por Papillomavirus/genética , Pronóstico , Neoplasias del Cuello Uterino/genética

RESUMEN

Mesenchymal stem cells (MSC) favour a scenario where leukemic cells survive. The protein kinase C (PKC) is essential to confer MSC support to leukemic cells and may be responsible for the intrinsic leukemic cell growth. Here we have evaluated the capacity of a chimeric peptide (HKPS), directed against classical PKC isoforms, to inhibit leukemic cell growth. HKPS was able to strongly inhibit viability of different leukemic cell lines, while control HK and PS peptides had no effect. Further testing showed that 30% of primary samples from paediatric B-cell acute lymphoblastic leukaemia (B-ALL) were also strongly affected by HKPS. We showed that HKPS disrupted the supportive effect of MSC that promote leukemic cell survival. Interestingly, ICAM-1 and VLA-5 expression increased in MSC during the co-cultures with B-ALL cells, and we found that HKPS inhibited the interaction between MSC and B-ALL cells due to a reduction in the expression of these adhesion molecules. Of note, the susceptibility of B-ALL cells to dexamethasone increased when MSC were treated with HKPS. These results show the relevance of these molecular interactions in the leukemic niche. The use of HKPS may be a new strategy to disrupt intercellular communications, increasing susceptibility to therapy, and at the same time, directly affecting the growth of PKC-dependent leukemic cells.

Asunto(s)

Antineoplásicos/farmacología , Linfocitos B/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Oligopéptidos/farmacología , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Linfocitos B/metabolismo , Adhesión Celular , Proliferación Celular , Células Cultivadas , Niño , Humanos , Integrinas/genética , Integrinas/metabolismo , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Células Jurkat , Células K562 , Células Madre Mesenquimatosas/metabolismo , Proteínas Recombinantes/farmacología

RESUMEN

Fungi that belong to the genus Paracoccidioides are the etiologic agents of paracoccidioidomycosis, a human systemic mycosis, which occurs in Latin America. Epithelial cell is one of the first cells that interact with these fungi and responds by secreting inflammatory mediators such as cytokines. In the present study, we demonstrate that yeasts of different isolates of Paracoccidioides brasiliensis (Pb18 and Pb03) and Paracoccidioides lutzii (Pb01) distinctly promoted interleukin (IL)-8 secretion by the lung epithelial cell line A549. Depending on the isolate, this cytokine release may rely on the epithelial cell interaction with fungal secreted components or direct contact with the pathogen. In addition, adhesion of yeasts to the pulmonary epithelial cells was also different among Paracoccidioides isolates, and the highest percentage of A549 cells with adhered fungi was observed with P. lutzii. All Paracoccidioides isolates induced an expression increase of α3 and α5 integrins in A549 cells and, using small interfering RNA, we observed that the integrin silencing promoted a reduction of P. lutzii adhesion, which suggests the involvement of integrins in this event. Together, these results indicate that host epithelial cell response may depend on the isolate of Paracoccidioides.

Asunto(s)

Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/microbiología , Interleucina-8/biosíntesis , Paracoccidioides/fisiología , Paracoccidioidomicosis/metabolismo , Paracoccidioidomicosis/microbiología , Células A549 , Adhesión Celular , Supervivencia Celular , Células Cultivadas , Citocinas/metabolismo , Silenciador del Gen , Humanos , Integrinas/genética

RESUMEN

Class 1 myosins (Myo1s) were the first unconventional myosins identified and humans have eight known Myo1 isoforms. The Myo1 family is involved in the regulation of gene expression, cytoskeletal rearrangements, delivery of proteins to the cell surface, cell migration and spreading. Thus, the important role of Myo1s in different biological processes is evident. In this study, we have investigated the effects of pentachloropseudilin (PClP), a reversible and allosteric potent inhibitor of Myo1s, on angiogenesis. We demonstrated that treatment of cells with PClP promoted a decrease in the number of vessels. The observed inhibition of angiogenesis is likely to be related to the inhibition of cell proliferation, migration and adhesion, as well as to alteration of the actin cytoskeleton pattern, as shown on a PClP-treated HUVEC cell line. Moreover, we also demonstrated that PClP treatment partially prevented the delivery of integrins to the plasma membrane. Finally, we showed that PClP caused DNA strand breaks, which are probably repaired during the cell cycle arrest in the G1 phase. Taken together, our results suggest that Myo1s participate directly in the angiogenesis process.

Asunto(s)

Citoesqueleto de Actina/efectos de los fármacos , Inhibidores de la Angiogénesis/farmacología , Ciclo Celular/efectos de los fármacos , Hidrocarburos Clorados/farmacología , Integrinas/metabolismo , Pirroles/farmacología , Inhibidores de la Angiogénesis/toxicidad , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Hidrocarburos Clorados/toxicidad , Integrinas/genética , Miosina Tipo I/metabolismo , Pirroles/toxicidad , ARN Mensajero/metabolismo

RESUMEN

SUMMARY: An alternative superovulator to replace clomiphene citrate is needed as clomiphene citrate is associated with low pregnancy rates. Anastrozole is an effective superovulator, but it has not been well researched. In order to determine the effectiveness of anastrozole as a superovulator and to compare it with clomiphene citrate in similar situations, this study ascertained the effects of these drugs on the expression of the focal adhesion proteins, vinculin and integrin β5, which are uterine receptivity markers, in the uterine epithelial cells of day 1 and day 6 pregnant Wistar rats. The results show that vinculin and integrin β5 are co-localized at the base of the uterine epithelium at day 1 of pregnancy whereas at day 6, they disassemble from the basal focal adhesions and co-localize and significantly increase their expression apically (p≤0.0001). Moreover, there is a significant difference in the protein expression levels of vinculin and integrin b5 in uterine luminal epithelial cells between untreated (control) and chlomiphene citrate treated rats (p≤0.0001), anastrozole and chlomiphene citrate treated rats at day 6 (p≤0.0001) suggesting the interpretation that anastrozole seems to enhance their expression in order to perhaps assist in the implantation process of the blastocyst. The immunofluorescence experiments agree with the vinculin and integrin β5 gene expression findings in which at day 6 of pregnancy, vinculin and integrin β5 gene expression are significantly upregulated in uterine luminal epithelial cells in the anastrozole treated group relative to the calibrator sample (p≤0.0001). These findings suggest that anastrozole is implantation friendly.

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RESUMEN: Es necesario un superovulador alternativo para reemplazar el citrato de clomifeno, debido a que está asociado con bajas tasas de preñez. El anastrozol es un superovulador eficaz, sin embargo es poca su investigación. Con el fin de determinar la efectividad del anastrozol como superovulador y compararlo con citrato de clomifeno en situaciones similares, se determinaron los efectos de estos fármacos sobre la expresión de las proteínas de adhesión focal, vinculina e integrina β5, en marcadores de receptividad uterina en días 1 y 6, en las células epiteliales uterinas de ratas Wistar preñadas. Los resultados muestran que la vinculina y la integrina β5 se co-localizan en la base del epitelio uterino al día 1 de la gravidez mientras que al día 6 se desmontan de las adherencias focales basales, co-localizan y aumentan significativamente su expresión apicalmente (p≤0.0001). Además, existe una diferencia significativa en los niveles de expresión de proteína de vinculina e integrina β5 en células epiteliales luminales uterinas entre ratas no tratadas (control) y tratadas con citrato declomifeno (p≤0.0001), ratas tratadas con anastrozol y citrato declomifeno al día 6 (p≤0,0001) sugiriendo la interpretación de que el anastrozol parece mejorar su expresión con el fin de ayudar en el proceso de implantación del blastocisto. Los experimentos de inmunofluorescencia coinciden con los resultados de la expresión de los genes vinculina e integrina β5 en los cuales al día 6 de la preñez, la vinculina y la integrina β5 están significativamente reguladas en células epiteliales luminales uterinas en el grupo tratado con anastrozol con respecto a la muestra del calibrador (p<0,0001). Estos hallazgos sugieren que el anastrozol es favorable para la implantación.

Asunto(s)

Animales , Femenino , Embarazo , Ratas , Integrinas/efectos de los fármacos , Nitrilos/farmacología , Triazoles/farmacología , Útero/efectos de los fármacos , Vinculina/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Adhesiones Focales/efectos de los fármacos , Integrinas/genética , Integrinas/fisiología , Microscopía Confocal , Microscopía Fluorescente , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Vinculina/genética , Vinculina/fisiología

RESUMEN

Anoikis is a programmed cell death induced upon cell detachment from extracellular matrix. Anoikis resistance is a critical mechanism in tumor metastasis. Cancer cells deregulate and adapt their metabolism to survive in the absence of adhesion, spreading metastases to distant organs. These adaptations include abnormal regulation of growth factor receptors activating prosurvival signaling pathways, such as the Ras/ERK and PI3K/Akt pathways, and extracellular matrix remodeling, leading to metastasis by an increase of invasiveness and inhibiting anoikis. This study investigates the possible involvement of ECM components and signaling pathways in the regulation of resistance to anoikis in endothelial cells (EC). Endothelial cells submitted to stressful conditions by blocking adhesion to substrate (anoikis resistance) display an up-regulation of Ras/ERK and PI3k/Akt pathways by high expression of Ras, ERK, PI3K (p110α) and Akt (Thr 308). After ERK and PI3K inhibiting, all EC-derived cell lines studied showed lower growth, a decrease in invasive potential and a higher rate of apoptosis. Furthermore, anoikis-resistant cell lines display a decrease in the expression of fibronectin, collagen IV and hyaluronic acid and an increase in the expression of laminin, perlecan, αv, ß3, α5 and ß1 integrins subunits, hyaluronidades 1, 2 and 3 and metalloproteinases 2 and 9. These results indicate that the acquisition of anoikis resistance induced remodeling of the extracellular matrix and overexpression of the PI3K/Akt and Ras/ERK pathway components. Acquisition of resistance to anoikis is a potentially crucial step in endothelial cell transformation.

Asunto(s)

Anoicis/genética , Fosfatidilinositol 3-Quinasa Clase Ib/genética , Células Endoteliales/citología , Proteínas de la Matriz Extracelular/genética , Genes ras/genética , Sistema de Señalización de MAP Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Apoptosis , Adhesión Celular , Línea Celular , Activación Enzimática/genética , Integrinas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Conejos , Transducción de Señal/genética , Regulación hacia Arriba/genética

RESUMEN

Vascular remodeling, i.e. whole-vessel structural reshaping, determines lumen caliber in (patho)physiology. Here we review mechanisms underlying vessel remodeling, with emphasis in redox regulation. First, we discuss confusing terminology and focus on strictu sensu remodeling. Second, we propose a mechanobiological remodeling paradigm based on the concept of tensional homeostasis as a setpoint regulator. We first focus on shear-mediated models as prototypes of remodeling closely dominated by highly redox-sensitive endothelial function. More detailed discussions focus on mechanosensors, integrins, extracellular matrix, cytoskeleton and inflammatory pathways as potential of mechanisms potentially coupling tensional homeostasis to redox regulation. Further discussion of remodeling associated with atherosclerosis and injury repair highlights important aspects of redox vascular responses. While neointima formation has not shown consistent responsiveness to antioxidants, vessel remodeling has been more clearly responsive, indicating that despite the multilevel redox signaling pathways, there is a coordinated response of the whole vessel. Among mechanisms that may orchestrate redox pathways, we discuss roles of superoxide dismutase activity and extracellular protein disulfide isomerase. We then discuss redox modulation of aneurysms, a special case of expansive remodeling. We propose that the redox modulation of vascular remodeling may reflect (1) remodeling pathophysiology is dominated by a particularly redox-sensitive cell type, e.g., endothelial cells (2) redox pathways are temporospatially coordinated at an organ level across distinct cellular and acellular structures or (3) the tensional homeostasis setpoint is closely connected to redox signaling. The mechanobiological/redox model discussed here can be a basis for improved understanding of remodeling and helps clarifying mechanisms underlying prevalent hard-to-treat diseases.

Asunto(s)

Aneurisma de la Aorta/metabolismo , Vasos Sanguíneos/metabolismo , Células Endoteliales/metabolismo , Mecanotransducción Celular , Neointima/metabolismo , Remodelación Vascular , Animales , Aneurisma de la Aorta/genética , Aneurisma de la Aorta/patología , Vasos Sanguíneos/patología , Citoesqueleto/metabolismo , Citoesqueleto/patología , Células Endoteliales/patología , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Regulación de la Expresión Génica , Homeostasis/genética , Humanos , Integrinas/genética , Integrinas/metabolismo , Neointima/genética , Neointima/patología , Oxidación-Reducción , Proteína Disulfuro Isomerasas/genética , Proteína Disulfuro Isomerasas/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Terminología como Asunto

RESUMEN

Neutrophils (PMN) are the most abundant leukocytes in the blood. PMN migrate from the circulation to sites of infection, where they are responsible for antimicrobial functions. PMN use phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. NETs are fibers composed of chromatin and neutrophil-granule proteins. Several pathogens, including bacteria, fungi, and parasites, and also some pharmacological stimuli such as phorbol 12-myristate 13-acetate (PMA) are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. However the particular Fcγ receptor involved in triggering this function is a matter of controversy. In order to provide some insight into what Fcγ receptor is responsible for NET formation, each of the two human Fcγ receptors was stimulated individually by specific monoclonal antibodies and NET formation was evaluated. FcγRIIa cross-linking did not promote NET formation. Cross-linking other receptors such as integrins also did not promote NET formation. In contrast FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. NET formation was dependent on NADPH-oxidase, PKC, and ERK activation. These data show that cross-linking FcγRIIIb is responsible for NET formation by the human neutrophil.

Asunto(s)

Trampas Extracelulares/inmunología , Neutrófilos/inmunología , Receptores de IgG/inmunología , Cromatina/inmunología , Trampas Extracelulares/química , Proteínas Ligadas a GPI/inmunología , Humanos , Integrinas/genética , Integrinas/inmunología , Sistema de Señalización de MAP Quinasas/inmunología , Fagocitosis , Especies Reactivas de Oxígeno , Transducción de Señal , Acetato de Tetradecanoilforbol/farmacología

RESUMEN

CTX-M-131 is a natural Asp240Gly variant from the CTX-M-2 group detected in a Providencia rettgeri clinical strain from Brazil. Molecular analysis showed that blaCTX-M-131 was inserted in a complex class 1 integron harbored by a 112-kb plasmid, which has not been previously described as a platform for CTX-M-encoding genes with the Asp240Gly mutation. Steady-state kinetic parameters showed that the enzyme has a typical cefotaximase catalytic profile and an enhanced activity against ceftazidime.

Asunto(s)

Providencia/genética , beta-Lactamasas/genética , Antibacterianos/farmacología , Brasil , Ceftazidima/farmacología , Integrinas/genética , Cinética , Plásmidos/genética , beta-Lactamasas/metabolismo

RESUMEN

Platelets are cytoplasmatic fragments from bone marrow megakaryocytes present in blood. In this work, we review the basis of platelet mechanisms, their participation in syndromes and in arterial thrombosis, and their potential as a target for designing new antithrombotic agents. The option of new biotechnological sources is also explored.

Asunto(s)

Plaquetas/metabolismo , Trastornos Hemostáticos/patología , Aspirina/farmacología , Aspirina/uso terapéutico , Plaquetas/efectos de los fármacos , Trastornos Hemostáticos/metabolismo , Humanos , Integrinas/genética , Integrinas/metabolismo , Inhibidores de Agregación Plaquetaria/farmacología , Inhibidores de Agregación Plaquetaria/uso terapéutico , Deficiencia de Almacenamiento del Pool Plaquetario/metabolismo , Deficiencia de Almacenamiento del Pool Plaquetario/patología , Trombosis/tratamiento farmacológico , Trombosis/patología

RESUMEN

Objective To analyze innovative contents on Early Child Development Promotion. Method This action-research involves nine faculties from four Higher Education Institutions at inner-state of São Paulo, Brazil.Data were collected by syllabi analyses (2009-2011), interviews and focus group. We have adopted an ECDP underpinning from international consensus, thus evaluating KT Results We have found relevant incorporation between teaching and extension in Nursing (87,5%) and Psychology (75%) undergraduate courses, while Pedagogy was restricted to teaching. Conclusion This KT evaluation has evinced innovative potential of extension, regardless teaching and research, for a better Early Childhood.
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Objetivo Analizar la incorporación de contenidos innovadores en la promoción del desarrollo infantil. Método Investigación-acciónque involucró nueve representantes de cuatro Instituciones Enseñanza Superior. Los datos fueran obtenidos del análisis de planes de cursos (2009-2011),entrevistas y grupo de foco. Se basó en consenso internacional sobre la Primera Infancia para evaluar de la traslación del conocimiento. Resultados La incorporación de ocho temáticas sobre Primera Infancia ocurrió de modo relevante en la enseñanza y extensión del curso de graduación en Enfermería (87,5%) y de Psicología (75%). Se observó la limitación a la enseñanza en el curso de Pedagogía. Conclusión la evaluación traslacionalha evidenciado el potencial innovador de la extensión universitaria, integrada a la enseñanza e investigación en laInfancia Temprana. .

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Objetivo Analisar a incorporação de conteúdos inovadores na promoção do desenvolvimento infantil com ênfase na extensão universitária. Método Pesquisa-ação, descritiva e exploratória, envolvendo nove representantes de quatro instituições de ensino superior paulistas. Os dados foram obtidos por meio de análise de ementas (2009-2011), entrevistas e grupo focal. Embasou-se em consenso internacional relacionado à primeira infância para a avaliação da translação do conhecimento. Resultados A incorporação de oito temáticas da promoção do desenvolvimento infantil ocorreu de modo relevante entre ensino e extensão dos cursos de graduação de Enfermagem (87,5%) e de Psicologia (75%). Observou-se restrição ao ensino no curso de Pedagogia. Conclusão A avaliação da translação do conhecimento evidencia o potencial inovador da extensão universitária, integrada ao ensino e à pesquisa, em prol da primeira infância.


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Asunto(s)

Humanos , Carcinoma Hepatocelular/genética , Integrinas/genética , Neoplasias Hepáticas/genética , /genética , Perfilación de la Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos

RESUMEN

Using a consensus epitope prediction approach, three rotavirus (RV) peptides that induce cytokine secretion by CD4 T cells from healthy volunteers were identified. The peptides were shown to bind HLA-DRB1*0101 and then used to generate MHC II tetramers. RV specific T cell lines specific for one of the three peptides studied were restricted by MHC class II molecules and contained T cells that bound the tetramer and secreted cytokines upon activation with the peptide. The majority of RV and Flu tetramer(+) CD4 T cells in healthy volunteers expressed markers of antigen experienced T cells, but only RV specific CD4 T cells expressed intestinal homing receptors. CD4 T cells from children that received a RV vaccine, but not placebo recipients, were stained with the RV-VP6 tetramer and also expressed intestinal homing receptors. Circulating RV-specific CD4 T cells represent a unique subset that expresses intestinal homing receptors.

Asunto(s)

Linfocitos T CD4-Positivos/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Integrinas/genética , Intestinos/inmunología , Receptores CCR/genética , Receptores Virales/genética , Infecciones por Rotavirus/inmunología , Rotavirus/inmunología , Adolescente , Adulto , Niño , Femenino , Antígenos de Histocompatibilidad Clase II/química , Antígenos de Histocompatibilidad Clase II/genética , Humanos , Integrinas/inmunología , Intestinos/virología , Masculino , Receptores CCR/inmunología , Receptores Virales/inmunología , Rotavirus/genética , Infecciones por Rotavirus/genética , Infecciones por Rotavirus/virología , Especificidad de la Especie , Proteínas Virales/genética , Proteínas Virales/inmunología , Adulto Joven

RESUMEN

The α4ß7 is a lymphocyte homing receptor to the gut-associated lymphoid tissue (GALT). HIV-1 gp120 binds to α4ß7 integrin through a mimetic tripeptide in V2 and ensures successful infection of GALT. In the present report, we investigated the presence of polymorphisms in the α4ß7 cytoplasmic and α4 N-terminal binding domains and their potential association with susceptibility to HIV infection or disease progression. Subjects displaying distinct categories of disease progression or transmission routes (HIV-positive adults, vertically infected infants, and seronegative subjects) had their ITGA4 and ITGB7 gene segments corresponding to virus binding sites and C-terminal domains PCR amplified and sequenced. An absolute conservation of the studied regions was observed in all patients and controls. Albeit polymorphisms in α4ß7 may exist in other regions not tracked in this study, α4ß7 activation and binding domains do not seem to be polymorphic and are not correlated with distinct patterns of HIV transmission or disease progression.

Asunto(s)

Secuencia Conservada , Transmisión de Enfermedad Infecciosa , Variación Genética , Infecciones por VIH/genética , Infecciones por VIH/inmunología , Integrinas/genética , Adulto , Animales , Niño , Preescolar , Progresión de la Enfermedad , Femenino , Genotipo , Infecciones por VIH/transmisión , VIH-1 , Humanos , Lactante , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN

RESUMEN

Dopamine and estradiol interact in the regulation of lactotroph cell proliferation and prolactin secretion. Ablation of the dopamine D2 receptor gene (Drd2(-/-)) in mice leads to a sexually dimorphic phenotype of hyperprolactinemia and pituitary hyperplasia, which is stronger in females. TGF-ß1 is a known inhibitor of lactotroph proliferation. TGF-ß1 is regulated by dopamine and estradiol, and it is usually down-regulated in prolactinoma experimental models. To understand the role of TGF-ß1 in the gender-specific development of prolactinomas in Drd2(-/-) mice, we compared the expression of different components of the pituitary TGF-ß1 system, including active cytokine content, latent TGF-ß-binding protein isoforms, and possible local TGF-ß1 activators, in males and females in this model. Furthermore, we evaluated the effects of dopamine and estradiol administration to elucidate their role in TGF-ß1 system regulation. The expression of active TGF-ß1, latent TGF-ß-binding protein isoforms, and several putative TGF-ß1 activators evaluated was higher in male than in female mouse pituitary glands. However, Drd2(-/-) female mice were more sensitive to the decrease in active TGF-ß1 content, as reflected by the down-regulation of TGF-ß1 target genes. Estrogen and dopamine caused differential regulation of several components of the TGF-ß1 system. In particular, we found sex- and genotype- dependent regulation of active TGF-ß1 content and a similar expression pattern for 2 of the putative TGF-ß1 activators, thrombospondin-1 and kallikrein-1, suggesting that these proteins could mediate TGF-ß1 activation elicited by dopamine and estradiol. Our results indicate that (1) the loss of dopaminergic tone affects the pituitary TGF-ß1 system more strongly in females than in males, (2) males express higher levels of pituitary TGF-ß1 system components including active cytokine, and (3) estradiol negatively controls most of the components of the system. Because TGF-ß1 inhibits lactotroph proliferation, we propose that the higher levels of the TGF-ß1 system in males could protect or delay the development of prolactinomas in Drd2(-/-) male mice.

Asunto(s)

Hipófisis/metabolismo , Prolactinoma/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Femenino , Regulación de la Expresión Génica/fisiología , Genotipo , Integrinas/genética , Integrinas/metabolismo , Masculino , Ratones , Ratones Noqueados , Neoplasias Hipofisarias/metabolismo , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Factores Sexuales , Trombospondina 1/genética , Trombospondina 1/metabolismo , Calicreínas de Tejido/genética , Calicreínas de Tejido/metabolismo , Factor de Crecimiento Transformador beta1/genética

RESUMEN

Genome-wide association studies and meta-analysis, as well as our own previous family-based association results, have pointed to chromosome (ch) 3p22.3 and 3p21.1 as candidate regions to contain a susceptibility gene for bipolar affective disorder (BPAD). In the present study, we further refined the region of interest on ch 3p22.3. We genotyped 94 SNPs within the candidate region in 74 families and performed family-based association analysis using a transmission disequilibrium test. One single SNP (rs166508) was associated with the BPAD phenotype (P = 0.0187). This SNP is located within intron 15 of the integrin alpha 9 (ITGA9) gene. ITGA9 encodes the α9 subunit of the α9ß1 integrin, a membrane glycoprotein receptor for neurotrophins, such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Quantification of ITGA9 transcripts in the peripheral blood of patients with BPAD and controls showed an upregulation of ITGA9 (Kruskal-Wallis P = 0.0339) in patients with the disease-associated genotype (rs166508*A/A), compared to those with rs166508*G/G and rs166508*G/A genotypes. Sequencing of the ITGA9 cDNA revealed a sequence variant (r.1689_1839del) in rs166508*A carriers, which leads to loss of the entire exon 16. In silico analysis revealed that the deleted region contains three putative microRNA binding sites, which may be involved in the negative regulation of ITGA9. In conclusion, our results confirm previous evidence pointing to a candidate region for BPAD on ch 3p.22.3. In addition, we suggest a molecular substrate that could explain the increase of ITGA9 mRNA levels in probands with BPAD, proposing a new mechanism that could be involved in the genetic susceptibility to the disease.

Asunto(s)

Trastorno Bipolar/genética , Cromosomas Humanos Par 3/genética , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Secuencia de Bases , ADN Complementario/genética , Regulación de la Expresión Génica , Frecuencia de los Genes/genética , Humanos , Integrinas/genética , Integrinas/metabolismo , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo

RESUMEN

The study of spermatogonial stem cells (SSCs) provides a model to better understand adult stem cell biology. Besides the biomedical potential to perform studies of infertility in many species, SSCs hold a promising application at animal transgenesis. Because stem cells are thought to be associated with basement membranes, expression of α-6 integrin has been investigated as a marker of type A spermatogonial cells, which are considered SSCs because of their undifferentiated status and self-renewal ability. In this manner, the aim of this study was to isolate type A SSCs from adult bulls by a two-step enzymatic procedure followed by a discontinuous Percoll density gradient purification and verify the expression of α-6 integrin by flow cytometry and real-time RT-PCR before and after Percoll purification. Spermatogonial cells were successfully obtained using the two-step enzymatic digestion. An average of 1 × 10(5) viable cells per gram of testis was isolated. However, the discontinuous Percoll did not purify isolated cells regarding α-6 integrin expression. Flow cytometry analysis demonstrated no differences in the α-6 integrin expression between cell samples before and after Percoll purification (p = 0.5636). The same was observed in the real-time PCR analysis (p > 0.05). In addition to α-6 integrin, the expression of GFRa-1 and PGP9.5, known bovine SSCs markers, was detected in all samples studied. Considering that Percoll can reduce cell viability, it is possible to conclude that Percoll density gradient is not suitable to purify bovine SSC, according to α-6 integrin expression.

Asunto(s)

Bovinos/fisiología , Centrifugación por Gradiente de Densidad/veterinaria , Regulación de la Expresión Génica/fisiología , Integrinas/metabolismo , Povidona/química , Dióxido de Silicio/química , Espermatogonias/metabolismo , Animales , Separación Celular/métodos , Separación Celular/veterinaria , Centrifugación por Gradiente de Densidad/métodos , Integrinas/genética , Masculino

RESUMEN

The α4 integrin subunit associates with ß7 and ß1 and plays important roles in immune function and cell trafficking. The gut-homing receptor α4ß7 has been recently described as a new receptor for HIV. Here, we describe polymorphisms of ITGA4 gene in New World primates (NWP), and tested their impact on the binding to monoclonal antibodies, natural ligands (MAdCAM and VCAM), and several gp120 HIV-1 envelope proteins. Genomic DNA of NWP specimens comprising all genera of the group had their exons 5 and 6 (encoding the region of binding to the ligands studied) analyzed. The polymorphisms found were introduced into an ITGA4 cDNA clone encoding the human α4 subunit. Mutant α4 proteins were co-expressed with ß7 and were tested for binding of mAbs, MAdCAM, VCAM and gp120 of HIV-1, which was compared to the wild-type (human) α4. Mutant α4 proteins harboring the K201E/I/N substitution had reduced binding of all ligands tested, including HIV-1 gp120 envelopes. The mAbs found with reduced biding included one from which a clinically-approved drug for the treatment of neurological disorders has been derived. α4 polymorphisms in other primate species may influence outcomes in the development and treatment of infectious and autoimmune diseases in humans and in non-human primates.

Asunto(s)

Proteína gp120 de Envoltorio del VIH/metabolismo , Integrinas/genética , Integrinas/metabolismo , Polimorfismo Genético , Molécula 1 de Adhesión Celular Vascular/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Bovinos , Genotipo , Células HEK293 , Humanos , Integrinas/inmunología , Ligandos , Ratones , Platirrinos , Unión Proteica

RESUMEN

Thy-1, an abundant mammalian glycoprotein, interacts with αvß3 integrin and syndecan-4 in astrocytes and thus triggers signaling events that involve RhoA and its effector p160ROCK, thereby increasing astrocyte adhesion to the extracellular matrix. The signaling cascade includes calcium-dependent activation of protein kinase Cα upstream of Rho; however, what causes the intracellular calcium transients required to promote adhesion remains unclear. Purinergic P2X7 receptors are important for astrocyte function and form large non-selective cation pores upon binding to their ligand, ATP. Thus, we evaluated whether the intracellular calcium required for Thy-1-induced cell adhesion stems from influx mediated by ATP-activated P2X7 receptors. Results show that adhesion induced by the fusion protein Thy-1-Fc was preceded by both ATP release and sustained intracellular calcium elevation. Elimination of extracellular ATP with Apyrase, chelation of extracellular calcium with EGTA, or inhibition of P2X7 with oxidized ATP, all individually blocked intracellular calcium increase and Thy-1-stimulated adhesion. Moreover, Thy-1 mutated in the integrin-binding site did not trigger ATP release, and silencing of P2X7 with specific siRNA blocked Thy-1-induced adhesion. This study is the first to demonstrate a functional link between αvß3 integrin and P2X7 receptors, and to reveal an important, hitherto unanticipated, role for P2X7 in calcium-dependent signaling required for Thy-1-stimulated astrocyte adhesion.

Asunto(s)

Adenosina Trifosfato/metabolismo , Adhesiones Focales/metabolismo , Integrinas/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Antígenos Thy-1/metabolismo , Animales , Astrocitos/metabolismo , Western Blotting , Calcio/metabolismo , Línea Celular , Técnica del Anticuerpo Fluorescente Indirecta , Integrinas/genética , Ratas , Receptores Purinérgicos P2X7/genética , Antígenos Thy-1/genética

RESUMEN

Alternative approaches to improve the treatment of advanced melanomas are highly needed. The disintegrin domain of metalloproteinases binds integrin receptors on tumor cells, blocking migration, invasion, and metastatization. Previous studies showed that jararhagin, from the Bothrops jararaca snake venom, induces changes in the morphology and viability of SK-Mel-28 human melanoma cells, and decreases the number of metastases in mice injected with pre-treated cells. The purpose of this study was to evaluate the molecular effects of jararhagin on SK-Mel-28 cells and fibroblasts, concerning the expression of integrins, cadherins, caspases, and TP53 genes. Sub-toxic doses of jararhagin were administered to confluent cells. RT-PCR was performed following extraction of total RNA. Jararhagin treatments induced similar morphological alterations in both normal and tumor cells, with higher IC50 values for fibroblasts. Integrin genes were downregulated in untreated cells, except for ITGA6a,b, ITGAv, and ITGB3 which were highly expressed in SK-Mel-28. The integrin expression profiles were not affected by the toxin. However, jararhagin 30ng/µl upregulated genes TP53, CDKN1A, CDKN2A, CASP3, CASP5, CASP6, CASP8, and E-CDH in SK-Mel-28, and genes ITGB6, ITGB7, CASP3, TP53, and CDKN1B in fibroblasts. Appropriate jararhagin concentration can have apoptotic and suppressant effects on SK-Mel-28 cells, rather than on fibroblasts, and can be used to develop potential anti-cancer drugs.

Asunto(s)

Venenos de Crotálidos/farmacología , Expresión Génica/efectos de los fármacos , Integrinas/genética , Melanoma/tratamiento farmacológico , Metaloendopeptidasas/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Neoplasias Cutáneas/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Cadherinas/genética , Cadherinas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Venenos de Crotálidos/aislamiento & purificación , Regulación hacia Abajo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Integrinas/metabolismo , Melanoma/genética , Metaloendopeptidasas/aislamiento & purificación , ARN Mensajero/metabolismo , Neoplasias Cutáneas/genética , Ensayo de Tumor de Célula Madre , Veneno de Bothrops Jararaca