RGD peptide inhibits tumor growth by affecting angiogenesis related signaling pathway of laryngeal cancer stem cells / El péptido RGD inhibe el crecimiento tumoral al afectar la vía de señalización relacionada con la angiogénesis de las células madre del cáncer de laringe

SUMMARY: This study is to investigate the role and mechanism of RGD peptide in laryngeal cancer stem cells (CSCs). Laryngeal cancer CD133+Hep-2 CSCs were sorted by flow cytometry. RGD peptide was co-cultured with sorted laryngeal CSCs. Cell proliferation was detected with CCK-8 assay. The mRNA levels of VEGF/VEGFR2/STAT 3/HIF-1α were detected with RT-PCR. The proteins of VEGF/ VEGFR2/STAT 3/HIF-1α were detected with Western blot. The sorted CSCs were inoculated into nude mice. Tumor volume was measured. Integrin αvβ3 expression in tumor tissues was analyzed with immunohistochemistry. The results showed that the ratio of CD133+ CSCs to the total number of cells was 1.34±0.87 %, while CD133-non-tumor stem cells accounted for 95.0±5.76 %. The sorted cancer stem cells grew well. The RGD peptide significantly inhibited the proliferation of CD133+Hep-2 laryngeal CSCs in a dose-dependent manner. The RGD peptide significantly inhibited the mRNA of VEGFR2, STAT3 and HIF-1α in laryngeal CSCs in a concentration-dependent manner. Consistently, the RGD peptide significantly inhibited the protein expression of VEGFR2, STAT3 and HIF-1α in laryngeal CSCs in a dose-dependent manner. At the same time, in vivo tumor experiments showed that the RGD peptide significantly inhibited tumor volume but not the body weight. Furthermore, RGD peptide significantly inhibited the expression of tumor angiogenesis-related protein integrin αvβ3. Our findings demonstrate that RGD peptide inhibits tumor cell proliferation and tumor growth. The underlying mechanism may that RGD inhibits tumor angiogenesis-related signaling pathways, thus affecting the tumor angiogenesis, and decreasing the progression of human laryngeal CSCs.

Este estudio se realizó para investigar el papel y el mecanismo del péptido RGD en las células madre del cáncer de laringe (CSC). Las CSC CD133+Hep-2 de cáncer de laringe se clasificaron mediante citometría de flujo. El péptido RGD se cocultivó con CSC laríngeas clasificadas. La proliferación celular se detectó con el ensayo CCK-8. Los niveles de ARNm de VEGF/VEGFR2/ STAT 3/HIF-1α se detectaron con RT-PCR. Las proteínas de VEGF/ VEGFR2/STAT 3/HIF-1α se detectaron con Western blot. Las CSC clasificadas se inocularon en ratones nudos. Se midió el volumen del tumor. La expresión de integrina αvβ3 en tejidos tumorales se analizó con inmunohistoquímica. Los resultados mostraron que la proporción de CSC CD133+ con respecto al número total de células fue de 1,34 ± 0,87 %, mientras que las células madre no tumorales CD133 representaron el 95,0 ± 5,76 %. Las células madre cancerosas clasificadas crecieron bien. El péptido RGD inhibió significativamente la proliferación de CSC laríngeas CD133+Hep-2 de una manera dependiente de la dosis. El péptido RGD inhibió significativamente el ARNm de VEGFR2, STAT3 y HIF-1α en CSC laríngeas de manera dependiente de la concentración. De manera consistente, el péptido RGD inhibió significativamente la expresión proteica de VEGFR2, STAT3 y HIF-1α en CSC laríngeas, de manera dependiente de la dosis. Al mismo tiempo, los experimentos con tumores in vivo mostraron que el péptido RGD inhibía significativamente el volumen del tumor pero no el peso corporal. Además, el péptido RGD inhibió significativamente la expresión de la proteína integrina αvβ3 relacionada con la angiogénesis tumoral. Nuestros hallazgos demuestran que el péptido RGD inhibe la proliferación de células tumorales y el crecimiento tumoral. El mecanismo subyacente puede ser que RGD inhiba las vías de señalización relacionadas con la angiogénesis tumoral, afectando así la angiogénesis tumoral y disminuyendo la progresión de las CSC laríngeas humanas.

Decreased rotavirus infection of MA104 cells via probiotic extract binding to Hsc70 and ß3 integrin receptors / Disminución de la infección por rotavirus en células MA104 por la unión de proteínas de extractos probióticos a receptores celulares integrina ß3 y Hsc70 / Diminuição da infecção por rotavírus em células MA104 pela união de proteínas de extratos probióticos a receptores integrina ß3 e Hsc70

Abstract Probiotic bacteria are microorganisms beneficial to human health, useful to improving biological conditions. Thanks to probiotic bacteria the symptoms of viral infections can be alleviated. Different mechanisms whereby probiotic bacteria exert they antiviral effect have been proposed. The aim of this study was to determine whether probiotic bacteria extracts bind to receptors of host cells susceptible of rotavirus (RV) infection. To accomplish this objective, four probiotic bacterial strains of Lactobacillus spp. and Bifidobacterium spp. were tested. Probiotic extracts were obtained after bacterial growth, cell lysis and centrifugation. Obtained probiotic extracts were used in assays to interfere with adhesion and penetration of a RV strain in the mammal cell line MA104. Furthermore, the interaction between probiotic extracts and MA104 cell receptors was evaluated by co-immunoprecipitation assays using anti-β3-integrins and anti-Hsc70 antibodies. All four probiotic, protein-rich, extracts reduced RV infections in MA104 cells, suggesting a successful antiviral activity mediated by these probiotic extracts. All probiotic extracts significantly exerted their antiviral activity by interfering with RV adhesion on MA104 cell receptors, with proteins in probiotic extracts competitively interacting with cell surface receptors necessary to RV infection. Co-immunoprecipitation assay results showed that proteins in probiotic extracts were able to bind to β3-integrinsand Hsc70, which are two cellular receptors required to viral infection. The most significant contribution of this study is an insight into the mechanisms of probiotic antiviral activity, thus expanding current probiotics fundamental knowledge.

Resumen Las bacterias probióticas son microorganismos con efectos positivos en la salud humana, gracias a las bacterias probióticas los síntomas de infecciones virales pueden mitigarse. Al respecto, varios mecanismos antivirales de las bacterias probióticas han sido propuestos. El propósito de este estudio fue determinar, de manera experimental, si extractos de bacterias probióticas reducen la infección rotavírica al interferir con la unión entre el rotavirus y sus receptores celulares blanco. Extractos de cuatro cepas probióticas de Lactobacillus spp. y Bifidobacterium spp. fueron obtenidos a partir de cultivos bacterianos lisados y centrifugados. Cada uno de los extractos fue usado en experimentos para determinar si estos interfieren con la adhesión y penetración del rotavirus en células de mamífero MA104. Además, la interacción entre extractos probióticos y receptores de las células MA104 fue evaluada con ensayos de co-inmunoprecipitación, usando anticuerpos anti-integrina β3 y anti-Hsc70. Se observó que los cuatro extractos probióticos, ricos en proteínas, redujeron significativamente la infección de rotavirus en las células MA104. También se estableció que la que la actividad antiviral de los extractos probióticos es mediada por la interacción competitiva de sus proteínas con los receptores integrina β3 y Hsc70 de las células MA104, necesarios para iniciar la infección por rotavirus. Estos hallazgos constituyen un aporte al conocimiento de los mecanismos básicos de acción antiviral de las bacterias probióticas.

Resumo Bactérias probióticas são microrganismos com efeitos positivos na saúde humana, úteis na melhora de certas condições biológicas. Gracas a bactérias probióticas os sintomas de uma infecção viral podem ser aliviados. Diferentes mecanismos pelos quais as bactérias probióticas exercem seus efeitos antivirales têm sido propostos. O objetivo de este estudo foi determinar se extratos de bactérias probióticas reduzem a infecção de rotavírus (RV) ao interferir com a união entre o RV e seus receptores celulares alvo. Quatro cepas probióticas de Lactobacillus spp. e Bifidobacterium spp. foram testadas. Os extratos probióticos foram obtidos após o crescimento bacteriano, lise celular e centrifugação. Os extratos probióticos obtidos foram utilizados em ensaios para determinar se interferem com a adesão e penetração de uma cepa de RV em células de mamífero MA104. Adicionalmente, a interação entre os extratos probióticos e os receptores das células MA104 foi avaliada por ensaios de co-imunoprecipitação usando anticorpos anti-integrina β3 e anti- Hsc70. Os quatro extratos probióticos, ricos em proteínas, reduziram as infecções por RV em células MA104, sugerindo uma atividade antiviral mediada por estes extratos. Todos os extratos interferiram na adesão do RV aos receptores de células MA104, sendo que as proteínas presentes nos extratos mostraram uma interação competitiva com os receptores integrina β3 e Hsc70 das células MA104, necessários para iniciar a infecção por RV. Estes resultados contribuem para o conhecimento dos mecanismos básicos de ação antiviral de bactérias probióticas.

Focal adhesion proteins, vinculin and integrin ß5, during early pregnancy in rat uterine epithelial cells: anastrozole favors their normal distribution / Proteínas de adhesión focales, vinculina e integrina ß5, durante el embarazo temprano en células epiteliales uterinas de rata: anastrozol favorece su distribución normal

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.

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.

Characterization of inflammatory markers associated with systemic lupus erythematosus patients undergoing treatment / Caracterização de marcadores inflamatórios associados a pacientes com lúpus eritematoso sistêmico em tratamento

ABSTRACT Objective: To characterize the inflammatory profiles of patients with systemic lupus erythematosus receiving standard treatment compared to healthy controls. Patients and methods: Peripheral venous blood was collected from systemic lupus erythematosus patients (n = 14) and controls (n = 18) at enrollment. Blood samples were used for quantification, by flow cytometry, of CD11b (integrin) and Chemokine receptor CXCR2 expression surface antigen in neutrophils and lymphocytes, while cytokines were assayed in serum samples. Purified neutrophils were assayed by their ability to phagocytize human plasma-opsonized zymosan. Results: Patients had a median (interquartile range) disease activity index of 1.0 (0-2.0) characteristic of patients in remission. Interleukin-6 and interleukin-10 serum concentrations were significantly higher in the patient group compared to controls and the phagocytic index of circulating neutrophils was significantly reduced in patients compared to controls. The levels of interleukin-2, interleukin-5, interleukin-8 and tumor necrosis factor alpha did not significantly differ between patients and controls. Flow cytometric analysis revealed that the integrin expression levels were reduced in lymphocytes (but not in neutrophils) obtained from systemic lupus erythematosus patients, while surface expression of the chemokine receptor 2 was similar in both neutrophils and lymphocytes. Conclusion: Systemic lupus erythematosus patients receiving standard treatment presented with elevated systemic levels of interleukin-6 and interleukin-10, reduced neutrophil phagocytic capacity, and reduced lymphocyte expression of integrin even when symptoms were in remission. These alterations to innate immune components may put these individuals at a greater risk for acquiring infections.

RESUMO Objetivo: Caracterizar os perfis inflamatórios de pacientes com lúpus eritematoso sistêmico (LES) que recebiam o tratamento padrão em comparação com controles saudáveis. Pacientes e métodos: Coletou-se o sangue venoso periférico de pacientes com LES (n = 14) e controles (n = 18) no momento da entrada no estudo. As amostras de sangue foram usadas para quantificação, por citometria de fluxo, da expressão dos antígenos de superfície CD11b (integrina) e CXCR2 em neutrófilos e linfócitos, enquanto as citocinas foram avaliadas em amostras de soro. Avaliou-se a capacidade dos neutrófilos purificados de fagocitar zimosan opsonizado com plasma humano. Resultados: Os pacientes apresentavam uma pontuação mediana (intervalo interquartil) no Sledai de 1 (0-2), característica de pacientes em remissão. As concentrações séricas de IL-6 e IL-10 foram significativamente maiores no grupo de pacientes em comparação com os controles; o índice de fagocitose de neutrófilos circulantes estava significativamente reduzido nos pacientes em comparação com os controles. Os níveis de IL-2, IL-5, IL-8 e TNF-α não diferiram significativamente entre pacientes e controles. A análise da citometria de fluxo revelou que os níveis de expressão de CD11b estavam reduzidos nos linfócitos (mas não nos neutrófilos) obtidos de pacientes com LES, enquanto a expressão do receptor de superfície CXCR2 foi semelhante em neutrófilos e linfócitos. Conclusão: Os pacientes com LES que recebiam tratamento padrão apresentaram níveis sistêmicos elevados de IL-6 e IL-10, redução na capacidade fagocítica dos neutrófilos e redução da expressão de CD11b em linfócitos, mesmo quando os sintomas estavam em remissão. Essas alterações nos componentes da imunidade inata podem colocar esses indivíduos em maior risco de adquirir infecções.

Estudio y análisis comparativo de interacciones entre la proteina integrina con fragmentos de la proteína fibrilina-1 y fragmentos mutados de esta utilizando la metodología de docking molecular / Study and comparative analysis of interactions protein between the integrina with fragments fibrillin-1 and mutated fragments of this by using molecular docking

Resumen Objetivo: Evaluar las interacciones proteína-proteína que pueden generarse entre fragmentos de la proteína fibrilina-1, cuyas mutaciones causan el síndrome de Marfan (SM). Materiales y métodos: Se realizó una serie de cálculos docking proteína-proteína entre las macromoléculas de interés; se empleó el programa Molsoft ICM; se utilizaron las estructuras cristalinas de la proteína integrina αVβ3 y los fragmentos de la proteína fibrilina-1; además se generó una sucesión de mutaciones en la fibrilina-1, las cuales son características de pacientes con síndrome de Marfan, y posteriormente se realizó el acoplamiento molecular. Adicionalmente se determinó los aminoácidos que con mayor frecuencia estaban presentes en el sitio de interacción y su hidrofobicidad. Resultados: Se cuantificó la cantidad de aminoácidos hidrófobos presentes en las zonas de interacción producidas por los acoplamientos, teniendo en cuenta la energía del sistema; esta ponderación estuvo entre el 40 y 50 % de los aminoácidos de la zona de interacción, con un porcentaje mayor con respecto a aminoácidos neutros o cargados. En los resultados obtenidos utilizando las mutaciones realizadas sobre los fragmentos cbEGF22-TB4-cbEGF23 y cbEGF9-hyb2-cbEGF10 de la fibrilina-1 se encontró que no se ubicaron en zonas cercanas al sitio de interacción en la mayoría de los casos. Conclusiones: Las interacciones entre los fragmentos de fibrilina-1, y estos con respecto a la integrina, mostraron en sus zonas de interacción la presencia mayoritariamente de aminoácidos hidrofóbicos, que es lo esperado normalmente.

Abstract Objective: To assess protein-protein interactions between fragments of fibrillin-1 protein, whose mutations cause Marfan syndrome (MS). Materials and Methods: We performed a series of protein-protein docking calculations between the macromolecules of interest for this purpose was used Molsoft ICM program. We used the crystal structures of αVβ3 Integrin protein and fragments of fibrillin-1 protein also were generated mutations in the fibrillin-1, which are characteristic in patients with Marfan syndrome and subsequently to the molecular docking. We determined the amino acids most often present at the site of interaction and its hydrophobicity. Results: The amount of hydrophobic amino acids present in the areas of interaction given by the couplings was quantified. Given the energy of the system, was between 40 and 50% of the amino acids of the interaction zone, with a higher proportion relative to charged or neutral amino acids. In the results obtained using the mutations performed on fragments cbEGF23 cbEGF22-TB4-and-cbEGF10 cbEGF9-HYB2 of fibrillin-1, was found they were not placed in areas near the site of interaction in most cases. Conclusion: The interaction between fragments of fibrillin-1, and those with respect to their integrin showed the presence interaction zones mostly hydrophobic amino acids, which are normally expected.

Avaliação da expressão de integrina alfa2, proteína de choque térmico 47, e mediadores pro-inflamatórios e imunorregulatórios em resposta à infecção endodôntica / To examine alpha 2 integrin, molecular mediators, cytokines, and chemokines from cells in periapical interstitial fluid from root canal infections

Objetivo: analisar a expressão de integrina alfa2, mediadores moleculares, citocinas e quiocinas, a partir de células presentes no líquido interstical periapical adjacente e dentes portadores de infecção dos canais radiculares. Metodologia: os 13 pacientes incluídos no estudo foram encaminhados à Faculdade de Odontologia da Universidade Federal de Minas Gerais (Belo Horizonte, MG, Brasil). As amostras foram retiradas de dentes com necrose pulpar e nenhum paciente apresentou sintomas periapicais agudos no momento das coletas (...) Resultados: Observaram-se níveis significativamente mais baixos de TNF-α, CCL5, CCL2 / MCP-1 e IL8 em dentes com cargas bacterianas redusidas...

Desarrollo de un péptido cíclico marcado con 99mTc dirigido contra el receptor de la vitronectina / Development of a 99mTc-labeled cyclic peptide with affinity for the vitronectine receptor

El objetivo del presente trabajo fue desarrollar un péptido híbrido, que presente una secuencia capaz de quelatar al 99mTc y otra con afinidad por el receptor de la vitronectina, que permita la detección in vivo de tumores malignos. El marcaje del péptido PICIC3 con 99mTc se realizó de forma directa. Se estudió la estabilidad del complejo en exceso de L-cisteína y en plasma, la unión a proteínas plasmáticas, el coeficiente de partición en el sistema NaCl 0.9 por ciento:n-octanol, la carga del complejo mediante electroforesis y la afinidad por el receptor de la vitronectina se valoró a partir de un ensayo de saturación con membranas de células B16-F10. Se determinó la biodistribución en ratones C57BL/6 con injertos de melanoma B16-F10. Conclusiones: El péptido desarrollado mostró una afinidad satisfactoria por el receptor de la vitronectina y que permitió la detección in vivo de los melanomas múridos del tipo B16-F10 en los ratones injertados.

The aim of the present work was to develop a hybrid peptide, with a sequence for the chelation of 99mTc and other with affinity for the vitronectine receptor, to allow in vivo detection of malignant tumors. 99mTc-labeling of peptide PICIC3 was directly performed. The stability in presence of L-cysteine excess and plasma of the complex, its binding to plasma proteins, the partition coefficient in NaCl 0.9 percent:n-octanol, the charge of the chelate by electrophoresis and the peptide affinity for the vitronectine receptor by a saturation assay using membranes of B16-F10 cells, were studied. Biodistribution in C57BL/6 mice injerted with melanoma B16-F10 was assessed. Conclusions: Developed peptide showed a satisfactory affinity for the vitronectine receptor and allowed in vivo detection of murine melanomas in mice with allografts.

16S rRNA methylase and genetic relateness of multidrug-resistant Acinetobacter spp / 中华检验医学杂志

Objective To characterize 16S rRNA methylase encoding genes associated with aminoglycoaides resistance, gene cassettes of class Ⅰ integrons of the multidrug-resistant Acinetobctcter spp. The sixty one Acinetobacter isolates were collected at the Second Hospital of Shanxi Medical Uni versity from July, 2007 to May, 2008. Methods Species identification was confirmed by sequence analysis of the blaOXA-51-like gene and 16S-23S rRNA gene space-region. Antimierobial susceptibility tests were performed by agar dilution method. 16S rRNA methylaae encoding genes and gene cassettes associated with integrons were amplified by PCR method. Results Among the sixty one strains, there were fifty five of Acinetobacter baumannii, three genospecies 3TU, one 13TU, one Aeinetobaeter ealcoacetieus, and one Aeinetobaeter haemolytieus. Forty eight isolates showed high-level resistance to three aminoglyeosides, including amikaein, tobramyein and gentamicin. The armA gene was found in 47 isolates and all isolates were negative for rmtA, rmtB, rmtC and rmtD genes. The Intl gene was found in 27 isolates. The gene cassettes contained arr-3, accA4,ctacCI ,catB8, aadA1 or dfrA12 genes. According to the PFGE DNA patterns, 5 distinct clones of armA-pasitive strains were identified. Clone A and Clone B were the dominant clones, widely distributed among different divisions. Condnsions 16S rRNA methylase encoding gene (armA) distributed widely in muhidrug-resistant Acinetobacter spp. The armA gene is not located in class Ⅰintegron. The class Ⅰ integron carries multiple resistant genes associated with aminoglycosides and chloramphenieol resistance.PFGE analysis suggests that armA-pesitive strains are widely spread in our hospitaL Effective infection control measure should be conducted in order to control the outbreak of resistant bacteria.