Caracterización bioquímica y genética de la infantaricina A: una nueva bacteriocina antineumocócica producida por la cepa de origen lácteo "Streptococcus infantarius" subesp. "infantarius" LP90

En este trabajo se ha investigado la utilidad del empleo de bacterias lácticas, principalmente de origen alimentario, y/o sus bacteriocinas como estrategia alternativa o complementaria a los antibióticos para el control de las infecciones causadas por Streptococcus pneumoniae en humanos, empleando para ello diversas técnicas microbiológicas, bioquímicas y genéticas. En el Capítulo III se describe la evaluación de la actividad antimicrobiana de 38 BAL bacteriocinogénicas frente a cepas clínicas de S. pneumoniae de diversos serotipos y patrones de susceptibilidad a los antibióticos más empleados en medicina humana utilizando ensayos de antagonismo microbiano (i. e., inhibición por siembra en picadura [ISP], técnica de difusión en agar [TDA] y ensayo en placas microtituladoras [EPM]). Streptococcus infantarius subesp. infantarius LP90, una cepa aislada de leche de búfala de Venezuela, destacó por su amplio y potente espectro de acción frente a S. pneumoniae, por lo que fue seleccionada para su posterior caracterización. La purificación mediante un protocolo multicromatográfico de las bacteriocinas producidas por S. infantarius LP90, así como su análisis mediante espectrometría de masas MALDI-TOF, permitió determinar que la actividad antineumocócica de esta cepa es debida a la producción de, al menos, una nueva bacteriocina con una masa molecular de 3.963 Da a la que se denominó infantaricina A (InfA). La comparación de la secuencia aminoacídica de la región N-terminal de 19 aminoácidos de InfA, obtenida mediante degradación N-terminal de Edman, con las de otras proteínas depositadas en la base de datos Genbank reveló una elevada homología con una hipotética proteína de S. infantarius ATCCBAA-102..

A simple and robust high-performance liquid chromatography coupled to a diode-array detector method for the analysis of genistein in mouse tissues

A simple liquid-liquid extraction procedure and quantification by high-performance liquid chromatography (HPLC) method coupled to a diode-array detector (DAD) of genistein (GEN) was developed in various mouse biological matrices. 7-ethoxycoumarin was used as internal standard (IS) and peaks were optimally separated using a Kinetex C18 column (2.6 µm, 150 mm X 2.10 mm I.D.) at 40 ºC with an isocratic elution of mobile phase with sodium dihydrogen phosphate 0.01M in water at pH 2.5 and methanol (55:45, v/v), at a flow rate of 0.25 mL/min. The injection volume was 10 µL. In all cases, the range of GEN recovery was higher than 61%. The low limit of quantification (LLOQ) was 25 ng/mL. The linearity of the calibration curves was satisfactory in all cases as shown by correlation coefficients >0.996. The within-day and between-day precisions were <15% and the accuracy ranged in all cases between 90.14 and 106.05%. This method was successfully applied to quantify GEN in liver, spleen, kidney and plasma after intravenous administration of a single dose (30 mg/Kg) in female BALB/C mice

Development and Evaluation of a Microarray Platform for Detection of Serum Antibodies against Streptococcus pneumoniae Capsular Polysaccharides

7 pags., 1 fig., 2 tabs.Streptococcus pneumoniae is responsible for severe infections, causing millions of deaths yearly. Immunoglobulin G (IgG) antibodies against the capsular polysaccharide (CPS) offer S. pneumoniae serotype-specific protection. In this work, we examined the applicability of the microarray technology to detect CPS type-specific IgGs in serum, using a collection of 22 microarray-printed S. pneumoniae CPSs. First, printing of five CPSs onto nitrocellulose-coated glass slides was tested. Successful printing was only achieved for certain CPS types and concentrations. This behavior was tentatively related with diverse viscosities of the CPS solutions. Measurement of dynamic viscosities fully supported this assumption and helped to establish suitable CPS type-and concentration-dependent printing conditions. Next, the potential of CPS microarrays for detecting recognition by anti-CPS IgGs was examined using well-defined rabbit pneumococcal antisera. In all cases, the expected antiserum-CPS binding signals were detected, prompting a proof-of-concept analysis of human serum samples. Clearly distinct serum-and CPS-specific binding patterns and intensities were observed, evidencing selective detection of CPS type-specific IgGs. Compared to the ELISA assay commonly used to quantitate CPS type-specific IgGs in serum, the newly developed S. pneumoniae CPS microarrays offer the advantage of enabling the simultaneous analysis of multiple CPS-serum interactions using minute CPS amounts and significantly reduced serum volumes. Therefore, the approach could be particularly valuable for gauging the presence of CPS type-specific IgGs in human serum when sample volumes are limited and/or numerous serum samples are being examined.We gratefully acknowledge financial support from the Spanish Ministry of Economy, Industry, and Competitiveness (Grants BFU2015-70052-R and SAF2017-88664-R), the Spanish Ministry of Science, Innovation, and Universities, the Spanish State Research Agency, and the European Regional Development Fund (Grant RTI2018-099985-B-I00, MCIU/AEI/ FEDER, UE), and the CIBER of Respiratory Diseases (CIBERES), an initiative from the Spanish Institute of Health Carlos III (ISCIII)

The role of collectins and galectins in lung innate immune defense

10 pags, 2 figsDifferent families of endogenous lectins use complementary defense strategies against pathogens. They may recognize non-self glycans typically found on pathogens and/or host glycans. The collectin and galectin families are prominent examples of these two lectin categories. Collectins are C-type lectins that contain a carbohydrate recognition domain and a collagen-like domain. Members of this group include surfactant protein A (SP-A) and D (SP-D), secreted by the alveolar epithelium to the alveolar fluid. Lung collectins bind to several microorganisms, which results in pathogen aggregation and/or killing, and enhances phagocytosis of pathogens by alveolar macrophages. Moreover, SP-A and SP-D influence macrophage responses, contributing to resolution of inflammation, and SP-A is essential for tissue-repair functions of macrophages. Galectins also function by interacting directly with pathogens or by modulating the immune system in response to the infection. Direct binding may result in enhanced or impaired infection of target cells, or can have microbicidal effects. Immunomodulatory effects of galectins include recruitment of immune cells to the site of infection, promotion of neutrophil function, and stimulation of the bactericidal activity of infected macrophages. Moreover, intracellular galectins can serve as danger receptors, promoting autophagy of the invading pathogen. This review will focus on the role of collectins and galectins in pathogen clearance and immune response activation in infectious diseases of the respiratory system.This study was supported by the Spanish Ministerio de Economía y Competitividad through Grants SAF2015-65307-R (to CC) and BFU2015-70052-R (to DS) and Instituto de Salud Carlos III (CIBERES-CB06/06/0002 to CC and CB06/06/1102 to DS)

A major role for RCAN1 in atherosclerosis progression

This is an open access article under the terms of the Creative Commons Attribution License.-- et al.Atherosclerosis is a complex inflammatory disease involving extensive vascular vessel remodelling and migration of vascular cells. As RCAN1 is implicated in cell migration, we investigated its contribution to atherosclerosis. We show RCAN1 induction in atherosclerotic human and mouse tissues. Rcan1 was expressed in lesional macrophages, endothelial cells and vascular smooth muscle cells and was induced by treatment of these cells with oxidized LDLs (oxLDLs). Rcan1 regulates CD36 expression and its genetic inactivation reduced atherosclerosis extension and severity in Apoe-/- mice. This effect was mechanistically linked to diminished oxLDL uptake, resistance to oxLDL-mediated inhibition of macrophage migration and increased lesional IL-10 and mannose receptor expression. Moreover, Apoe-/-Rcan1-/- macrophages expressed higher-than-Apoe-/- levels of anti-inflammatory markers. We previously showed that Rcan1 mediates aneurysm development and that its expression is not required in haematopoietic cells for this process. However, transplantation of Apoe-/-Rcan1-/- bone-marrow (BM) cells into Apoe-/- recipients confers atherosclerosis resistance. Our data define a major role for haematopoietic Rcan1 in atherosclerosis and suggest that therapies aimed at inhibiting RCAN1 expression or function might significantly reduce atherosclerosis burden. © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.The Spanish Ministry of Economy and Competitiveness (Ministerio de Economía y Competitividad) supports MRC, JMR and JM‐G with grants SAF2010‐15126, SAF2009‐10708 and SAF2012‐40127, respectively; MRC is also supported by the Spanish Council for Scientific Research (CSIC); JMR is also supported by Fundación La Marató TV3 (080731), and Fundación Genoma España (GENOMA). The Red de Investigacion Cardiovascular (RIC) of the Spanish Ministry of Health (Ministerio de Sanidad) supports the research of JMR, VA and JM‐G with grants RD12/0042/0022, RD12/0042/0028 and RD12/0042/0053, respectively. The Centro Nacional de Investigaciones Cardiovasculares (CNIC) is supported by the Spanish Ministry of Economy and Competitiveness and the Pro‐CNIC Foundation. TM was supported in part by the Leading‐edge Research Promotion Fund (LS038, TM). VE was an inve stigator of the Sara Borrell Program (CD06/00232), and NM‐B holds an FPU fellowship (FPU2008‐1500).Peer Reviewe

The Role of Collectins and Galectins in Lung Innate Immune Defense

Different families of endogenous lectins use complementary defense strategies against pathogens. They may recognize non-self glycans typically found on pathogens and/or host glycans. The collectin and galectin families are prominent examples of these two lectin categories. Collectins are C-type lectins that contain a carbohydrate recognition domain and a collagen-like domain. Members of this group include surfactant protein A (SP-A) and D (SP-D), secreted by the alveolar epithelium to the alveolar fluid. Lung collectins bind to several microorganisms, which results in pathogen aggregation and/or killing, and enhances phagocytosis of pathogens by alveolar macrophages. Moreover, SP-A and SP-D influence macrophage responses, contributing to resolution of inflammation, and SP-A is essential for tissue-repair functions of macrophages. Galectins also function by interacting directly with pathogens or by modulating the immune system in response to the infection. Direct binding may result in enhanced or impaired infection of target cells, or can have microbicidal effects. Immunomodulatory effects of galectins include recruitment of immune cells to the site of infection, promotion of neutrophil function, and stimulation of the bactericidal activity of infected macrophages. Moreover, intracellular galectins can serve as danger receptors, promoting autophagy of the invading pathogen. This review will focus on the role of collectins and galectins in pathogen clearance and immune response activation in infectious diseases of the respiratory system.Ministerio de Economía y CompetitividadInstituto de Salud Carlos IIIDepto. de Bioquímica y Biología MolecularFac. de Ciencias QuímicasTRUEpu