RESUMEN
Surfactant and its components have multiple functions. The so called collectins are surfactant proteins which opsonize bacteria and improve pulmonary host defense via the phagocytosis and clearance of microorganisms and particles. In this special issue of the Annals of Anatomy a new surfactant protein, Surfactant Associated 3, is highlighted. As outlined in this mini review Surfactant Associated 3 is regarded as an enhancer of phagocytosis. In addition, the role played by SP-A is updated and open research questions raised.
Asunto(s)
Inmunidad Innata/inmunología , Pulmón/inmunología , Macrófagos/inmunología , Modelos Inmunológicos , Fagocitosis/inmunología , Proteínas Asociadas a Surfactante Pulmonar/inmunología , HumanosRESUMEN
In phagocytes, pathogen recognition is followed by Ca(2+) mobilization and NADPH oxidase 2 (NOX2)-mediated "oxidative burst," which involves the rapid production of large amounts of reactive oxygen species (ROS). We showed that ORAI Ca(2+) channels control store-operated Ca(2+) entry, ROS production, and bacterial killing in primary human monocytes. ROS inactivate ORAI channels that lack an ORAI3 subunit. Staphylococcal infection of mice reduced the expression of the gene encoding the redox-sensitive Orai1 and increased the expression of the gene encoding the redox-insensitive Orai3 in the lungs or in bronchoalveolar lavages. A similar switch from ORAI1 to ORAI3 occurred in primary human monocytes exposed to bacterial peptides in culture. These alterations in ORAI1 and ORAI3 abundance shifted the channel assembly toward a more redox-insensitive configuration. Accordingly, silencing ORAI3 increased the redox sensitivity of the channel and enhanced oxidation-induced inhibition of NOX2. We generated a mathematical model that predicted additional features of the Ca(2+)-redox interplay. Our results identified the ORAI-NOX2 feedback loop as a determinant of monocyte immune responses.
Asunto(s)
Canales de Calcio Activados por la Liberación de Calcio/inmunología , Calcio/inmunología , Modelos Biológicos , Monocitos/inmunología , Neumonía Estafilocócica/inmunología , Especies Reactivas de Oxígeno/inmunología , Staphylococcus aureus/inmunología , Animales , Calcio/metabolismo , Canales de Calcio Activados por la Liberación de Calcio/genética , Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Femenino , Humanos , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Ratones , Monocitos/metabolismo , Monocitos/patología , NADPH Oxidasa 2 , NADPH Oxidasas/genética , NADPH Oxidasas/inmunología , NADPH Oxidasas/metabolismo , Oxidación-Reducción , Neumonía Estafilocócica/genética , Neumonía Estafilocócica/metabolismo , Neumonía Estafilocócica/patología , Especies Reactivas de Oxígeno/metabolismo , Staphylococcus aureus/metabolismoRESUMEN
Hyperhydricity is a syndrome that causes morpho-physiological malformations in tissue culture plantlets. Micro-RNAs (miRNA) are small non-coding RNAs that play important regulatory roles in plant development, stress response, and adaptation to environmental conditions. In this study, differential expression analysis indicated that miRNAs play an underlying role in the responses to the hyperhydricity syndrome in peach Prunus persica (L.) leaves. 24 known and three novel potential miRNAs were characterized in hyperhydric and non-hyperhydric transcriptome libraries. The miRNA-target transcript analyses indicated that transport, plant cuticle development, intracellular part, and stress response are regulated by miRNAs in hyperhydric leaves. It is also suggested that miR5021 and miRnovel2 might play critical regulatory roles in hyperhydricity regarding miRNA-based response to stress. This study went one step further to advance understanding of molecular miRNA-based regulatory mechanisms regarding responses to hyperhydricity in peach.
Asunto(s)
MicroARNs/genética , Prunus persica/genética , Prunus persica/fisiología , Secuencia de Bases , Biología Computacional , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Ontología de Genes , MicroARNs/química , MicroARNs/metabolismo , Anotación de Secuencia Molecular , Conformación de Ácido Nucleico , Hojas de la Planta/genéticaRESUMEN
BACKGROUND: The influence of extracellular calcium on phagocytosis is limited, and the involvement of extracellular magnesium is unclear. AIMS: The role of extracellular calcium and magnesium on phagocytosis efficiency was investigated. RESULTS: Extracellular calcium had no influence on the internalization of 1 µm polystyrene particles by primary monocytes as has been shown before for the human lymphoma-derived, differentiated cell line U937 and murine alveolar macrophage-derived MH-S cells. In contrast, the phagocytosis by differentiated U937 cells was positively affected by the presence of extracellular magnesium whereas that of MH-S cells was not. An extracellular increase in the magnesium level did not cause an increase in the free cytosolic magnesium concentration in either cell line. In contrast to magnesium, extracellular calcium caused an increase in intracellular divalent cation levels in differentiated U937 cells. CONCLUSIONS: A phagocytosis-enhancing effect in the extracellular space was observed in relation to extracellular magnesium rather than with an intracellular increase in magnesium levels, indicating that murine and human immune cells might be regulated differently.
Asunto(s)
Citosol/metabolismo , Espacio Extracelular/metabolismo , Magnesio/metabolismo , Magnesio/farmacología , Fagocitosis/efectos de los fármacos , Animales , Calcio/metabolismo , Cationes Bivalentes/metabolismo , Línea Celular , Citometría de Flujo , Humanos , Macrófagos/efectos de los fármacos , Ratones , Monocitos/efectos de los fármacos , Células U937RESUMEN
BACKGROUND: Surfactant proteins (SP) secreted by alveolar type 2 cells, play an essential role in maintaining the air-liquid barrier of the lung and are also involved in the opsonisation and clearance of bacteria by phagocytes. We have recently described a novel surfactant protein, SP-H (SFTA3). Expression of SP-H was earlier demonstrated to be upregulated by LPS and negatively regulated by IL-1ß and IL-23 in vitro. The influence of SP-H on phagocytosis was measured using a murine and a human phagocytic cell line and fluorescent latex beads. FINDINGS: SP-H markedly increases phagocytosis in vitro in the murine-derived alveolar macrophage cell lines MH-S and in human-derived differentiated U937 cells. CONCLUSION: It can be assumed that SP-H is involved in regulating phagocytic activity of macrophages. SP-H is a new player in pulmonary host defence.
Asunto(s)
Linfocitos/efectos de los fármacos , Macrófagos Alveolares/efectos de los fármacos , Fagocitosis , Proteínas Asociadas a Surfactante Pulmonar/farmacología , Animales , Línea Celular , Relación Dosis-Respuesta a Droga , Fluorescencia , Humanos , Linfocitos/citología , Linfocitos/inmunología , Macrófagos Alveolares/citología , Macrófagos Alveolares/inmunología , Ratones , Microesferas , Especificidad de Órganos , Especificidad de la EspecieRESUMEN
BACKGROUND: Macrophages are equipped with several receptors for the recognition of foreign particles and pathogens. Upon binding to these receptors, particles become internalized. An interaction of particles with macrophage surface receptors is accompanied by an increase in cytosolic calcium concentration. This calcium is provided by intracellular stores and also by an influx of external calcium upon activation of the calcium channels. Nevertheless, the role of calcium in phagocytosis remains controversial. Some researchers postulate the necessity of calcium in Fc-receptor-mediated phagocytosis and a calcium-independent phagocytosis of complement opsonized particles. Others refute the need for calcium in Fc-receptor-mediated phagocytosis by macrophages. METHODS: In this study, the influence of external calcium concentrations and thapsigargin on the phagocytosis of polystyrene latex beads by the macrophage-like cell lines MH-S (murine) and differentiated U937 (human) was analyzed. The phagocytosis efficiency was determined by flow cytometry and was evaluated statistically by ANOVA test and Dunett's significance test, or ANOVA and Bonferroni's Multiple Comparison. RESULTS: Acquired data revealed an external calcium-independent way of internalization of non-functionalized polystyrene latex beads at free calcium concentrations ranging from 0 mM to 3 mM. The phagocytosis efficiency of the cells is not significantly decreased by a complete lack of external calcium. Furthermore, the presence of thapsigargin, known to lead to an increase of cytosolic calcium levels, did not have a significant enhancing influence on bead uptake by MH-S cells and only an enhancing effect on bead uptake by macrophage-like U937 cells at an external calcium concentration of 4 mM. CONCLUSION: The calcium-independent phagocytosis process and the decrease of phagocytosis efficiency in the presence of complement receptor inhibitor staurosporine lead to the assumption that besides other calcium independent receptors, complement receptors are also involved in the uptake of polystyrene beads. The comparison of the phagocytosis efficiencies of both cell types in bivalent cation-free HBSS buffer and in cell medium, leads to the conclusion that it is more likely that other media ingredients such as magnesium are of greater importance for phagocytosis of non-functionalized polystyrene beads than calcium.
Asunto(s)
Calcio/farmacología , Macrófagos/efectos de los fármacos , Poliestirenos/farmacología , Tapsigargina/farmacología , Animales , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Línea Celular , Humanos , Macrófagos/fisiología , Ratones , Fagocitosis/efectos de los fármacos , Células U937RESUMEN
As a response to environmental stress, bacterial cells can enter a physiological state called viable but noncultivable (VBNC). In this state, bacteria fail to grow on routine bacteriological media. Consequently, standard methods of contamination detection based on bacteria cultivation fail. Although they are not growing, the cells are still alive and are able to reactivate their metabolism. The VBNC state and low bacterial densities are big challenges for cultivation-based pathogen detection in drinking water and the food industry, for example. In this context, a new molecular-biological separation method for bacteria using point-mutated lysozymes immobilised on magnetic beads for separating bacteria is described. The immobilised mutated lysozymes on magnetic beads serve as bait for the specific capture of bacteria from complex matrices or water due to their remaining affinity for bacterial cell wall components. Beads with bacteria can be separated using magnetic racks. To avoid bacterial cell lysis by the lysozymes, the protein was mutated at amino acid position 35, leading to the exchange of the catalytic glutamate for alanine (LysE35A) and glutamine (LysE35Q). As proved by turbidity assay with reference bacteria, the muramidase activity was knocked out. The mutated constructs were expressed by the yeast Pichia pastoris and secreted into expression medium. Protein enrichment and purification were carried out by SO(3)-functionalised nanoscale cationic exchanger particles. For a proof of principle, the proteins were biotinylated and immobilised on streptavidin-functionalised, fluorescence dye-labelled magnetic beads. These constructs were used for the successful capture of Syto9-marked Microccocus luteus cells from cell suspension, as visualised by fluorescence microscopy, which confirmed the success of the strategy.