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BackgroundNeutrophils are involved in the pathophysiology of allergic asthma, where the Eosinophil Cationic Protein(ECP) is a critical inflammatory mediator. Although ECP production is attributed to eosinophils, we reported that ECP is also present in neutrophils from allergic patients where, in contrast to eosinophils, it is produced in an IgE-dependent manner. Given the key role of ECP in asthma, we investigated the molecular mechanisms involved in ECP production as well as the effects induced by agonists and widely used clinical approaches. We also analyzed the correlation between ECP production and lung function.MethodsNeutrophils from allergic asthmatic patients were challenged with allergens, alone or in combination with cytokines, in the presence of cell-signaling inhibitors and clinical drugs. We analyzed ECP levels by ELISA and confocal microscopy. Lung function was assessed by spirometry.ResultsIgE-mediated ECP release is dependent on phosphoinositide 3-kinase, the extracellular signal-regulated kinase (ERK1/2) and the production of reactive oxygen species by NADPH-oxidase. Calcineurin phosphatase and the transcription factor NFAT are also involved. ECP release is enhanced by the cytokines interleukin (IL)-5 and granulocyte macrophage-colony stimulating factor, and inhibited by interferon-γ, IL-10, clinical drugs (formoterol, tiotropium and budesonide) and allergen-specific IT. We also found an inverse correlation between asthma severity and ECP levels.ConclusionsOur results suggest the molecular pathways involved in ECP production and potential therapeutic targets. We also provide a new method to evaluate disease severity in asthmatic patients based on the quantification ofin vitroECP production by peripheral neutrophils.

Histamine is a critical inflammatory mediator in allergic diseases. We showed in a previous work that neutrophils from allergic patients produce histamine in response to allergens to which the patients were sensitized. Here, we investigate the molecular mechanisms involved in this process using peripheral blood neutrophils. We challenged these cells in vitro with allergens and analyzed histamine release in the culture supernatants. We also explored the effect of common therapeutic drugs that ameliorate allergic symptoms, as well as allergen-specific immunotherapy. Additionally, we examined the expression of histidine decarboxylase and diamine oxidase, critical enzymes in the metabolism of histamine, under allergen challenge. We show that allergen-induced histamine release is dependent on the activation of the phosphoinositide 3-kinase, mitogen-activated protein kinase p38, and extracellular signal-regulated kinase 1/2 signaling pathways. We also found a contribution of the phosphatase calcineurin to lesser extent. Anti-histamines, glucocorticoids, anti-M3-muscarinic receptor antagonists, and mainly β2 -receptor agonists abolished the allergen-dependent histamine release. Interestingly, allergen-specific immunotherapy canceled the histamine release through the downregulation of histidine decarboxylase expression. Our observations describe novel molecular mechanisms involved in the allergen-dependent histamine release by human neutrophils and provide new targets to inhibit histamine production.

The aim of our work was the isolation and characterization of bacteria from the rhizosphere of Spartina maritima in the metal contaminated Odiel estuary (Huelva, SW Spain). From 25 strains, 84 % were identified as gram-positive, particularly Staphylococcus and Bacillus. Gram-negative bacteria were represented by Pantoea and Salmonella. Salt and heavy metal tolerance, metal bioabsorption, plant growth promoting (PGP) properties, and biofilm formation were investigated in the bacterial collection. Despite the higher abundance of gram-positive bacteria, gram-negative isolates displayed higher tolerance toward metal(loid)s (As, Cu, Zn, and Pb) and greater metal biosorption, as deduced from ICP-OES and SEM-EDX analyses. Besides, they exhibited better PGP properties, which were retained in the presence of metals and the ability to form biofilms. Gram-negative strains Pantoea agglomerans RSO6 and RSO7, together with gram-positive Bacillus aryabhattai RSO25, were selected for a bacterial consortium aimed to inoculate S. maritima plants in metal polluted estuaries for phytoremediation purposes.

The rhizobia-legume interaction has been proposed as an interesting and appropriate tool for rhizostabilization of soils contaminated with heavy metals. One of the main requirements to use this symbiosis is the availability of tolerant and symbiotically effective rhizobia. The aim of this work was to improve the symbiotic properties of the arsenic-resistant wild-type strain Ensifer medicae MA11 in Cu-contaminated substrates. The copAB genes from a Cu-resistant Pseudomonas fluorescens strain were expressed in E. medicae MA11 under the control of the nifH promoter. The resulting strain E. medicae MA11-copAB was able to alleviate the toxic effect of Cu in Medicago truncatula. At 300 µM Cu, root and shoot dry matter production, nitrogen content, number of nodules and photosynthetic rate were significantly reduced in plants inoculated with the wild-type strain. However, these parameters were not altered in plants inoculated with the genetically modified strain. Moreover, nodules elicited by this strain were able to accumulate twofold the Cu measured in nodules formed by the wild-type strain. In addition, the engineered E. medicae strain increased Cu accumulation in roots and decreased the content in shoots. Thus, E. medicae MA11-copAB increased the capacity of M. truncatula to rhizostabilize Cu, decreasing the translocation factor and avoiding metal entry into the food chain. The plasmid containing the nifH promoter-copAB construct could be a useful biotool for Cu rhizostabilization using legumes, since it can be transferred to different rhizobia microsymbionts of authoctonous legumes growing in Cu-contaminated soils.

Metal contamination of estuaries is a severe environmental problem, for which phytoremediation is gaining momentum. In particular, the associations between halophytes-autochthonous rhizobacteria have proven useful for metal phytostabilization in salt marshes. In this work, three bacterial strains (gram-negative and gram-positive) were used for Spartina densiflora inoculation. All three bacteria, particularly Pantoea strains, promoted plant growth and mitigated metal stress on polluted sediments, as revealed from functionality of the photosynthetic apparatus (PSII) and maintenance of nutrient balance. Pantoea strains did not significantly affect metal accumulation in plant roots, whereas the Bacillus strain enhanced it. Metal loading to shoots depended on particular elements, although in all cases it fell below the threshold for animal consumption. Our results confirm the possibility of modulating plant growth and metal accumulation upon selective inoculation, and the suitability of halophyte-rhizobacteria interactions as biotechnological tools for metal phytostabilization in salt marshes, preventing metal transfer to the food chain.

Excess copper (Cu) in soils has deleterious effects on plant growth and can pose a risk to human health. In the last decade, legume-rhizobium symbioses became attractive biotechnological tools for metal phytostabilization. For this technique being useful, metal-tolerant symbionts are required, which can be generated through genetic manipulation.In this work, a double symbiotic system was engineered for Cu phytostabilization: On the one hand, composite Medicago truncatula plants expressing the metallothionein gene mt4a from Arabidopsis thaliana in roots were obtained to improve plant Cu tolerance. On the other hand, a genetically modified Ensifer medicae strain, expressing copper resistance genes copAB from Pseudomonas fluorescens driven by a nodulation promoter, nifHp, was used for plant inoculation. Our results indicated that expression of mt4a in composite plants ameliorated plant growth and nodulation and enhanced Cu tolerance. Lower levels of ROS-scavenging enzymes and of thiobarbituric acid reactive substances (TBARS), such as malondialdehyde (a marker of lipid peroxidation), suggested reduced oxidative stress. Furthermore, inoculation with the genetically modified Ensifer further improved root Cu accumulation without altering metal loading to shoots, leading to diminished values of metal translocation from roots to shoots. The double modified partnership is proposed as a suitable tool for Cu rhizo-phytostabilization.

The aim of this work was to express the seed-specific metallothionein 4 (mt4a) gene in Arabidopsis thaliana vegetative tissues, in order to study its effect on plant Cu and Zn tolerance and accumulation. In that way, mt4a gene was expressed under the regulation of the 35S CaMV promoter in A. thaliana plants. Expression of mt4a gene in vegetative tissues at different developmental stages conferred increased plant tolerance towards Cu and Zn, as concluded from root length measurement and shoot biomass determination. However, our results suggest that expression of mt4a gene in vegetative tissues of A. thaliana plants leads to a slight increase in plant Cu accumulation and do not increase Zn content in the plants.

The aim of this work was to establish the conditions for using Ochrobactrum cytisi Azn6.2 as a metal biosorbent. Azn6.2 is a novel strain from the legume symbiont O. cytisi that has been isolated from nodules of Medicago polymorpha plants grown on heavy metal-polluted soils. Compared with the strain ESC1, Azn6.2 showed some biochemical differences, as well as antibiotic susceptibility, Azn6.2 was multi-resistant to heavy metals, such as Cu, Cd and Zn, and bacterial pellets were able to biosorb high amounts of Cd and Zn. As shown by scanning electron microscopy coupled to energy dispersive X-ray, most of Cd was attached to the cell surface. Optimal conditions for Cd biosorption were established, being 1 mM Cd ions in solution and 2 h of contact with the biosorbent at room temperature. At these conditions, maximal Cd loading capacity reached 32–34 mg/g. Cd desorption from bacterial pellets was achieved after washing with EDTA or, at higher efficiency, at pH 1.0. These results indicated that biosorption/desorption on O. cytisi Azn6.2 biomass should be a cost-effective method for Cd recovery from contaminated solutions.

La survenue d'une grossesse en hémodialyse chronique (HDC) est rare, mais depuis la description du premier cas par Confortini en 1971, plusieursobservations ont été rapportées. L'hémodialyse a considérablement amélioré la fertilité de ces patientes. Nous rapportons l'expérience de douze grossesses survenues entre 1999 et 2014, chez douze patientes d'âge médian 34 ans (22-44), en hémodialyse (HD) depuis 40 mois (3-72), l'âge gestationnel moyen de diagnostic est de 16 semaines d'aménorrhée, la grossesse était compliquée dans 50% des cas par un hydramnios. Le terme moyen est de 35 semaine d'aménorrhée (SA) et l'accouchement a été réalisé dans 90% des grossesses par voie basse. Le poids moyen des nouveau-nés est de 1800g. De telles grossesses sont à haut risque du fait de la fréquence des complications. Elles devraient être contrôlées par les équipes multidisciplinaires, et la consultation prénatal ne devrait pas être négligée. L'objectif de ce travail est de rapporter notre expérience concernant la survenue d'une grossesse chez les patientes dialysées et de la confronter aux données de la littérature.

Summary The genetic regulation underlying the effect of arsenic (As(III)) on the model symbiosis Medicago–Ensifer was investigated using a combination of physiological (split-roots), microscopy and genetic (microarrays, qRT-PCR and composite plants) tools. Nodulation was very sensitive to As(III) (median inhibitory dose (ID50) = 20 μM). The effect on root elongation and on nodulation was local (nonsystemic). A battery of stress (salt, drought, heat shock, metals, etc.)-related genes were induced. Glutathione played a pivotal role in tolerance/detoxification, together with secondary metabolites ((iso)flavonoids and phenylpropanoids). However, antioxidant enzymes were not activated. Concerning the symbiotic interaction, molecular evidence suggesting that rhizobia alleviate As stress is for the first time provided. Chalcone synthase (which is involved in the first step of the legume–rhizobia cross-talk) was strongly enhanced, suggesting that the plants are biased to establish symbiotic interactions under As(III) stress. In contrast, 13 subsequent nodulation genes (involved in nodulation factors (Nod factors) perception, infection, thread initiation and progression, and nodule morphogenesis) were repressed. Overexpression of the ethylene responsive factor ERN in composite plants reduced root stress and partially restored nodulation, whereas overexpression of the early nodulin ENOD12 enhanced nodulation both in the presence and, particularly, in the absence of As, without affecting root elongation. Several transcription factors were identified, which could be additional targets for genetic engineering aiming to improve nodulation and/or alleviate root stress induced by this toxic.

In the salt marshes of the joint estuary of Tinto and Odiel rivers (SW Spain), one of the most polluted areas by heavy metals in the world, Spartina densiflora grows on sediments with high concentrations of heavy metals. Furthermore, this species has shown to be useful for phytoremediation. The total bacterial population of the rhizosphere of S. densiflora grown in two estuaries with different levels of metal contamination was analyzed by PCR denaturing gradient gel electrophoresis. Results suggested that soil contamination influences bacterial population in a greater extent than the presence of the plant. Twenty-two different cultivable bacterial strains were isolated from the rhizosphere of S. densiflora grown in the Tinto river estuary. Seventy percent of the strains showed one or more plant growth-promoting (PGP) properties, including phosphate solubilization and siderophores or indolacetic acid production, besides a high resistance towards Cu. A bacterial consortium with PGP properties and very high multiresistance to heavy metals, composed by Aeromonas aquariorum SDT13, Pseudomonas composti SDT3, and Bacillus sp. SDT14, was selected for further experiments. This consortium was able to two-fold increase seed germination and to protect seeds against fungal contamination, suggesting that it could facilitate the establishment of the plant in polluted estuaries.

In this work, engineering Cu-hyperaccumulation in plants was approached. First, the copC gene from Pseudomonas sp. Az13, encoding a periplasmic Cu-binding protein, was expressed in Arabidopsis thaliana driven by the CaMV35S promoter (transgenic lines 35S-copC). 35S-copC lines showed up to 5-fold increased Cu accumulation in roots (up to 2000 μg Cu. g(-1)) and shoots (up to 400 μg Cu. g(-1)), compared to untransformed plants, over the limits established for Cu-hyperaccumulators. 35S lines showed enhanced Cu sensitivity. Second, copC was engineered under the control of the cab1 (chlorophyll a/b binding protein 1) promoter, in order to drive copC expression to the shoots (transgenic lines cab1-copC). cab1-copC lines showed increased Cu translocation factors (twice that of wild-type plants) and also displayed enhanced Cu sensitivity. Finally, subcellular targeting the CopC protein to plant vacuoles was addressed by expressing a modified copC gene containing specific vacuole sorting determinants (transgenic lines 35S-copC-V). Unexpectedly, increased Cu-accumulation was not achieved-neither in roots nor in shoots-when compared to 35S-copC lines. Conversely, 35S-copC-V lines did display greatly enhanced Cu-hypersensitivity. Our results demonstrate the feasibility of obtaining Cu-hyperaccumulators by engineering a prokaryotic Cu-binding protein, but they highlight the difficulty of altering the exquisite Cu homeostasis in plants.