NGF Modulates trkANGFR/p75NTR in αSMA-Expressing Conjunctival Fibroblasts from Human Ocular Cicatricial Pemphigoid (OCP)

Objective

In a previous study, we reported the upregulation of Nerve Growth Factor (NGF) and trkA^NGFR expression in Ocular Cicatricial Pemphigoid (OCP), an inflammatory and remodeling eye disease. Herein, we hypothesize a potential NGF-driven mechanism on fibroblasts (FBs) during OCP remodeling events. To verify, human derived OCP-FBs were isolated and characterized either at baseline or after NGF exposure.

Materials and Methods

Conjunctival biopsies were obtained from 7 patients having OCP and 6 control subjects (cataract surgery). Both conjunctivas and primary FB cultures were characterised for αSMA, NGF and trkA^NGFR/p75^NTR expression. Subcultures were exposed to NGF and evaluated for αSMA, NGF, trkA^NGFR/p75^NTR expression as well as TGFβ1/IL4 release. For analysis, early and advanced subgroups were defined according to clinical parameters.

Results

OCP-conjunctivas showed αSMA-expressing FBs and high NGF levels. Advanced OCP-FBs showed higher αSMA expression associated with higher p75^NTR and lower trkA^NGFR expression, as compared to early counterparts. αSMA expression was in keeping with disease severity and correlated to p75^NTR. NGF exposure did not affect trkA^NGFR levels in early OCP-FBs while decreased both αSMA/p75^NTR expression and TGFβ1/IL4 release. These effects were not observed in advanced OCP-FBs.

Conclusions

Taken together, these data are suggestive for a NGF/p75^NTR task in the potential modulation of OCP fibrosis and encourages further studies to fully understand the underlying mechanism occurring in fibrosis. NGF/p75^NTR might be viewed as a potential therapeutic target.     

Intranasal Delivery of Influenza rNP Adjuvanted with c-di-AMP Induces Strong Humoral and Cellular Immune Responses and Provides Protection against Virus Challenge

There is a critical need for new influenza vaccines able to protect against constantly emerging divergent virus strains. This will be sustained by the induction of vigorous cellular responses and humoral immunity capable of acting at the portal of entry of this pathogen. In this study we evaluate the protective efficacy of intranasal vaccination with recombinant influenza nucleoprotein (rNP) co-administrated with bis-(3′,5′)-cyclic dimeric adenosine monophosphate (c-di-AMP) as adjuvant. Immunization of BALB/c mice with two doses of the formulation stimulates high titers of NP-specific IgG in serum and secretory IgA at mucosal sites. This formulation also promotes a strong Th1 response characterized by high secretion of INF-γ and IL-2. The immune response elicited promotes efficient protection against virus challenge. These results suggest that c-di-AMP is a potent mucosal adjuvant which may significantly contribute towards the development of innovative mucosal vaccines against influenza.     

Properties of Ca2+ sparks revealed by four-dimensional confocal imaging of cardiac muscle

Parameters (amplitude, width, kinetics) of Ca(2+) sparks imaged confocally are affected by errors when the spark source is not in focus. To identify sparks that were in focus, we used fast scanning (LSM 5 LIVE; Carl Zeiss) combined with fast piezoelectric focusing to acquire x-y images in three planes at 1-μm separation (x-y-z-t mode). In 3,000 x-y scans in each of 34 membrane-permeabilized cat atrial cardiomyocytes, 6,906 sparks were detected. 767 sparks were in focus. They had greater amplitude, but their spatial width and rise time were similar compared with all sparks recorded. Their distribution of amplitudes had a mode at ΔF/F(0) = 0.7. The Ca(2+) release current underlying in-focus sparks was 11 pA, requiring 20 to 30 open channels, a number at the high end of earlier estimates. Spark frequency was greater than in earlier imaging studies of permeabilized ventricular cells, suggesting a greater susceptibility to excitation, which could have functional relevance for atrial cells. Ca(2+) release flux peaked earlier than the time of peak fluorescence and then decayed, consistent with significant sarcoplasmic reticulum (SR) depletion. The evolution of fluorescence and release flux were strikingly similar for in-focus sparks of different rise time (T). Spark termination involves both depletion of Ca(2+) in the SR and channel closure, which may be synchronized by depletion. The observation of similar flux in sparks of different T requires either that channel closure and other termination processes be independent of the determinants of flux (including [Ca(2+)](SR)) or that different channel clusters respond to [Ca(2+)](SR) with different sensitivity.     

Structural analysis of ConBr reveals molecular correlation between the carbohydrate recognition domain and endothelial NO synthase activation

Diocleinae lectins are highly homologous in their primary structure which features metal binding sites and a carbohydrate recognition domain (CRD). Differences in the biological activity of legume lectins have been widely investigated using hemagglutination inhibition assays, isothermal titration microcalorimetry and co-crystallization with mono- and oligosaccharides. Here we report a new lectin crystal structure (ConBr) extracted from seeds of Canavalia brasiliensis, predict dimannoside binding by docking, identify the α-aminobutyric acid (Abu) binding pocket and compare the CRD of ConBr to that of homologous lectins. Based on the hypothesis that the carbohydrate affinity of lectins depends on CRD configuration, the relationship between tridimensional structure and endothelial NO synthase activation was used to clarify differences in biological activity. Our study established a correlation between the position of CRD amino acid side chains and the stimulation of NO release from endothelium.     

Hypothyroidism provides resistance to kidney mitochondria against the injury induced by renal ischemia-reperfusion

Massive Ca(2+) accumulation in mitochondria, plus the stimulating effect of an inducing agent, i.e., oxidative stress, induces the so-called permeability transition, which is characterized by the opening of a nonspecific pore. This work was aimed at studying the influence of thyroid hormone on the opening of such a nonspecific pore in kidney mitochondria, as induced by an oxidative stress. To meet this objective, membrane permeability transition was examined in mitochondria isolated from kidney of euthyroid and hypothyroid rats, after a period of ischemia/reperfusion. It was found that mitochondria from hypothyroid rats were able to retain accumulated Ca(2+) to sustain a transmembrane potential after Ca(2+) addition, as well as to maintain matrix NAD(+) and membrane cytochrome c content. The protective effect of hypothyroidism was clearly opposed to that occurring in ischemic reperfused mitochondria from euthyroid rats. Our findings demonstrate that these mitochondria were unable to preserve selective membrane permeability, except when cyclosporin A was added. It is proposed that the protection is conferred by the low content of cardiolipin found in the inner membrane. This phospholipid is required to switch adenine nucleotide translocase from specific carrier to a non-specific pore.     

Mathematical modelling and kinetic study for CD production catalysed by Toruzyme® and CGTase from Bacillus firmus strain 37

A new mathematical model was developed for the kinetics of α-, β- and γ-cyclodextrin production, expanding an existing model that only included the production of β- and γ-cyclodextrins, because a detailed kinetic modelling of the reactions involved allows the manipulation of the process yields. The kinetic behaviour of the commercial enzyme Toruzyme^® was studied with maltodextrin as substrate at different concentrations and for CGTase from Bacillus firmus strain 37 at a concentration of 100 g L⁻¹. The mathematical model showed a proper fit to the experimental data, within the 24-h period studied, confirming that the considered hypotheses represent the kinetic behaviour of the enzymes in the reaction medium. The kinetic parameters generated by the model allowed reproducing previous observed qualitative tendencies as it can be seen that changing experimental conditions in the reaction process such as enzyme and substrate concentrations results in large changes in the enzyme kinetics and using high substrate concentrations does not guarantee the highest conversion rates due to enzyme inhibition and reverse reactions. In addition, this new mathematical model complements previous qualitative observations enabling the manipulation of the direct and reverse reactions catalysed by the enzyme by adjusting the reaction conditions, to target quantitative results of increased productivity and better efficiency in the production of a desired cyclodextrin.

     

Mechanisms maintaining biodiversity in Mediterranean pine-oak forests: insights from a spatial simulation model

Recurrent anthropogenic and natural perturbations, resource limitations and heterogeneous environments contribute to the maintenance of a remarkable biodiversity in Mediterranean plant communities. Yet, the essential mechanisms of community assembly in these systems remain largely unexplored. In the current paper we investigate the coexistence of Aleppo pine (Pinus halepensis Mill.) and Holm oak (Quercus ilex L.), two of the most widely distributed species in the Iberian Peninsula, in relation to gradients in water availability and disturbance. A spatial model of landscape forest dynamics was implemented, calibrated with experimental data, with stands arranged on a heterogeneous lattice and coupled by dispersal. It was found that pine population persistence in the landscape can be explained independently by a competition-colonization tradeoff (in mesic homogeneous environments) and by a tradeoff between shade and drought tolerance (in heterogeneous low disturbed environments). Both mechanisms reinforce mutually to maintain a shifting mosaic of both taxa along disturbance and aridity gradients. This view is consistent with palynological, historical and forest inventory records. In turn, equilibrium theories of vegetation dynamics (phytosociological), that neglect the role of heterogeneity and disturbances may be inadequate for Mediterranean forests, and have been shown to result in mismanagement practices. We claim that biologically informed models of forest dynamics are desperately needed as diagnosis tools for sustainable forest management.     

Hydrolysis of triacetin catalyzed by immobilized lipases: effect of the immobilization protocol and experimental conditions on diacetin yield

The effect of the immobilization protocol and some experimental conditions (pH value and presence of acetonitrile) on the regioselective hydrolysis of triacetin to diacetin catalyzed by lipases has been studied. Lipase B from Candida antarctica (CALB) and lipase from Rhizomucor miehei (RML) were immobilized on Sepabeads (commercial available macroporous acrylic supports) activated with glutaraldehyde (covalent immobilization) or octadecyl groups (adsorption via interfacial activation). All the biocatalysts accumulated diacetin. Covalently immobilized RML was more active towards rac-methyl mandelate than the adsorbed RML. However, this covalent RML preparation presented the lowest activity towards triacetin. For this reason, this preparation was discarded as biocatalyst for this reaction. At pH 7, acyl migration occurred giving a mixture of 1,2 and 1,3 diacetin, but at pH 5.5, only 1,2 diacetin was produced. Yields were improved at acidic pH values and in the presence of 20% acetonitrile (to over 95%). RML immobilized on octadecyl Sepabeads was proposed as optimal preparation, mainly due to its higher specific activity. Each enzyme preparation presented very different properties. Moreover, changes in the reaction conditions affected the various immobilized enzymes in a different way.