Gene Deficiency in Activating Fcγ Receptors Influences the Macrophage Phenotypic Balance and Reduces Atherosclerosis in Mice

Immunity contributes to arterial inflammation during atherosclerosis. Oxidized low-density lipoproteins induce an autoimmune response characterized by specific antibodies and immune complexes in atherosclerotic patients. We hypothesize that specific Fcγ receptors for IgG constant region participate in atherogenesis by regulating the inflammatory state of lesional macrophages. In vivo we examined the role of activating Fcγ receptors in atherosclerosis progression using bone marrow transplantation from mice deficient in γ-chain (the common signaling subunit of activating Fcγ receptors) to hyperlipidemic mice. Hematopoietic deficiency of Fcγ receptors significantly reduced atherosclerotic lesion size, which was associated with decreased number of macrophages and T lymphocytes, and increased T regulatory cell function. Lesions of Fcγ receptor deficient mice exhibited increased plaque stability, as evidenced by higher collagen and smooth muscle cell content and decreased apoptosis. These effects were independent of changes in serum lipids and antibody response to oxidized low-density lipoproteins. Activating Fcγ receptor deficiency reduced pro-inflammatory gene expression, nuclear factor-κB activity, and M1 macrophages at the lesion site, while increasing anti-inflammatory genes and M2 macrophages. The decreased inflammation in the lesions was mirrored by a reduced number of classical inflammatory monocytes in blood. In vitro, lack of activating Fcγ receptors attenuated foam cell formation, oxidative stress and pro-inflammatory gene expression, and increased M2-associated genes in murine macrophages. Our study demonstrates that activating Fcγ receptors influence the macrophage phenotypic balance in the artery wall of atherosclerotic mice and suggests that modulation of Fcγ receptor-mediated inflammatory responses could effectively suppress atherosclerosis.     

Partitioning of water and nitrogen in co-occurring Mediterranean woody shrub species of different evolutionary history

We studied the interspecific and intraspecific variation in the development of water stress and in the use of different water and nitrogen sources during the spring (wet season) and summer (dry season) in a shrub community in NE Spain. We measured shoot water potentials, stable deuterium isotopic composition (D) of xylem sap, leaf mass per area, leaf N and C concentrations, gas exchange, leaf ¹³C, and leaf ¹⁵N of the dominant species (Quercus coccifera, Arbutus unedo, Pistacia lentiscus, Erica multiflora, Globularia alypum). The D, the ¹³C and the shoot water potential values showed diurnal, seasonal, intraspecific and interspecific variation in the source and use of water. There was also seasonal, intraspecific and interspecific variation in the foliar ¹⁵N and N concentrations. In summer, some species (A. unedo, P. lentiscus and E. multiflora) presented significantly different D values in morning and afternoon measurements likely indicating that they used different sources of water during the day, and a dual root system in these species. We conjecture that dew may be one of these water sources. Species predawn water potential was negatively correlated with species xylem water D. There was also a positive correlation between ¹³C and D in P. lentiscus, species for which we took additional samples from nearby sites. These results suggest that the access to water from greater depths allowed the maintenance of more favourable plant water supply. Multivariate principal component analysis based on the studied hydrological and isotope variables clearly separated the seasons (wet spring and dry summer) and the species. The species resulted separated according to their evolutionary history (Pre-Mediterranean and Mediterranean) and the associated root and functional traits. These results show water (and nitrogen) partitioning among coexisting species of the same functional type (Mediterranean woody shrubs). They also show the great intraspecific plasticity of responses to resource availability.     

Diurnal and seasonal variations in the photosynthetic performance and water relations of two co-occurring Mediterranean shrubs,Erica multiflora and Globularia alypum

Diurnal and seasonal fluctuations in the photosynthetic performance and water relations of two co-occurring Mediterranean shrubs, Erica multiflora and Globularia alypum were monitored throughout two consecutive years at Garraf Natural Park in north-east Spain. Leaf gas exchange rates, chlorophyll fluorescence and shoot water potentials were measured once each season. Leaf nitrogen and carbon concentrations, leaf delta13C and delta15N and specific leaf area (SLA) were also measured once a year (August) on well developed mature leaves. Globularia alypum experienced seasonal fluctuations in their water potential, with the lowest values recorded in summer, whereas E. multiflora did not show significant differences in water potential among seasons. Moreover, lower water potentials were found in G. alypum than in E. multiflora throughout the entire study, suggesting that the latter behaved as a drought-avoiding species, whereas the former tolerated lower water potentials. In both species, maximum leaf gas exchange rates were observed in autumn and secondarily in spring; in contrast, photosynthetic and transpiration rates reached absolute minima in summer. The stronger fluctuations in water potential and leaf gas exchange rates found in G. alypum compared to E. multiflora, suggest that G. alypum is, sensu Levitt (1980), a water spender, whereas E. multiflora is a water conservative. This hypothesis is further supported by a higher integrated water-use efficiency (higher delta13C values) and a higher degree of sclerophylly (lower SLA) in E. multiflora in comparison with G. alypum. Globularia alypum showed higher leaf gas exchange rates and higher predawn potential photochemical efficiency (Fv/Fm) than E. multiflora during most of the study. In spring and autumn, predawn Fv/Fm values were within the optimal range, whereas chronic photoinhibition in summer and winter was detected in both species. However, whereas both species could maintain positive photosynthetic rates in winter, frequent negative values were found in summer, suggesting higher levels of stress during the drought period. These results together with the high correlations that were found between the net photosynthetic rates and several parameters of water availability (accumulated rainfall, soil moisture or midday water potential) provided further evidence of the key role of water availability in the regulation of the photosynthetic rates in these Mediterranean species. Warmer and drier conditions in future decades, as a consequence of climate change, may alter the present, slight competitive advantage of G. alypum and the fitness of both shrub species within semi-arid Mediterranean environments.     

Endogenous lipid- and peptide-derived anti-inflammatory pathways generated with glucocorticoid and aspirin treatment activate the lipoxin A4 receptor

Aspirin (ASA) and dexamethasone (DEX) are widely used anti-inflammatory agents yet their mechanism(s) for blocking polymorphonuclear neutrophil (PMN) accumulation at sites of inflammation remains unclear. Here, we report that inhibition of PMN infiltration by ASA and DEX is a property shared by aspirin-triggered lipoxins (ATL) and the glucocorticoid-induced annexin 1 (ANXA1)-derived peptides that are both generated in vivo and act at the lipoxin A(4) receptor (ALXR/FPRL1) to halt PMN diapedesis. These structurally diverse ligands specifically interact directly with recombinant human ALXR demonstrated by specific radioligand binding and function as well as immunoprecipitation of PMN receptors. In addition, the combination of both ATL and ANXA1-derived peptides limited PMN infiltration and reduced production of inflammatory mediators (that is, prostaglandins and chemokines) in vivo. Together, these results indicate functional redundancies in endogenous lipid and peptide anti-inflammatory circuits that are spatially and temporally separate, where both ATL and specific ANXA1-derived peptides act in concert at ALXR to downregulate PMN recruitment to inflammatory loci.     

Non-adenine based purines accelerate wound healing

Wound healing is a complex sequence of cellular and molecular processes that involves multiple cell types and biochemical mediators. Several growth factors have been identified that regulate tissue repair, including the neurotrophin nerve growth factor (NGF). As non-adenine based purines (NABPs) are known to promote cell proliferation and the release of growth factors, we investigated whether NABPs had an effect on wound healing. Full-thickness, excisional wound healing in healthy BALB/c mice was significantly accelerated by daily topical application of NABPs such as guanosine (50% closure by days 2.5–2.8). Co-treatment of wounds with guanosine plus anti-NGF reversed the guanosine-promoted acceleration of wound healing, indicating that this effect of guanosine is mediated, at least in part, by NGF. Selective inhibitors of the NGF-inducible serine/threonine protein kinase (protein kinase N), such as 6-methylmercaptopurine riboside abolished the acceleration of wound healing caused by guanosine, confirming that activation of this enzyme is required for this effect of guanosine. Treatment of genetically diabetic BKS.Cg-m+/+lepr db mice, which display impaired wound healing, with guanosine led to accelerated healing of skin wounds (25% closure by days 2.8–3.0). These results provide further confirmation that the NABP-mediated acceleration of cutaneous wound healing is mediated via an NGF-dependent mechanism. Thus, NABPs may offer an alternative and viable approach for the treatment of wounds in a clinical setting.     

Glucokinase (GCK) mutations and their characterization in MODY2 children of southern Italy

Type 2 Maturity Onset Diabetes of the Young (MODY2) is a monogenic autosomal disease characterized by a primary defect in insulin secretion and hyperglycemia. It results from GCK gene mutations that impair enzyme activity. Between 2006 and 2010, we investigated GCK mutations in 66 diabetic children from southern Italy with suspected MODY2. Denaturing High Performance Liquid Chromatography (DHPLC) and sequence analysis revealed 19 GCK mutations in 28 children, six of which were novel: p.Glu40Asp, p.Val154Leu, p.Arg447Glyfs, p.Lys458_Cys461del, p.Glu395_Arg397del and c.580-2A>T. We evaluated the effect of these 19 mutations using bioinformatic tools such as Polymorphism Phenotyping (Polyphen), Sorting Intolerant From Tolerant (SIFT) and in silico modelling. We also conducted a functional study to evaluate the pathogenic significance of seven mutations that are among the most severe mutations found in our population, and have never been characterized: p.Glu70Asp, p.His137Asp, p.Phe150Tyr, p.Val154Leu, p.Gly162Asp, p.Arg303Trp and p.Arg392Ser. These seven mutations, by altering one or more kinetic parameters, reduced enzyme catalytic activity by >40%. All mutations except p.Glu70Asp displayed thermal-instability, indeed >50% of enzyme activity was lost at 50°C/30 min. Thus, these seven mutations play a pathogenic role in MODY2 insurgence. In conclusion, this report revealed six novel GCK mutations and sheds some light on the structure-function relationship of human GCK mutations and MODY2.     

Oxidation of the Tryptophan 32 Residue of Human Superoxide Dismutase 1 Caused by Its Bicarbonate-dependent Peroxidase Activity Triggers the Non-amyloid Aggregation of the Enzyme

The role of oxidative post-translational modifications of human superoxide dismutase 1 (hSOD1) in the amyotrophic lateral sclerosis (ALS) pathology is an attractive hypothesis to explore based on several lines of evidence. Among them, the remarkable stability of hSOD1^WT and several of its ALS-associated mutants suggests that hSOD1 oxidation may precede its conversion to the unfolded and aggregated forms found in ALS patients. The bicarbonate-dependent peroxidase activity of hSOD1 causes oxidation of its own solvent-exposed Trp³² residue. The resulting products are apparently different from those produced in the absence of bicarbonate and are most likely specific for simian SOD1s, which contain the Trp³² residue. The aims of this work were to examine whether the bicarbonate-dependent peroxidase activity of hSOD1 (hSOD1^WT and hSOD1^G93A mutant) triggers aggregation of the enzyme and to comprehend the role of the Trp³² residue in the process. The results showed that Trp³² residues of both enzymes are oxidized to a similar extent to hSOD1-derived tryptophanyl radicals. These radicals decayed to hSOD1-N-formylkynurenine and hSOD1-kynurenine or to a hSOD1 covalent dimer cross-linked by a ditryptophan bond, causing hSOD1 unfolding, oligomerization, and non-amyloid aggregation. The latter process was inhibited by tempol, which recombines with the hSOD1-derived tryptophanyl radical, and did not occur in the absence of bicarbonate or with enzymes that lack the Trp³² residue (bovine SOD1 and hSOD1^W32F mutant). The results support a role for the oxidation products of the hSOD1-Trp³² residue, particularly the covalent dimer, in triggering the non-amyloid aggregation of hSOD1.     

HLA supratypes in an Italian population

A supratype analysis of a North Italian population was performed, using 16 polymorphisms in the HLA region spanning the HLA-A-DP segment. Fourteen supratypes were identified, mostly corresponding to those found in other Caucasiod populations. The degree of their conservation both within the B-DR/DQ region and in the regions telomeric and centromeric from HLA-A and DP was evaluated and linkage disequilibria among several DR and DP alleles were identified. Notably, the degree of association with DP increased when the DR marker was part of a conserved B-DR/DQ supratype. These data are relevant to the definition of the genetic structure of the population and to the prediction of probabilities of histocompatibility matching between unrelated individuals.     

Methanol as a signal triggering isoprenoid emissions and photosynthetic performance in <Emphasis Type="Italic">Quercus ilex</Emphasis>

Several volatile organic compounds (VOCs) have been reported as having a communication role between plants and also between plants and animals. We aimed to test whether methanol, a short-chain oxygenated VOC, could also have a signalling role between plants. We monitored photosynthetic performance and VOC exchange rates of Quercus ilex L. saplings before and after two different treatments: (a) clipping of some leaves to simulate an attack by herbivores and (b) fumigation with gaseous methanol for 5 h to simulate the amount of methanol a plant could receive from surrounding plants if those had been already attacked by herbivores. The clipping treatment enhanced the photosynthetic rates, the chlorophyll a to b ratio and the carotenoid to chlorophyll ratio of non-clipped leaves, suggesting an activation of plant protective metabolism. Also, a small but interesting systemic (in non-clipped leaves) increase in methanol emission rates was observed, which agrees with the possibility that methanol may act as a signalling cue. The methanol fumigation treatment induced an increase in the actual photochemical efficiency of PSII and also in the carotenoid to chlorophyll ratio. Methanol fumigation also promoted a 14% increase in the monoterpene emission rate, 1 day after the treatment, a similar response to the ones induced by other signalling VOCs. The enhanced monoterpene emissions could add to the blend of VOCs emitted after stress and be part of further signalling pathways, thus forwarding the message started by methanol. This study suggests that clipping and methanol fumigation at natural concentrations elicit significant neighbour plant physiological responses and further BVOC emissions.