Synergy between Ficolin-2 and Pentraxin 3 Boosts Innate Immune Recognition and Complement Deposition

The long pentraxin 3 (PTX3) is a multifunctional soluble pattern recognition molecule that is crucial in innate immune protection against opportunistic fungal pathogens such as Aspergillus fumigatus. The mechanisms that mediate downstream effects of PTX3 are largely unknown. However, PTX3 interacts with C1q from the classical pathway of the complement. The ficolins are recognition molecules of the lectin complement pathway sharing structural and functional characteristics with C1q. Thus, we investigated whether the ficolins (Ficolin-1, -2, and -3) interact with PTX3 and whether the complexes are able to modulate complement activation on A. fumigatus. Ficolin-2 could be affinity-isolated from human plasma on immobilized PTX3. In binding studies, Ficolin-1 and particularly Ficolin-2 interacted with PTX3 in a calcium-independent manner. Ficolin-2, but not Ficolin-1 and Ficolin-3, bound A. fumigatus directly, but this binding was enhanced by PTX3 and vice versa. Ficolin-2-dependent complement deposition on the surface of A. fumigatus was enhanced by PTX3. A polymorphism in the FCN2 gene causing a T236M amino acid change in the fibrinogen-like binding domain of Ficolin-2, which affects the binding to GlcNAc, reduced Ficolin-2 binding to PTX3 and A. fumigatus significantly. These results demonstrate that PTX3 and Ficolin-2 may recruit each other on pathogens. The effect was alleviated by a common amino acid change in the fibrinogen-like domain of Ficolin-2. Thus, components of the humoral innate immune system, which activate different complement pathways, cooperate and amplify microbial recognition and effector functions.The ficolins are multimeric collagen-like proteins consisting of an N-terminal domain, a collagen-like domain (CD),² and a C-terminal fibrinogen-like (FBG) domain involved in innate immune defense (1, 2). In humans, three types of ficolins have been identified as follows: Ficolin-1 (M-ficolin), Ficolin-2 (L-ficolin), and Ficolin-3 (H-ficolin/Hakata antigen). They function as recognition molecules in the lectin complement pathway along with mannose-binding lectin but with differentiated complement activating capacity (3). Ficolin-2 and Ficolin-3 circulate in the blood with a median concentration of 5 and 25 μg/ml, respectively (4, 5). Ficolin-2 is mainly produced in the liver, whereas Ficolin-3 is synthesized in both the liver and lungs, with the highest expression in the lungs (3). Ficolin-1 is primarily expressed by bone marrow-derived cells and lung epithelial cells (3, 6–8) and has recently been shown to be present in the blood with a median plasma concentration of 60 ng/ml (9). The ficolin genes (FCN1, -2, and -3) are polymorphic, and particularly polymorphisms in FCN2 regulate both the level and function of Ficolin-2 (4, 10, 11). In this respect, a base substitution in exon 8 at position 6359 (C→T) causing a threonine to be replaced by a methionine (T236M) in the FBG domain of Ficolin-2 has been shown to cause decreased binding activity toward GlcNAc (10).Ficolin-1 has been reported to bind to GlcNAc, GalNAc, and sialic acid (8, 12). It may opsonize Staphylococcus aureus via GlcNAc and interact with a smooth-type strain of Salmonella typhimurium through an unknown ligand, the binding of which is not diminished by GlcNAc (8). Ficolin-2 has been shown to recognize specific pathogen-associated molecular patterns, which are typically located in pathogen cell membranes, such as lipoteichoic acid and peptidoglycan in Gram-positive bacteria cell walls, lipopolysaccharide in Gram-negative bacteria cell walls, and 1,3-β-d-glucan in yeast and fungal cell walls (13, 14). The ligand specificity of Ficolin-2 has also been defined as acetyl groups, including those of N-acetylmannosamine, GlcNAc, GalNAc as well as acetyl groups on cysteine, glycine, and choline (15). Ficolin-2 recognizes clinically important pathogens, like S. typhimurium, S. aureus, and Streptococcus pneumoniae (13, 16, 17). Ficolin-3 shows affinity for GlcNAc, GalNAc, and d-fucose and may interact with S. typhimurium, Salmonella minnesota, and Aerococcus viridans (17, 18).The long pentraxin 3 (PTX3) is a soluble pattern recognition molecule mediating innate immune recognition (19). PTX3 is a glycoprotein of 45 kDa, which assembles into an octameric structure through protomer linkage by disulfide bonds (20). PTX3 shares C-terminal structural similarity with the classic short pentraxins, C-reactive protein (CRP), and serum amyloid P component, whereas the N-terminal sequence differs from the other proteins (21). Myeloid cells are a major source of PTX3, but PTX3 has also been shown in vitro to be produced by a variety of cells in response to inflammatory signals (21). During inflammation PTX3 is rapidly up-regulated and released into the surrounding tissue and into the bloodstream. PTX3 interacts with C1q and participates in activation of the classical complement pathway (22, 23). Moreover, it has also been shown that PTX3 binds the complement regulatory factor H and that this interaction regulates the alternative pathway of complement (24).PTX3 can interact with a number of different pathogens, bacteria as well as fungi and viruses. A specific binding has been observed for selected Gram-positive and Gram-negative bacteria, including S. aureus, Pseudomonas aeruginosa, S. typhimurium, Klebsiella pneumoniae, S. pneumoniae, and Neisseria meningitidis (21). PTX3 also binds zymosan and conidia from Aspergillus fumigatus) (25). Furthermore, it has been shown that ptx3 knock-out mice are extremely susceptible to invasive pulmonary aspergillosis. The phenotypic defect can be completely reversed by treatment with recombinant PTX3 (25, 26). These data indicate that PTX3 is important in protection against A. fumigatus, which has become a major cause of morbidity in medical institutions because of the increasing number of immunosuppressed patients (27).Based on the knowledge of the structural and functional similarities between C1q and the ficolins, this study was designed to characterize a possible interaction between the ficolins and PTX3 using A. fumigatus as a model. Based on our data, we propose an important role for previously unlinked collaboration of PTX3 and Ficolin-2, but not Ficolin-1 and Ficolin-3, in the recognition of A. fumigatus and amplification of complement activation. Moreover, our results demonstrate functional consequences of the Ficolin-2 T236M substitution in the interaction between PTX3 and A. fumigatus.     

Protective effects of β-glucan extracted from Agaricus brasiliensis against chemically induced DNA damage in human lymphocytes

β-Glucans (BGs) are polysaccharides that are found in the cell walls of organisms such as bacteria, fungi, and some cereals. The objective of the present study was to investigate the genotoxic and antigenotoxic effects of BG extracted from the mushroom Agaricus brasiliensis (= Agaricus blazei Murrill ss. Heinemann). The mutagenic activity of BG was tested in single-cell gel electrophoresis assays with human peripheral lymphocytes. In addition, the protective effects against the cooked food mutagen 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and (+/−)-anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE), which is the main metabolite of B[a]P, and against ROS (H₂O₂)-induced DNA damage, were studied. The results showed that the compound itself was devoid of mutagenic activity, and that a significant dose-dependent protective effect against damage induced by hydrogen peroxide and Trp-P-2 occurred in the dose range 20–80 μg/ml. To investigate the prevention of Trp-P-2-induced DNA damage, a binding assay was carried out to determine whether BG inactivates the amine via direct binding. Since no such interactions were observed, it is likely that BG interacts with enzymes involved in the metabolism of the amine.     

Preparation of ready-to-use, storable and reconstituted type I collagen from rat tail tendon for tissue engineering applications

Collagen is a widely investigated extracellular matrix material with extensive potentials in the field of tissue engineering. This protocol describes a method to prepare reconstituted collagen that can be ready-to-use, storable and suitable for further in vitro and in vivo investigations. Type I collagen was extracted from rat tail tendons and processed in acetic acid solution to obtain sterile soluble collagen. At first, crude collagen was dissolved in acetic acid, then frozen at -20 degrees C and lyophilized to obtain a sponge, which could be stored at -80 degrees C. Lyophilized collagen was then dispersed in acetic acid to obtain a sterile solution of collagen at targeted concentrations. The whole low-cost process from the extraction to the final sterile solution takes around 2-3 weeks. The collagen solution, once neutralized, has the potential to be used to produce gels or scaffolds, to deposit thin films on supports and to develop drug delivery systems.     

5-HT uptake blockade prevents the increasing effect of K(ATP) channel blockers on electrically evoked [3H]-5-HT release in rat and mouse neocortical slices

To explore if prolonged--as opposed to acute--5-HT uptake blockade can lead to changes in the function of ATP-dependent potassium (K(ATP)) channels, we investigated in rat and mouse neocortical slices the effects of K(ATP) channel blockers on electrically evoked [3H]-serotonin ([3H]-5-HT) release after short- and long-term exposure to 5-HT uptake blockers. Glibenclamide (1 microM), a K(ATP) channel blocker, enhanced the electrically evoked [3H]-5-HT release by 66 and by 77%, respectively, in rat and in mouse neocortex slices. This effect was confirmed in the rat by tolbutamide (1 microM), another K(ATP) channel antagonist. After short-term blockade (45 min) of 5-HT uptake, glibenclamide still increased the release of [3H]-5-HT in the rat. Glibenclamide, however, failed to enhance [3H]-5-HT release after long-term uptake blockade (210 min). In the mouse, however, both short- and long-term inhibition of 5-HT reuptake by citalopram (1 microM) prevented the facilitatory effect of glibenclamide. The Na(+)/K(+)-ATPase inhibitor ouabain (3.2 microM) abolished the glibenclamide-induced increase in [3H]-5-HT release in both rat and mouse, suggesting that an operative Na(+)/K(+)-ATPase is a prerequisite for activation of K(ATP) channels. The terminal 5-HT(1B) autoreceptor-mediated feedback control was involved in the glibenclamide-induced increase in [(3)H]-5-HT release only in mouse neocortical tissue, as evident from the use of the 5-HT(1B) autoreceptor ligands metitepin (1 microM) and cyanopindolol (1 microM). These results suggest that in the rat long-term uptake blockade leads to an impaired activity of the Na(+)/K(+)-ATPase, which increases intracellular ATP and consequently closes K(ATP) channels. In the mouse, however, short-term uptake blockade seems to already reduce the activity of the Na(+)/K(+)-ATPase and thereby the consumption of ATP. Blockade of 5-HT transporters thus may close K(ATP) channels through increased intracellular ATP. The following slight depolarisation seems to facilitate 5-HT release. These results may contribute to a better understanding of the mechanisms involved in the clinical time latency of antidepressant efficacy of monoamine uptake blockers.     

A high throughput method for genome-wide analysis of retroviral integration

Retroviral and lentiviral vectors integrate their DNA into the host cell genome leading to stable transgene expression. Integration preferentially occurs in the proximity of active genes, and may in some case disturb their activity, with adverse toxic consequences. To efficiently analyze high numbers of lentiviral insertion sites in the DNA of transduced cells, we developed an improved high-throughput method called vector integration tag analysis (VITA). VITA is based on the identification of Genomic Tags associated to the insertion sites, which are used as signatures of the integration events. We use the capacity of MmeI to cleave DNA at a defined distance of its recognition site, in order to generate 21 bp long tags from libraries of junction fragments between vector and cellular DNA. The length of the tags is sufficient in most cases, to identify without ambiguity an unique position in the human genome. Concatenation, cloning and sequencing of the tags allow to obtain information about 20–25 insertion sites in a single sequencing reaction. As a validation of this method, we have characterized 1349 different lentiviral vector insertion sites in transduced HeLa cells, from only 487 sequencing reactions, with a background of <2% false positive tags.     

Rare canopy species in communities within the Atlantic Coastal Forest in Rio de Janeiro State, Brazil

Rare species are one of the principal components of the species richness and diversity encountered in Dense Ombrophilous Tropical Forests. This study sought to analyze the rare canopy species within the Atlantic Coastal Forest in Rio de Janeiro State, Brazil. Six different communities were examined: Dense Ombrophilous alluvial Forest; Dense sub-montane Ombrophilous Forest; Dense Montane Ombrophilous in Serra do Mar and Serra da Mantiqueira. In each area the vegetation was sampled within forty 10 × 25 m plots alternately distributed along a linear transect. All trees with DBH (1.3 m above ground level) ≥5 cm were sampled. The canopy was characterized using the allometric relationship between diameter and height, and included all trees with BDH ≥10 cm and height ≥10 m. A total of 64 families, 206 genera, and 542 species were sampled, of which 297 (54.8%) represented rare species (less than one individual per hectare). The percentage of rare species varied from 34 to 50% in each of the different communities sampled. A majority of these rare trees belonged to the Rosidae, and a smaller proportion to the Dilleniidae. It was concluded that there was no apparent pattern to rarity among families, that rarity was probably derived from a number of processes (such as gap formation), and that a great majority of the rare species sampled were consistently rare. This indicates that the restricted geographic distribution and high degree of endemism of many arboreal taxa justifies the conservation of even small fragments of Atlantic Forest.     

Fine-scale spatial genetic structure and allozymic diversity in natural populations of <Emphasis Type="Italic">Ocotea catharinensis</Emphasis> Mez. (Lauraceae)

In order to establish a strategy for conservation, the distribution of genetic diversity in four natural populations of Ocotea catharinensis in the Brazilian Atlantic rain forest was investigated using 18 allozyme loci. Estimates of the average number of alleles per loci (2.2), percentage of polymorphic loci (83.3%) and expected genetic diversity (0.427) in adult individuals were high; suggesting that all populations have genetic potential for conservation. The inbreeding within populations ([^(f)] = - 0.0 1 1 \hat{f} = - 0.0 1 1 ) and the total inbreeding ([^(F)] = 0. 1 3 3 \hat{F} = 0. 1 3 3 ) suggest population structure, since a high level of divergence among populations ( [^(q)]_\textP = 0. 1 4 3 \hat{\theta }_{\text{P}} = 0. 1 4 3 ) was also detected. Significant values of spatial genetic structure were found inside the four populations. This study demonstrates that the realized gene flow among the remaining populations of O. catharinensis is not sufficient to stop population divergence due to genetic drift and local selection, which threatens the future viability of this species.