Pharmacological analysis of the neutrophil migration induced by D. rostrata lectin: Involvement of cytokines and nitric oxide

In the present study, we investigated the involvement of resident cell and inflammatory mediators in the neutrophil migration induced by chemotactic activity of a glucose/mannose-specific lectin isolated from Dioclea rostrata seeds (DrosL). Rats were injected i.p. with DrosL (125-1000 mu g/cavity), and at 2-96 h thereafter the leukocyte counts in peritoneal fluid were determined. DrosL-induced a dose-dependent neutrophil migration accumulation, which reached maximal response at 24 h after injection and declines thereafter. The carbohydrate ligand nearly abolished the neutrophil influx. Pre-treatment of peritoneal cavities with thioglycolate which increases peritoneal macrophage numbers, enhanced neutrophil migration induced by DrosL by 303%. However, the reduction of peritoneal mast cell numbers by treatment of the cavities with compound 48/80 did not modify DrosL-induced neutrophil migration. The injection into peritoneal cavities of supernatants from macrophage cultures stimulated with DrosL (125, 250 and 500 mu g/ml) induced neutrophil migration. In addition, DrosL treatment induced cytokines (TNF-alpha, IL-1 beta and CINC-1) and NO release into the peritoneal cavity of rats. Finally, neutrophil chemotaxis assay in vitro showed that the lectin (15 and 31 mu g/ml) induced neutrophil chemotaxis by even 180%. In conclusion, neutrophil migration induced by D. rostrata lectin occurs by way of the release of NO and cytokines such as IL-1 beta, TNF-alpha and CINC-1. (C) 2009 Elsevier Ltd. All rights reserved.

Immuno-gluorescence study of five zygomycetous fungi with two chitin-binding probes

A polyclonal antiserum was prepared against a purified microsomal chitinase isolated from the fungus Choanephora cucurbitarum. Indirect immunofluorescence was used to localize chitinase at various developmental stages of five zygomycetous fungi and during abiotrophic mycoparasite interaction with a susceptible and resistant host. This was compared to localization of oligomers of N-acetylglucosamine with the lectin wheat germ agglutinin (WGA). Dotimmunoblot and Western blot techniques revealed that the anti-serum reacted strongly with the antigen from which it was derived. Cross reactivity of the antiserum was found with WGA and another chitin binding lectin, Phyto/acca americana agglutinin (PAA). Immuno-fluorescence results showed the direct involvement of chitinase in spore swelling, germination, sporangium development and response during mechanical injury. There appeared to be no involvement of chitinase during apical hyphal growth or new branch initiation in any of the fungi tested despite mild proteolysis and permeabilization of the cell surface prior to labelling. Binding with WGA revealed similar patterns of fluorescence to that of chitinase localization but differed by showing fluorescence and therefore chitin localization at the apex and new branch initiation when tested at different developmental stages. There was no difference between chitinase localization and binding with WGA in a susceptible host and resistant host challenged with the mycoparasite, Piptocephalis virginiana. Differences in binding ability of antichitinase and lectin WGA suggests that the latter is not a suitable indicator for indirect localization of the lytic enzyme, chitinase.

Isolation of a lectin and a galactoxyloglucan from Mucuna sloanei seeds

A lectin and a galactoxyloglucan were characterized from Mucuna sloanei seed cotyledons. The galactoxyloglucan, isolated by water extraction and ethanol precipitation, had Glc:Xyl:Gal proportions in a molar ratio of 1.8:1.7:1.0 and a molar mass (M(w)) of 1.6 x 10(6) g mol(-1). The lectin (sloanin), isolated from the same seed by affinity chromatography on cross-linked Adenanthera pavonina galactomannan, gave two protein bands by SDS-PAGE (36 and 34 kDa) and one peak by gel filtration (63.6 kDa). Its N-terminal sequence indicated similar to 69% identity with soybean agglutinin to leguminous lectins. Circular dichroism (CD) spectra established that sloanin predominantly contains beta-sheet structures. Sloanin has similar to 5.5% carbohydrate and displayed hemagglutinating activity against rabbit and enzyme treated human erythrocytes, inhibited only by D-Gal containing sugars. The interaction between sloanin and storage cell-wall galactoxyloglucan was tested by affinity chromatography and fluorescence spectroscopy. (C) 2009 Elsevier Ltd. All rights reserved.

The specificity of frutalin lectin using biomembrane models

Frutalin is a homotetrameric alpha-D-galactose (D-Gal)-binding lectin that activates natural killer cells in vitro and promotes leukocyte migration in vivo. Because lectins are potent lymphocyte stimulators, understanding the interactions that occur between them and cell surfaces can help to the action mechanisms involved in this process. In this paper, we present a detailed investigation of the interactions of frutalin with phospho- and glycolipids using Langmuir monolayers as biomembrane models. The results confirm the specificity of frutalin for D-Gal attached to a biomembrane. Adsorption of frutalin was more efficient for the galactose polar head lipids, in contrast to the one for sulfated galactose, in which a lag time is observed, indicating a rearrangement of the monolayer to incorporate the protein. Regarding ganglioside GM1 monolayers, lower quantities of the protein were adsorbed, probably due to the farther apart position of D-galactose from the interface. Binary mixtures containing galactocerebroside revealed small domains formed at high lipid packing in the presence of frutalin, suggesting that lectin induces the clusterization and the forming of domains in vitro, which may be a form of receptor internalization. This is the first experimental evidence of such lectin effect, and it may be useful to understand the mechanism of action of lectins at the molecular level. (C) 2010 Elsevier B.V. All rights reserved.

Good agreement between capillary and venous sampling for lectin pathway proteins

Background: The lectin pathway of complement activation, in particular mannose-binding lectin (MBL), has been extensively investigated over recent years. So far, studies were exclusively based on venous samples. The aim of this study was to investigate whether measurements of lectin pathway proteins obtained by capillary sampling are in agreement with venous samples. Methods: Prospective study including 31 infants that were admitted with suspected early-onset sepsis. Lectin pathway proteins were measured in simultaneously obtained capillary and venous samples. Bland–Altman plots of logarithmized results were constructed, and the mean capillary to venous ratios (ratiocap/ven) were calculated with their 95% confidence intervals (CI). Results: The agreement between capillary and venous sampling was very high for MBL (mean ratiocap/ven, 1.01; 95% CI, 0.85–1.19). Similarly, high agreement was observed for H-ficolin (mean ratiocap/ven, 1.02; 95% CI, 0.72–1.44), MASP-2 (1.04; 0.59–1.84), MASP-3 (0.96; 0.71–1.28), and MAp44 (1.01; 0.82–1.25), while the agreement was moderate for M-ficolin (mean ratiocap/ven, 0.78; 95% CI, 0.27–2.28). Conclusions: The results of this study show an excellent agreement between capillary and venous samples for most lectin pathway proteins. Except for M-ficolin, small volume capillary samples can thus be used when assessing lectin pathway proteins in neonates and young children.

Activation of the lectin pathway of complement in pig-to-human xenotransplantation models

BACKGROUND Natural IgM containing anti-Gal antibodies initiates classic pathway complement activation in xenotransplantation. However, in ischemia-reperfusion injury, IgM also induces lectin pathway activation. The present study was therefore focused on lectin pathway as well as interaction of IgM and mannose-binding lectin (MBL) in pig-to-human xenotransplantation models. METHODS Activation of the different complement pathways was assessed by cell enzyme-linked immunosorbent assay using human serum on wild-type (WT) and α-galactosyl transferase knockout (GalTKO)/hCD46-transgenic porcine aortic endothelial cells (PAEC). Colocalization of MBL/MASP2 with IgM, C3b/c, C4b/c, and C6 was investigated by immunofluorescence in vitro on PAEC and ex vivo in pig leg xenoperfusion with human blood. Influence of IgM on MBL binding to PAEC was tested using IgM depleted/repleted and anti-Gal immunoabsorbed serum. RESULTS Activation of all the three complement pathways was observed in vitro as indicated by IgM, C1q, MBL, and factor Bb deposition on WT PAEC. MBL deposition colocalized with MASP2 (Manders' coefficient [3D] r=0.93), C3b/c (r=0.84), C4b/c (r=0.86), and C6 (r=0.80). IgM colocalized with MBL (r=0.87) and MASP2 (r=0.83). Human IgM led to dose-dependently increased deposition of MBL, C3b/c, and C6 on WT PAEC. Colocalization of MBL with IgM (Pearson's coefficient [2D] rp=0.88), C3b/c (rp=0.82), C4b/c (rp=0.63), and C6 (rp=0.81) was also seen in ex vivo xenoperfusion. Significantly reduced MBL deposition and complement activation was observed on GalTKO/hCD46-PAEC. CONCLUSION Colocalization of MBL/MASP2 with IgM and complement suggests that the lectin pathway is activated by human anti-Gal IgM and may play a pathophysiologic role in pig-to-human xenotransplantation.

Association of lectin pathway proteins with intra-abdominal Candida infection in high-risk surgical intensive-care unit patients. A prospective cohort study within the fungal infection network of Switzerland

OBJECTIVES Human studies on the role of mannose-binding lectin (MBL) in patients with invasive candidiasis have yielded conflicting results. We investigated the influence of MBL and other lectin pathway proteins on Candida colonization and intra-abdominal candidiasis (IAC) in a cohort of high-risk patients. METHODS Prospective observational cohort study of 89 high-risk intensive-care unit (ICU) patients. Levels of lectin pathway proteins at study entry and six MBL2 single-nucleotide polymorphisms were analyzed by sandwich-type immunoassays and genotyping, respectively, and correlated with development of heavy Candida colonization (corrected colonization index (CCI) ≥0.4) and occurrence of IAC during a 4-week period. RESULTS Within 4 weeks after inclusion a CCI ≥0.4 and IAC was observed in 47% and 38% of patients respectively. Neither serum levels of MBL, ficolin-1, -2, -3, MASP-2 or collectin liver 1 nor MBL2 genotypes were associated with a CCI ≥0.4. Similarly, none of the analyzed proteins was found to be associated with IAC with the exception of lower MBL levels (HR 0.74, p = 0.02) at study entry. However, there was no association of MBL deficiency (<0.5 μg/ml), MBL2 haplo- or genotypes with IAC. CONCLUSION Lectin pathway protein levels and MBL2 genotype investigated in this study were not associated with heavy Candida colonization or IAC in a cohort of high-risk ICU patients.

Genome-wide analysis of G-type lectin genes in Fragaria vesca and functional characterization of FaMBL1 gene in defense response of F. × ananassa to fungal pathogens

Strawberry (Fragaria × ananassa) is an important soft fruit but easily to be infected by pathogens. Anthracnose and gray mold are two of the most destructive diseases of strawberry which lead to serious fruit rot. The first chapter introduced strawberry anthracnose caused by Colletotrichum acutatum. The infection strategy, disease cycle and management of C. acutatum on strawberry were reported. Likewise, the second chapter summarized the infection strategy of Botrytis cinerea and the defense responses of strawberry. As we already know white unripe strawberry fruits are more resistant to C. acutatum than red ripe fruits. During the interaction between strawberry white/red fruit and C. acutaum, a mannose binding lectin gene, FaMBL1, was found to be the most up-regulated gene and induced exclusively in white fruit. FaMBL1 belongs to the G-type lectin family which has important roles in plant development and defense process. To get insight into the role of FaMBL1, genome-wide identification was carried out on G-type lectin gene family in Fragaria vesca and the results were showed in chapter 3. G-type lectin genes make up a large family in F. vesca. Active expression upon biotic/abiotic stresses suggested a potential role of G-lectin genes in strawberry defenses. Hence, stable transgenic strawberry plants with FaMBL1 gene overexpressed were generated. Transformed strawberry plants were screened and identified. The results were showed in chapter 4, content of disease-related phytohormone, jasmonic acid, was found decreased in overexpressing lines compared with wild type (WT). Petioles inoculated by C. fioriniae of overexpressing lines had lower disease incidence than WT. Leaves of overexpressing lines challenged by B. cinerea showed remarkably smaller lesion diameters compared with WT. The chitinase 2-1 (FaChi2-1) showed higher expression in overexpressing lines than in WT during the interaction with B. cinerea, which could be related with the lower susceptibility of overexpressing lines.

Galectin-1 Induces Reversible Phosphatidylserine Exposure at the Plasma Membrane

Cells normally undergo physiological turnover through the induction of apoptosis and phagocytic removal, partly through exposure of cell surface phosphatidylserine (PS). In contrast, neutrophils appear to possess apoptosis-independent mechanisms of removal. Here we show that Galectin-1 (Gal-1) induces PS exposure independent of alterations in mitochondrial potential, caspase activation, or cell death. Furthermore, Gal-1-induced PS exposure reverts after Gal-1 removal without altering cell viability. Gal-1-induced PS exposure is uniquely microdomain restricted, yet cells exposing PS do not display evident alterations in membrane morphology nor do they exhibit bleb formation, typically seen in apoptotic cells. Long-term exposure to Gal-1 prolongs PS exposure with no alteration in cell cycle progression or cell growth. These results demonstrate that Gal-1-induced PS exposure and subsequent phagocytic removal of living cells represents a new paradigm in cellular turnover.

Immunological Basis for the Gender Differences in Murine Paracoccidioides brasiliensis Infection

This study aimed to investigate the immunological mechanisms involved in the gender distinct incidence of paracoccidioidomycosis (pcm), an endemic systemic mycosis in Latin America, which is at least 10 times more frequent in men than in women. Then, we compared the immune response of male and female mice to Paracoccidioides brasiliensis infection, as well as the influence in the gender differences exerted by paracoccin, a P. brasiliensis component with carbohydrate recognition property. High production of Th1 cytokines and T-bet expression have been detected in the paracoccin stimulated cultures of spleen cells from infected female mice. In contrast, in similar experimental conditions, cells from infected males produced higher levels of the Th2 cytokines and expressed GATA-3. Macrophages from male and female mice when stimulated with paracoccin displayed similar phagocytic capability, while fungicidal activity was two times more efficiently performed by macrophages from female mice, a fact that was associated with 50% higher levels of nitric oxide production. In order to evaluate the role of sexual hormones in the observed gender distinction, we have utilized mice that have been submitted to gonadectomy followed by inverse hormonal reconstitution. Spleen cells derived from castrated males reconstituted with estradiol have produced higher levels of IFN-gamma (1291+/-15 pg/mL) and lower levels of IL-10 (494+/-38 pg/mL), than normal male in response to paracoccin stimulus. In contrast, spleen cells from castrated female mice that had been treated with testosterone produced more IL-10 (1284+/-36 pg/mL) and less IFN-gamma (587614 pg/mL) than cells from normal female. In conclusion, our results reveal that the sexual hormones had a profound effect on the biology of immune cells, and estradiol favours protective responses to P. brasiliensis infection. In addition, fungal components, such as paracoccin, may provide additional support to the gender dimorphic immunity that marks P. brasiliensis infection.

Bioinsecticidal activity of Talisia esculenta reserve protein on growth and serine digestive enzymes during larval development of Anticarsia gemmatalis

Plants synthesize a variety of molecules to defend themselves against an attack by insects. Talisin is a reserve protein from Talisia esculenta seeds, the first to be characterized from the family Sapindaceae. In this study, the insecticidal activity of Talisin was tested by incorporating the reserve protein into an artificial diet fed to the velvetbean caterpillar Anticarsia gemmatalis, the major pest of soybean crops in Brazil. At 1.5% (w/w) of the dietary protein, Talisin affected larval growth, pupal weight, development and mortality, adult fertility and longevity, and produced malformations in pupae and adult insects. Talisin inhibited the trypsin-like activity of larval midgut homogenates. The trypsin activity in Talisin-fed larvae was sensitive to Talisin, indicating that no novel protease-resistant to Talisin was induced in Talisin-fed larvae. Affinity chromatography showed that Talisin bound to midgut proteinases of the insect A. gemmatalis, but was resistant to enzymatic digestion by these larval proteinases. The transformation of genes coding for this reserve protein could be useful for developing insect resistant crops. (C) 2010 Elsevier Inc. All rights reserved.

Ligand Reduces Galectin-1 Sensitivity to Oxidative Inactivation by Enhancing Dimer Formation

Galectin-1 (Gal-1) regulates leukocyte turnover by inducing the cell surface exposure of phosphatidylserine (PS), a ligand that targets cells for phagocytic removal, in the absence of apoptosis. Gal-1 monomer- dimer equilibrium appears to modulate Gal-1-induced PS exposure, although the mechanism underlying this regulation remains unclear. Here we show that monomer- dimer equilibrium regulates Gal-1 sensitivity to oxidation. A mutant form of Gal-1, containing C2S and V5D mutations (mGal-1), exhibits impaired dimerization and fails to induce cell surface PS exposure while retaining the ability to recognize carbohydrates and signal Ca(2+) flux in leukocytes. mGal-1 also displayed enhanced sensitivity to oxidation, whereas ligand, which partially protected Gal-1 from oxidation, enhanced Gal-1 dimerization. Continual incubation of leukocytes with Gal-1 resulted in gradual oxidative inactivation with concomitant loss of cell surface PS, whereas rapid oxidation prevented mGal-1 from inducing PS exposure. Stabilization of Gal-1 or mGal-1 with iodoacetamide fully protected Gal-1 and mGal-1 from oxidation. Alkylation-induced stabilization allowed Gal-1 to signal sustained PS exposure in leukocytes and mGal-1 to signal both Ca(2+) flux and PS exposure. Taken together, these results demonstrate that monomer-dimer equilibrium regulates Gal-1 sensitivity to oxidative inactivation and provides a mechanism whereby ligand partially protects Gal-1 from oxidation.

Differential expression of immunomodulatory galectin-1 in peripheral leukocytes and adult tissues and its cytosolic organization in striated muscle

Galectin-1 (Gal-1) is important in immune function and muscle regeneration, but its expression and localization in adult tissues and primary leukocytes remain unclear. To address this, we generated a specific monoclonal antibody against Gal-1, termed alpha hGal-1, and defined a sequential peptide epitope that it recognizes, which is preserved in human and porcine Gal-1, but not in murine Gal-1. Using alpha hGal-1, we found that Gal-1 is expressed in a wide range of porcine tissues, including striated muscle, liver, lung, brain, kidney, spleen, and intestine. In most types of cells, Gal-1 exhibits diffuse cytosolic expression, but in cells within the splenic red pulp, Gal-1 showed both cytosolic and nuclear localization. Gal-1 was also expressed in arterial walls and exhibited prominent cytosolic and nuclear staining in cultured human endothelial cells. However, human peripheral leukocytes and promyelocytic HL60 cells lack detectable Gal-1 and also showed very low levels of Gal-1 mRNA. In striking contrast, Gal-1 exhibited an organized cytosolic staining pattern within striated muscle tissue of cardiac and skeletal muscle and colocalized with sarcomeric actin on I bands. These results provide insights into previously defined roles for Gal-1 in inflammation, immune regulation and muscle biology.

Expression of galectin-1 in the mouse olfactory system

Primary sensory olfactory axons arise from the olfactory neuroepithelium that lines the nasal cavity and then project via the olfactory nerve into the olfactory bulb. The P-galactoside binding lectin, galectin-1,and its laminin ligand have been implicated in the growth of these axons along this pathway. In galectin-1 null mutant mice, a subpopulation of primary sensory olfactory axons fails to reach its targets in the olfactory bulb. In the present study we examined the spatiotemporal expression pattern of galectin-1 in normal mice in order to understand its role in the development of the olfactory nerve pathway. At E15.5, when olfactory axons have already contacted the olfactory bulb, galectin-1 was expressed in the cartilage and mesenchyme surrounding the nasal cavity but was absent from the olfactory neuroepithelium, nerve and bulb. Between E16.5 and birth galectin-1 began to be expressed by olfactory nerve ensheathing cells in the lamina propria of the neuroepithelium and nerve fibre layer. Galectin-1 was neither expressed by primary sensory neurons in the olfactory neuroepithelium nor by their axons in the olfactory nerve. Laminin, a galectin-1 ligand, also exhibited a similar expression pattern in the embryonic olfactory nerve pathway. Our results reveal that galectin-1 is dynamically expressed by glial elements within the nerve fibre layer during a discrete period in the developing olfactory nerve pathway. Previous studies have reported galectin-1 acts as a substrate adhesion molecule by cross-linking primary sensory olfactory neurons to laminin. Thus, the coordinate expression of galectin-1 and laminin in the embryonic nerve fibre layer suggests that these molecules support the adhesion and fasciculation of axons en route to their glomerular targets.

Real-time monitoring and kinetic parameter estimation of the affinity interaction of jArtinM and rArtinM with peroxidase glycoprotein by the electrogravimetric technique

ArtinM is a D-mannose binding lectin that has been arousing increasing interest because of its biomedical properties, especially those involving the stimulation of Th1 immune response, which confers protection against intracellular pathogens The potential pharmaceutical applications of ArtinM have motivated the production of its recombinant form (rArtinM) so that it is important to compare the sugar-binding properties of jArtinM and rArtinM in order to take better advantage of the potential applications of the recombinant lectin. In this work, a biosensor framework based on a Quartz Crystal Microbalance was established with the purpose of making a comparative study of the activity of native and recombinant ArtinM protein The QCM transducer was strategically functionalized to use a simple model of protein binding kinetics. This approach allowed for the determination of the binding/dissociation kinetics rate and affinity equilibrium constant of both forms of ArtinM with horseradish peroxidase glycoprotein (HRP), a N-glycosylated protein that contains the trimannoside Man alpha 1-3[Man alpha 1-6]Man, which is a known ligand for jArtinM (Jeyaprakash et al, 2004). Monitoring of the real-time binding of rArtinM shows that it was able to bind HRP, leading to an analytical curve similar to that of jArtinM, with statistically equivalent kinetic rates and affinity equilibrium constants for both forms of ArtinM The lower reactivity of rArtinM with HRP than jArtinM was considered to be due to a difference in the number of Carbohydrate Recognition Domains (CRDs) per molecule of each lectin form rather than to a difference in the energy of binding per CRD of each lectin form. (C) 2010 Elsevier B V. All rights reserved