Immunomodulatory effects of cyanobacterial toxin cylindrospermopsin on innate immune cells.

Cylindrospermopsin (CYN), a cyanobacterial toxin, is an important water pollutant with broad biological activity. It has been known mainly from tropical areas, but the area of occurrence of its producers is spreading to temperate climates. It can be found in high concentrations in the environment as well as in purified drinking waters. The aim of the study is to bring a basic information on the ability of CYN to interfere with mammalian innate immunity cells and thus increase the understanding of the immunomodulatory potency of CYN. This study investigated whether immune cells can be a target of CYN either alone or in combination with a model immunomodulatory agent, lipopolysaccharide (LPS). We examined the effects on cellular viability and inflammation signaling of CYN on murine macrophage-like RAW 264.7 cells. Macrophages were treated either with pure toxin (1 µM) or together with a known stimulator of immunologically active cells, bacterial or cyanobacterial LPS. CYN has had a significant effect on production on pro-inflammatory mediator tumor necrosis factor α (TNF-α) which correlates with its effect on reactive oxygen species (ROS) production. We found that CYN potentiated the effect of bacterial and cyanobacterial LPS that was documented by activation of inflammatory signaling pathways including mitogen-activated protein kinase p38 as well as consequent expression of inducible nitric oxide synthase (iNOS) and increased production of pro-inflammatory mediators such as nitric oxide (NO), TNF-α, interleukin-6 (IL-6). Our study brings one of the first information that contributes to the elucidation of immunomodulatory role of CYN in macrophages under normal and pro-inflammatory conditions.

Microcystin-LR Enrichment from Freshwater by a Recombinant Plant-derived Antibody Using Sol-Gel-Glass Immunoextraction.

Eutrophication of water bodies can promote cyanobacterial (blue-green algae) blooms, which has become a source of increasing concern for both recreational and drinking water use. Many bacterial species can produce toxins that pose threats to wildlife, domestic animals and humans. Microcystin-leucine-arginine (MC-LR) is the most frequent and most toxic microcystin congener. For the first time, lab-scale investigations were performed to test the application of a recombinant plant-derived anti-MC-LR antibody immobilized on an immunoaffinity support material to selectively extract the toxin from spiked freshwater samples. As a comparison, its hybridoma-derived counterpart (murine monoclonal antibody) was evaluated. The antibody-doped material was prepared via an optimized sol-gel process; its stability and binding efficiency of MC-LR in spiked freshwater samples were thoroughly tested using the ELISA and orthogonal LC-MS methods. For removal, two column-based procedures with sequential or continuous cyclic sample addition and a suspension mode (moving adsorbent) were tested. Noteworthy the results obtained with a crude antibody fraction were fully compatible with the highly purified preparation. This study paves the way for further investigation being focused on novel applications of plant-derived anti-MC-LR antibodies in bioremediation to selectively deplete the toxin from freshwater: a green and promising technology without secondary pollution.

An aptamer-based immunoassay in microchannels of a portable analyzer for detection of microcystin-leucine-arginine.

The rapid detection of microcystin-leucine-arginine (MC-LR), the most highly toxic among MCs, is significantly important to environmental and human health protection and prevention of MC-LR from being used as a bioweapon. Although aptamers offer higher affinity, specificity, and stability with MC-LR than antibodies in the immunodetection of MC-LR due to steric hindrance between two antibodies and limited epitopes of MC-LR for use in a sandwich immunoassay, no sandwich immunoassay using an aptmer has been developed for MC-LR detection. This study is aimed at developing an aptamer-antibody immunoassay (AAIA) to detect MC-LR using a portable analyzer. The aptamers were immobilized onto the glass surface of a microchamber to capture MC-LR. MC-LR and horseradish peroxidase (HRP)-labeled antibody were pulled into the microchamber to react with the immobilized aptamer. The chemiluminescence (CL) catalyzed by HRP was tested by a photodiode-based portable analyzer. MC-LR at 0.5-4.0 µg/L was detected quantitatively by the AAIA, with a CL signal sensitivity of 0.3 µg/L. The assay took less than 35 min for a single sample and demonstrated a high specificity, detecting only MC-LR, but not MC-LA, MC-YR, or nodularin-R. The recovery of two spiked real environmental samples calculated as 94.5-112.7%. Therefore, this AAIA was proved to be a rapid and simple method to detect MC-LR in the field by a single analyst.

Two ScFv antibody libraries derived from identical VL-VH framework with different binding site designs display distinct binding profiles.

In directed evolution experiments, a single randomization scheme of an antibody gene does not provide optimal diversity for recognition of all sizes of antigens. In this study, we have expanded the recognition potential of our universal library, termed ScFvP, with a second distinct diversification scheme. In the second library, termed ScFvM, diversity was designed closer to the center of the antigen binding site in the same antibody framework as earlier. Also, the CDR-H3 loop structures were redesigned to be shorter, 5-12 aa and mostly without the canonical salt bridge between Arg106H and Asp116H to increase the flexibility of the loop and to allow more space in the center of the paratope for binding smaller targets. Antibodies were selected from the two libraries against various antigens separately and as a mixture. The origin and characteristics of the retrieved antibodies indicate that complementary diversity results in complementary functionality widening the spectrum of targets amenable for selection.

Effect of cyanobacteria on immune function of crucian carp (Carassius auratus) via chronic exposure in diet.

Cyanobacterial blooms caused by water eutrophication have become a worldwide problem. Microcystins (MCs) released during cyanobacterial blooms exert toxicity on fish. Up to now, immunotoxicity of MCs on fish has been rarely reported. The present study investigated immune response of crucian carp (Carassius auratus) to cyanobacteria via chronic exposure in diet. Fish were fed with diets containing 20% (low dose group) and 40% (high dose group) of cyanobacteria lyophilized powder. After exposure of 30 d, a batch of assays was determined for assessing immunotoxicity of MCs. The head kidney and spleen indexes significantly increased in high dose group. Blood nitroblue tetrazolium activity in high dose group was nearly twice as much as that in control group with no cyanobacteria additive. Marked haemorrhage and hyperemia were observed in kidney and spleen in high dose group. The edematous mitochondria, deformation of the nucleus and compaction of chromatin occurred in lymphocytes of head kidney and spleen in both cyanobacteria groups. Lysozyme activity showed an obvious increase in low dose group but a sharp decrease in high dose group. Significant increase of macrophage bactericidal activity was detected in low dose group. The present findings indicate that via chronic diet exposure of different cyanobacteria levels, fish exhibit various immune responses. Fish immunity tends to proceed toward the direction of immunostimulative response at low MCs concentrations but toward the trend of immunosuppressive answer at high MCs concentrations.

Generation of transgenic plants expressing plasma membrane-bound antibodies to the environmental pollutant microcystin-LR.

In this paper we describe the engineering and regeneration of transgenic tobacco plants expressing a recombinant plasma membrane-retained antibody specific to microcystin-LR (MC-LR), the environmental toxin pollutant produced by cyanobacteria. The antibody was created by a genetic fusion of the antigen binding regions of the microcystin-specific single chain antibody, 3A8, with the constant regions from the murine IgG1κ, Guy's 13, including a membrane retention sequence at the C-terminal end of the antibody heavy chain. The antibody produced in the leaves was shown to be functional by binding to MC-LR in an ELISA with antibody yields in transgenic plant leaves reaching a maximum of 1.2 µg g(-1) leaf f.wt (0.005% total soluble protein). Antibody-MC-LR complexes formed in leaves after addition of MC-LR to hydroponic medium around the roots of transgenic plant cultures.

Generation of transgenic plants expressing antibodies to the environmental pollutant microcystin-LR.

We describe the engineering, regeneration, and characterization of transgenic tobacco plants expressing a recombinant antibody specific to microcystin-LR (MC-LR), the environmental toxin pollutant produced by species of cyanobacteria. The antibody was created by a genetic fusion of the antigen-binding regions of the microcystin-specific single-chain antibody, 3A8, with constant regions from the murine IgG1kappa, Guy's 13. IgG transgenes were controlled by a leader peptide that targets the transgene products to the secretory pathway and also allows for rhizosecretion. The antibody, extracted from the leaves or rhizosecreted into hydroponic medium by transgenic plants, was shown to have functional binding to MC-LR. Antibody yields in transgenic plant leaves reached a maximum of 64 microg/g leaf fresh weight (0.6% total soluble protein), and the rate of antibody rhizosecretion reached a maximum of 5 microg/g root dry weight/24 h. Rhizosecreted antibody bound to MC-LR in hydroponic medium, and transgenic plants grew more efficiently on medium containing MC-LR compared to wild-type controls. This proof of concept paves the way for applications to produce diagnostic antibodies to microcystin-LR, remove it from the environment by phytoremediation, or enhance yields in crops exposed to MC-LR.-Drake, P. M. W., Barbi, T., Drever, M. R., van Dolleweerd, C. J., Porter, A. J. R., Ma, J. K.-C. Generation of transgenic plants expressing antibodies to the environmental pollutant microcystin-LR.

[Application of ELISA for microcystins detection].

Microcystins (MCs) may be group of closely related toxic cyclic heptapeptides produced by common cyanobacteria (blue-green algae). Their toxicity could be associated with specific inhibition of intracellular protein phosphatases type-1 and type-2A (PP1 and PP2A, respectively). There are some methods for analyzing and detecting MCs in aquatic environment, such as HPLC, LC-MS, PPIA, and ELISA. This review introduces advances on MCs determined by enzyme-linked immunosorbent assay (ELISA), and estimates the prospective development of ELISA for monitoring MCs in natural waters.

Highly sensitive amperometric immunosensors for microcystin detection in algae.

The presence of cyanobacterial toxins in water and algae pose a health hazard for animals and humans, due to their tumour-promoting activity and carcinogen effects. The use of simple, rapid and reliable tools for routine analysis is becoming a necessity. With this purpose, our group has developed two electrochemical immunosensors for the detection of microcystin-LR (MC-LR) based on the affinity between this cyanotoxin and the corresponding monoclonal and polyclonal antibodies. A competitive direct enzyme-linked immunosorbent assays (ELISAs) was designed and, after validation of the approach on microtiter wells, screen-printed graphite electrodes were used as supports. Colorimetry was used to optimise the experimental parameters and to compare the performance of monoclonal and polyclonal antibodies. Afterwards, electrochemical measurements were performed at -200 mV (versus Ag/AgCl) using 5-methyl-phenazinium methyl sulfate (MPMS) as mediator for horseradish peroxidase (HRP), the enzymatic label of the competitor. The IC(50) values were 0.10 and 1.73 microgL(-1) for MAb and PAb, respectively. Whereas Mab provided higher sensitivities, the reproducibility was better when using PAb. The developed amperometric immunosensors were applied to the analysis of cyanobacterial samples from the Tarn River (Midi-Pyrénées, France) and the presence of MC was confirmed by the colorimetric protein phosphatase inhibition (PPI) assay and high performance liquid chromatography (HPLC). The limits of detection attained from the calibration curves and the results obtained for the real samples demonstrate the potential use of the immunosensors as screening tools for routine use in the assessment of water quality and the control of toxins in algae.