IL-26 inhibits hepatitis C virus replication in hepatocytes.

BACKGROUND & AIMS: Interleukin-26 (IL-26) is a proinflammatory cytokine that has properties atypical for a cytokine, such as direct antibacterial activity and DNA-binding capacity. We previously observed an accumulation of IL-26 in fibrotic and inflammatory lesions in the livers of patients with chronic HCV infection and showed that infiltrating CD3+ lymphocytes were the principal source of IL-26. Surprisingly, IL-26 was also detected in the cytoplasm of hepatocytes from HCV-infected patients, even though these cells do not produce IL-26, even when infected with HCV. Based on this observation and possible interactions between IL-26 and nucleic acids, we investigated the possibility that IL-26 controlled HCV infection independently of the immune system. METHODS: We evaluated the ability of IL-26 to interfere with HCV replication in hepatocytes and investigated the mechanisms by which IL-26 exerts its antiviral activity. RESULTS: We showed that IL-26 penetrated HCV-infected hepatocytes, where it interacted directly with HCV double-stranded RNA replication intermediates, thereby inhibiting viral replication. IL-26 interfered with viral RNA-dependent RNA polymerase activity, preventing the de novo synthesis of viral genomic single-stranded RNA. CONCLUSIONS: These findings reveal a new role for IL-26 in direct protection against HCV infection, independently of the immune system, and increase our understanding of the antiviral defense mechanisms controlling HCV infection. Future studies should evaluate the possible use of IL-26 for treating other chronic disorders caused by RNA viruses, for which few treatments are currently available, or emerging RNA viruses. LAY SUMMARY: This study sheds new light on the body's arsenal for controlling hepatitis C virus (HCV) infection and identifies interleukin-26 (IL-26) as an antiviral molecule capable of blocking HCV replication. IL-26, which has unique biochemical and structural characteristics, penetrates infected hepatocytes and interacts directly with viral RNA, thereby blocking viral replication. IL-26 is, therefore, a new player in antiviral defenses, operating independently of the immune system. It is of considerable potential interest for treating HCV infection and other chronic disorders caused by RNA viruses for which few treatments are currently available, and for combating emerging RNA viruses.

Metabolomic Investigation of Staphylococcus aureus Antibiotic Susceptibility by Liquid Chromatography Coupled to High-Resolution Mass Spectrometry.

Staphylococcus aureus is a major human pathogen that can readily acquire antibiotic resistance. For instance, methicillin-resistant S. aureus represents a major cause of hospital- and community-acquired bacterial infections. In this chapter, we first provide a detailed protocol for obtaining unbiased and reproducible S. aureus metabolic profiles. The resulting intracellular metabolome is then analyzed in an untargeted manner by using both hydrophilic interaction liquid chromatography and pentafluorophenyl-propyl columns coupled to high-resolution mass spectrometry. Such analyses are done in conjunction with our in-house spectral database to identify with high confidence as many meaningful S. aureus metabolites as possible. Under these conditions, we can routinely monitor more than 200 annotated S. aureus metabolites. We also indicate how this protocol can be used to investigate the metabolic differences between methicillin-resistant and susceptible strains.

A Novel Subfamily of Bacterial AAT-Fold Basic Amino Acid Decarboxylases and Functional Characterization of Its First Representative: Pseudomonas aeruginosa LdcA.

Polyamines are small amino-acid derived polycations capable of binding negatively charged macromolecules. Bacterial polyamines are structurally and functionally diverse, and are mainly produced biosynthetically by pyridoxal-5-phosphate-dependent amino acid decarboxylases referred to as Lysine-Arginine-Ornithine decarboxylases (LAOdcs). In a phylogenetically limited group of bacteria, LAOdcs are also induced in response to acid stress. Here, we performed an exhaustive phylogenetic analysis of the AAT-fold LAOdcs which showcased the ancient nature of their short forms in Cyanobacteria and Firmicutes, and emergence of distinct subfamilies of long LAOdcs in Proteobacteria. We identified a novel subfamily of lysine decarboxylases, LdcA, ancestral in Betaproteobacteria and Pseudomonadaceae. We analyzed the expression of LdcA from Pseudomonas aeruginosa, and uncovered its role, intimately linked to cadaverine (Cad) production, in promoting growth and reducing persistence of this multidrug resistant human pathogen during carbenicillin treatment. Finally, we documented a certain redundancy in the function of the three main polyamines-Cad, putrescine (Put), and spermidine (Spd)-in P. aeruginosa by demonstrating the link between their intracellular level, as well as the capacity of Put and Spd to complement the growth phenotype of the ldcA mutant.

Low-Protein Diet Induces IRE1α-Dependent Anticancer Immunosurveillance.

Dietary restriction (DR) was shown to impact on tumor growth with very variable effects depending on the cancer type. However, how DR limits cancer progression remains largely unknown. Here, we demonstrate that feeding mice a low-protein (Low PROT) isocaloric diet but not a low-carbohydrate (Low CHO) diet reduced tumor growth in three independent mouse cancer models. Surprisingly, this effect relies on anticancer immunosurveillance, as depleting CD8+ T cells, antigen-presenting cells (APCs), or using immunodeficient mice prevented the beneficial effect of the diet. Mechanistically, we established that a Low PROT diet induces the unfolded protein response (UPR) in tumor cells through the activation of IRE1α and RIG1 signaling, thereby resulting in cytokine production and mounting an efficient anticancer immune response. Collectively, our data suggest that a Low PROT diet induces an IRE1α-dependent UPR in cancer cells, enhancing a CD8-mediated T cell response against tumors.

Rendomab B4, a monoclonal antibody that discriminates the human endothelin B receptor of melanoma cells and inhibits their migration.

Metastatic melanoma is an aggressive cancer with a poor prognostic, and the design of new targeted drugs to treat melanoma is a therapeutic challenge. A promising approach is to produce monoclonal antibodies (mAbs) against the endothelin B receptor (ETB), which is known to be overexpressed in melanoma and to contribute to proliferation, migration and vasculogenic mimicry associated with invasiveness of this cancer. We previously described rendomab-B1, a mAb produced by DNA immunization. It is endowed with remarkable characteristics in term of affinity, specificity and antagonist properties against human ETB expressed by the endothelial cells, but, surprisingly, had poor affinity for ETB expressed by melanoma cells. This characteristic strongly suggested the existence of a tumor-specific ETB form. In the study reported here, we identified a new mAb, rendomab-B4, which, in contrast to rendomab-B1, binds ETB expressed on UACC-257, WM-266-4 and SLM8 melanoma cells. Moreover, after binding to UACC-257 cells, rendomab-B4 is internalized and colocalizes with the endosomal protein EEA-1. Interestingly, rendomab-B4, despite its inability to compete with endothelin binding, is able to inhibit phospholipase C pathway and migration induced by endothelin. By contrast, rendomab-B4 fails to decrease ERK1/2 phosphorylation induced by endothelin, suggesting a biased effect on ETB. These particular properties make rendomab-B4 an interesting tool to analyze ETB-structure/function and a promising starting point for the development of new immunological tools in the field of melanoma therapeutics.

IL-26 is overexpressed in chronically HCV-infected patients and enhances TRAIL-mediated cytotoxicity and interferon production by human NK cells.

OBJECTIVE: Interleukin-26 (IL-26) is a member of the IL-10 cytokine family, first discovered based on its peculiar expression by virus-transformed T cells. IL-26 is overexpressed in chronic inflammation (rheumatoid arthritis and Crohn's disease) and induces proinflammatory cytokines by myeloid cells and some epithelial cells. We thus investigated the expression and potential role of IL-26 in chronic HCV infection, a pathology associated with chronic inflammation. DESIGN: IL-26 was quantified in a cohort of chronically HCV-infected patients, naive of treatment and its expression in the liver biopsies investigated by immunohistochemistry. We also analysed the ability of IL-26 to modulate the activity of natural killer (NK) cells, which control HCV infection. RESULTS: The serum levels of IL-26 are enhanced in chronically HCV-infected patients, mainly in those with severe liver inflammation. Immunohistochemistry reveals an intense IL-26 staining in liver lesions, mainly in infiltrating CD3+ cells. We also show that NK cells from healthy subjects and from HCV-infected patients are sensitive to IL-26. IL-26 upregulates membrane tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) expression on CD16- CD56(bright) NK cells, enabling them to kill HCV-infected hepatoma cells, with the same efficacy as interferon (IFN)-α-treated NK cells. IL-26 also induces the expression of the antiviral cytokines IFN-ß and IFN-γ, and of the proinflammatory cytokines IL-1ß and TNF-α by NK cells. CONCLUSIONS: This study highlights IL-26 as a new player in the inflammatory and antiviral immune responses associated with chronic HCV infection.

Fast and sensitive detection of enteropathogenic Yersinia by immunoassays.

Yersinia enterocolitica and Yersinia pseudotuberculosis, the two Yersinia species that are enteropathogenic for humans, are distributed worldwide and frequently cause diarrhea in inhabitants of temperate and cold countries. Y. enterocolitica is a major cause of foodborne disease resulting from consumption of contaminated pork meat and is further associated with substantial economic cost. However, investigation of enteropathogenic Yersinia species is infrequently performed routinely in clinical laboratories because of their specific growth characteristics, which make difficult their isolation from stool samples. Moreover, current isolation procedures are time-consuming and expensive, thus leading to underestimates of the incidence of enteric yersiniosis, inappropriate prescriptions of antibiotic treatments, and unnecessary appendectomies. The main objective of the study was to develop fast, sensitive, specific, and easy-to-use immunoassays, useful for both human and veterinary diagnosis. Monoclonal antibodies (MAbs) directed against Y. enterocolitica bioserotypes 2/O:9 and 4/O:3 and Y. pseudotuberculosis serotypes I and III were produced. Pairs of MAbs were selected by testing their specificity and affinity for enteropathogenic Yersinia and other commonly found enterobacteria. Pairs of MAbs were selected to develop highly sensitive enzyme immunoassays (EIAs) and lateral flow immunoassays (LFIs or dipsticks) convenient for the purpose of rapid diagnosis. The limit of detection of the EIAs ranged from 3.2 × 10(3) CFU/ml to 8.8 × 10(4) CFU/ml for pathogenic serotypes I and III of Y. pseudotuberculosis and pathogenic bioserotypes 2/O:9 and 4/O:3 of Y. enterocolitica and for the LFIs ranged from 10(5) CFU/ml to 10(6) CFU/ml. A similar limit of detection was observed for artificially contaminated human feces.

Detection of cresyl phosphate-modified butyrylcholinesterase in human plasma for chemical exposure associated with aerotoxic syndrome.

Flight crews complain of illness following a fume event in aircraft. A chemical in jet engine oil, the neurotoxicant tri-o-cresyl phosphate, after metabolic activation to cresyl saligenin phosphate makes a covalent adduct on butyrylcholinesterase (BChE). We developed a mass spectrometry method for detection of the cresyl phosphate adduct on human BChE as an indicator of exposure. Monoclonal mAb2, whose amino acid sequence is provided, was crosslinked to cyanogen bromide-activated Sepharose 4B and used to immunopurify plasma BChE treated with cresyl saligenin phosphate. BChE was released with acetic acid, digested with pepsin, and analyzed by liquid chromatography-tandem mass spectrometry (LC-MSMS) on the Triple TOF 5600 mass spectrometer. Peptide FGES198AGAAS with an added mass of 170 Da from cresyl phosphate on serine 198 (Ser198) was detected as parent ion 966.4 Da. When characteristic daughter ions were monitored in the MSMS spectrum, the limit of detection was 0.1% cresyl saligenin phosphate inhibited plasma BChE. This corresponds to 2×10(-9) g in 0.5 ml or 23×10(-15) moles of inhibited BChE in 0.5 ml of plasma. In conclusion, a sensitive assay for exposure to tri-o-cresyl phosphate was developed. Laboratories that plan to use this method are cautioned that a positive result gives no proof that tri-o-cresyl phosphate is toxic at low levels.

Molecular characterization of monoclonal antibodies that inhibit acetylcholinesterase by targeting the peripheral site and backdoor region.

The inhibition properties and target sites of monoclonal antibodies (mAbs) Elec403, Elec408 and Elec410, generated against Electrophorus electricus acetylcholinesterase (AChE), have been defined previously using biochemical and mutagenesis approaches. Elec403 and Elec410, which bind competitively with each other and with the peptidic toxin inhibitor fasciculin, are directed toward distinctive albeit overlapping epitopes located at the AChE peripheral anionic site, which surrounds the entrance of the active site gorge. Elec408, which is not competitive with the other two mAbs nor fasciculin, targets a second epitope located in the backdoor region, distant from the gorge entrance. To characterize the molecular determinants dictating their binding site specificity, we cloned and sequenced the mAbs; generated antigen-binding fragments (Fab) retaining the parental inhibition properties; and explored their structure-function relationships using complementary x-ray crystallography, homology modeling and flexible docking approaches. Hypermutation of one Elec403 complementarity-determining region suggests occurrence of antigen-driven selection towards recognition of the AChE peripheral site. Comparative analysis of the 1.9Å-resolution structure of Fab408 and of theoretical models of its Fab403 and Fab410 congeners evidences distinctive surface topographies and anisotropic repartitions of charges, consistent with their respective target sites and inhibition properties. Finally, a validated, data-driven docking model of the Fab403-AChE complex suggests a mode of binding at the PAS that fully correlates with the functional data. This comprehensive study documents the molecular peculiarities of Fab403 and Fab410, as the largest peptidic inhibitors directed towards the peripheral site, and those of Fab408, as the first inhibitor directed toward the backdoor region of an AChE and a unique template for the design of new, specific modulators of AChE catalysis.

Pathogen-free screening of bacteria-specific hybridomas for selecting high-quality monoclonal antibodies against pathogen bacteria as illustrated for Legionella pneumophila.

Antibodies are potent biological tools increasingly used as detection, diagnostic and therapeutic reagents. Many technological advances have optimized and facilitated production and screening of monoclonal antibodies. We report here an original method to screen for antibodies targeting biosafety level 2 or 3 pathogens without the fastidious handling inherent to pathogen use. A double ELISA screening was performed using as coated antigen transformed Escherichia coli expressing at its surface a protein specific to the pathogenic bacteria versus control untransformed E. coli. This method was applied to Legionella, using the surface-exposed Mip protein (macrophage infectivity potentiator). This screening proved to be an excellent means of selecting mAbs that bind Legionella pneumophila 1 surface-exposed Mip protein. This method also appears more biologically relevant than screening using the recombinant Mip protein alone and less tedious than a test performed directly on Legionella bacteria. We obtained 21 mAbs that bind strongly to L. pneumophila serogroups 1 to 13, and we validated their use in a rapid ELISA (performed in 4.5 h) and an immunochromatographic test (20 min).

Fast and simple detection of Yersinia pestis applicable to field investigation of plague foci.

Yersinia pestis, the plague bacillus, has a rodent-flea-rodent life cycle but can also persist in the environment for various periods of time. There is now a convenient and effective test (F1-dipstick) for the rapid identification of Y. pestis from human patient or rodent samples, but this test cannot be applied to environmental or flea materials because the F1 capsule is mostly produced at 37°C. The plasminogen activator (PLA), a key virulence factor encoded by a Y. pestis-specific plasmid, is synthesized both at 20°C and 37°C, making it a good candidate antigen for environmental detection of Y. pestis by immunological methods. A recombinant PLA protein from Y. pestis synthesized by an Escherichia coli strain was used to produce monoclonal antibodies (mAbs). PLA-specific mAbs devoid of cross-reactions with other homologous proteins were further cloned. A pair of mAbs was selected based on its specificity, sensitivity, comprehensiveness, and ability to react with Y. pestis strains grown at different temperatures. These antibodies were used to develop a highly sensitive one-step PLA-enzyme immunoassay (PLA-EIA) and an immunostrip (PLA-dipstick), usable as a rapid test under field conditions. These two PLA-immunometric tests could be valuable, in addition to the F1-disptick, to confirm human plague diagnosis in non-endemic areas (WHO standard case definition). They have the supplementary advantage of allowing a rapid and easy detection of Y. pestis in environmental and flea samples, and would therefore be of great value for surveillance and epidemiological investigations of plague foci. Finally, they will be able to detect natural or genetically engineered F1-negative Y. pestis strains in human patients and environmental samples.

Generation and characterization of rendomab-B1, a monoclonal antibody displaying potent and specific antagonism of the human endothelin B receptor.

Endothelin B receptor (ETBR) is a G protein-coupled receptor able to bind equally to the three identified human endothelin peptides. It is expressed primarily on vascular endothelial cells and involved in various physiological processes including vascular tone homeostasis, enteric nervous system development, melanogenesis and angiogenesis. Furthermore, overactivation or overexpression of ETBR have been associated with the development of various diseases such as cardiovascular disorders and cancers. Therefore, ETBR appears to be relevant target for the therapy or diagnosis of highly prevalent human diseases. In this study, we report the in vitro characterization of rendomab-B1, a monoclonal antibody (mAb) obtained by genetic immunization, which selectively recognizes the native form of human ETBR (hETBR). Rendomab-B1 is the first-reported mAb that behaves as a potent antagonist of hETBR. It recognizes an original extracellular conformational epitope on the receptor, distinct from the endothelin-1 (ET-1) binding site. Rendomab-B1 not only blocks ET-1-induced calcium signaling pathway and triggers rapid receptor internalization on recombinant hETBR-expressing cells, but also exerts pharmacological activities on human vascular endothelial cells, reducing both cell viability and ET-1-induced hETBR synthesis. In addition, binding experiments using rendomab-B1 on different melanoma cell lines reveal the structural and functional heterogeneity of hETBR expressed at the surface of these cancer cells, strongly suggesting the existence of tumor-specific receptors. Collectively, our results underscore the value of rendomab-B1 for research, therapeutic and diagnostic applications dealing with hETBR.

Fast and sensitive detection of Bacillus anthracis spores by immunoassay.

Bacillus anthracis is one of the most dangerous potential biological weapons, and it is essential to develop a rapid and simple method to detect B. anthracis spores in environmental samples. The immunoassay is a rapid and easy-to-use method for the detection of B. anthracis by means of antibodies directed against surface spore antigens. With this objective in view, we have produced a panel of monoclonal antibodies against B. anthracis and developed colorimetric and electrochemiluminescence (ECL) immunoassays. Using Meso Scale Discovery ECL technology, which is based on electrochemiluminescence (ECL) detection utilizing a sulfo-Tag label that emits light upon electrochemical stimulation (using a dedicated ECL plate reader, an electrical current is placed across the microplate with electrodes integrated into the bottom of the plate, resulting in a series of electrically induced reactions leading to a luminescent signal), a detection limit ranging between 0.3 × 10(3) and 10(3) CFU/ml (i.e., 30 to 100 spores per test), depending on the B. anthracis strain assayed, was achieved. In complex matrices (5 mg/ml of soil or simulated powder), the detection level (without any sample purification or concentration) was never altered more than 3-fold compared with the results obtained in phosphate-buffered saline.

Sensitive detection of Bacillus anthracis spores by immunocapture and liquid chromatography-tandem mass spectrometry.

Bacillus anthracis is one of the most dangerous agents of the bioterrorism threat. We present here a sensitive immuno-liquid chromatography-tandem mass spectrometry (immuno-LC-MS/MS) approach to spore detection in complex environmental samples. It is based on the combined specificity and sensitivity of two techniques: immunocapture and targeted mass spectrometry. The immunocapture step, realized directly on the intact spores, is essential for their selective isolation and concentration from complex environmental samples. After parallel trypsin and Glu-C digestions, proteotypic peptides corresponding to small acid-soluble spore protein-B (SASP-B) are specifically monitored in the multiple reaction monitoring (MRM) mass spectrometry mode. Peptide ratio is carefully monitored and provides an additional level of specificity, which is shown to be highly useful for distinguishing closely related samples and avoiding false-positive/negative results. Sensitivity at the level of the infectious dose is demonstrated, with limits of detection of 7 × 10(3) spores/mL of milk or 10 mg of soil. This mass spectrometry approach is thus complementary to polymerase chain reaction (PCR) techniques.

Neutralising antibodies against ricin toxin.

The Centers for Disease Control and Prevention have listed the potential bioweapon ricin as a Category B Agent. Ricin is a so-called A/B toxin produced by plants and is one of the deadliest molecules known. It is easy to prepare and no curative treatment is available. An immunotherapeutic approach could be of interest to attenuate or neutralise the effects of the toxin. We sought to characterise neutralising monoclonal antibodies against ricin and to develop an effective therapy. For this purpose, mouse monoclonal antibodies (mAbs) were produced against the two chains of ricin toxin (RTA and RTB). Seven mAbs were selected for their capacity to neutralise the cytotoxic effects of ricin in vitro. Three of these, two anti-RTB (RB34 and RB37) and one anti-RTA (RA36), when used in combination improved neutralising capacity in vitro with an IC(50) of 31 ng/ml. Passive administration of association of these three mixed mAbs (4.7 µg) protected mice from intranasal challenges with ricin (5 LD(50)). Among those three antibodies, anti-RTB antibodies protected mice more efficiently than the anti-RTA antibody. The combination of the three antibodies protected mice up to 7.5 hours after ricin challenge. The strong in vivo neutralising capacity of this three mAbs combination makes it potentially useful for immunotherapeutic purposes in the case of ricin poisoning or possibly for prevention.

Immunological and metabolomic impacts of administration of Cry1Ab protein and MON 810 maize in mouse.

We have investigated the immunological and metabolomic impacts of Cry1Ab administration to mice, either as a purified protein or as the Cry1Ab-expressing genetically modified (GM) MON810 maize. Humoral and cellular specific immune responses induced in BALB/cJ mice after intra-gastric (i.g.) or intra-peritoneal (i.p.) administration of purified Cry1Ab were analyzed and compared with those induced by proteins of various immunogenic and allergic potencies. Possible unintended effects of the genetic modification on the pattern of expression of maize natural allergens were studied using IgE-immunoblot and sera from maize-allergic patients. Mice were experimentally sensitized (i.g. or i.p. route) with protein extracts from GM or non-GM maize, and then anti-maize proteins and anti-Cry1Ab-induced immune responses were analyzed. In parallel, longitudinal metabolomic studies were performed on the urine of mice treated via the i.g. route. Weak immune responses were observed after i.g. administration of the different proteins. Using the i.p. route, a clear Th2 response was observed with the known allergenic proteins, whereas a mixed Th1/Th2 immune response was observed with immunogenic protein not known to be allergenic and with Cry1Ab. This then reflects protein immunogenicity in the BALB/c Th2-biased mouse strain rather than allergenicity. No difference in natural maize allergen profiles was evidenced between MON810 and its non-GM comparator. Immune responses against maize proteins were quantitatively equivalent in mice treated with MON810 vs the non-GM counterpart and no anti-Cry1Ab-specific immune response was detected in mice that received MON810. Metabolomic studies showed a slight "cultivar" effect, which represented less than 1% of the initial metabolic information. Our results confirm the immunogenicity of purified Cry1Ab without evidence of allergenic potential. Immunological and metabolomic studies revealed slight differences in mouse metabolic profiles after i.g. administration of MON810 vs its non-GM counterpart, but no significant unintended effect of the genetic modification on immune responses was seen.

Production and characterisation of a neutralising chimeric antibody against botulinum neurotoxin A.

Botulinum neurotoxins, produced by Clostridium botulinum bacteria, are the causative agent of botulism. This disease only affects a few hundred people each year, thus ranking it among the orphan diseases. However, botulinum toxin type A (BoNT/A) is the most potent toxin known to man. Due to their potency and ease of production, these toxins were classified by the Centers for Disease Control and Prevention (CDC) as Category A biothreat agents. For several biothreat agents, like BoNT/A, passive immunotherapy remains the only possible effective treatment allowing in vivo neutralization, despite possible major side effects. Recently, several mouse monoclonal antibodies directed against a recombinant fragment of BoNT/A were produced in our laboratory and most efficiently neutralised the neurotoxin. In the present work, the most powerful one, TA12, was selected for chimerisation. The variable regions of this antibody were thus cloned and fused with the constant counterparts of human IgG1 (kappa light and gamma 1 heavy chains). Chimeric antibody production was evaluated in mammalian myeloma cells (SP2/0-Ag14) and insect cells (Sf9). After purifying the recombinant antibody by affinity chromatography, the biochemical properties of chimeric and mouse antibody were compared. Both have the same very low affinity constant (close to 10 pM) and the chimeric antibody exhibited a similar capacity to its parent counterpart in neutralising the toxin in vivo. Its strong affinity and high neutralising potency make this chimeric antibody interesting for immunotherapy treatment in humans in cases of poisoning, particularly as there is a probable limitation of the immunological side effects observed with classical polyclonal antisera from heterologous species.

In vitro digestion of Cry1Ab proteins and analysis of the impact on their immunoreactivity.

A pepsin resistance test performed at pH 1.2 and with high pepsin to protein ratio is one of the steps of the weight-of-evidence approach used for assessment of allergenicity of new proteins. However, the use of other in vitro digestibility tests, performed in more physiologically relevant conditions and in combination with immunological assays so as to increase the value of the information gained from the studies of stability of a novel protein to digestion for the overall allergenicity assessment, has been proposed. This study then aimed to investigate the stability to digestion of Cry1Ab protoxin and toxin, insecticidal proteins expressed in genetically modified crops, using simulated gastric fluid (SGF) at different pH values and pepsin-to-substrate ratios, in the presence or absence of physiological surfactant phosphatidylcholine (PC). Electrophoresis and immunoblot patterns and residual immunoreactivity of digesta were analyzed. Although Cry1Ab protoxin is extensively degraded at pH 1.2 with high pepsin-to-protein ratio, it is only slightly degraded at pH 2.0 and conserved its immunoreactivity. Furthermore, Cry1Ab proteins were demonstrated to be stable in a more physiologically relevant in vitro digestibility test (pH 2.5, pepsin-to-substrate ratio 1:20 (w/w) with PC). Factors such as pH, SGF composition, and pepsin-to-substrate ratio then greatly influence the digestion of Cry1Ab proteins, confirming that new and more physiologically relevant in vitro digestibility tests should be also considered to study the relationship between the resistance of a protein to digestion and its allergenicity.

Immunotherapeutic effect of anti-PrP monoclonal antibodies in transmissible spongiform encephalopathy mouse models: pharmacokinetic and pharmacodynamic analysis.

Prion diseases are transmissible neurodegenerative disorders for which no therapeutic or prophylactic regimens exist. Passive immunization with appropriate antibodies directed against the cellular form of the prion protein (PrPC) can delay the onset of prion disease after peripheral infection, but mechanisms and parameters determining their in vivo efficacy remain unknown. In the present study, we characterized the main pharmacokinetic properties of anti-PrP antibodies in different mouse models expressing various levels of PrPC (Prnp(0/0), C57BL/6 and tga20 mice) in correlation with therapeutic effect. Plasma levels of free antibodies, total endogenous PrPC and PrPC-antibody complexes were monitored after a single intraperitoneal monoclonal antibody (mAb) injection. Efficacy in delaying PrPSc peripheral accumulation seemed to be associated with mAb capacity to form long-lasting complexes with endogenous PrPC in the plasma. In agreement with previous observations on cellular models of transmissible spongiform encephalopathy infection, we observed that injection of anti-PrP antibodies induced a large (up to 100-fold) increase in circulating PrPC. Finally, the most efficient antibody extended the lifespan of infected animals greatly. These results allowed us to define critical characteristics of anti-PrP mAbs associated with therapeutic efficacy and could constitute a useful reference for designing optimized passive immunotherapies for prion diseases.

A fluorescent immunochromatographic test using immunoliposomes for detecting microcystins and nodularins.

Microcystins (MCs), a group of cyclic heptapeptides produced by common cyanobacteria (blue green algae), cause both acute and chronic toxicity. Due to their toxicity, constant monitoring in drinking water, recreational waters as well as other potential exposure through ingestion of contaminated sea food, is very important. In this context, an immunochromatographic test (ICT) using a monoclonal antibody labeled with fluorescent liposomes (immunoliposomes) as tracer was developed, allowing a rapid and simple detection of a large number of MC and nodularin variants in field samples. The present ICT using immunoliposomes proved to be ten times more sensitive than the ICT using colloidal gold for labeling. To achieve quantitative measurement, this ICT was improved by including a stable signal on the control band allowing the expression of the results as a ratio of the fluorescence signals of the specific band versus the control band (SB/CB). Very low concentrations of MC-LR were detected in the analysis buffer (0.06 ng/ml), well below the guideline value of 1 ng/ml proposed by the World Health Organization (WHO), with a dynamic range from 0.06 to 1.5 ng/ml of MC-LR. This method was also validated using a hand-held commercial fluorometer (from ESE), providing the same performances obtained via the analysis station (from Kodak) used in our laboratory. Repeatability tests performed with both devices showed good accuracy (CV < 13%). Furthermore, quantification of MCs in natural samples (water bloom and Microcystis culture) was achieved using ICT, leading to similar results obtained via an EIA previously described. All these results demonstrate that this new fluorescent ICT could be used not only as a sensitive detection tool but also to quantify MCs in field samples.