Brigitte G. Dorner

MIP-1alpha, MIP-1beta, RANTES, and ATAC/lymphotactin function together with IFN-gamma as type 1 cytokines.

We analyzed for the first time the expression of chemokines in subpopulations of the murine immune system at the single-cell level. We demonstrate in vitro and in a model of murine listeriosis that macrophage inflammatory protein (MIP)-1α, MIP-1β, regulated on activation normal T cell expressed and secreted (RANTES), and activation-induced, T cell-derived, and chemokine-related cytokine (ATAC)/lymphotactin are cosecreted to a high degree with IFN-γ by activated individual natural killer (NK), CD8+ T, and CD4+ T helper 1 (Th1) cells. Functionally, ATAC and the CC chemokines cooperate with IFN-γ in the up-regulation of CD40, IL-12, and tumor necrosis factor-α, molecules playing a central role in the effector phase of macrophages. Our data indicate that (i) MIP-1α, MIP-1β, RANTES, and ATAC are not only chemoattractants but also coactivators of macrophages, (ii) MIP-1α, MIP-1β, RANTES, and ATAC constitute together with IFN-γ a group of “type 1 cytokines,” and (iii) these cytokines act together as a functional unit that is used by NK cells in the innate phase and then “handed over” to CD8+ T cells in the antigen-specific phase of the immune defense, thus bridging the two components of a Th1 immune reaction.

Selective Expression of the Chemokine Receptor XCR1 on Cross-presenting Dendritic Cells Determines Cooperation with CD8+ T Cells

The expression of the chemokine receptor XCR1 and the function of its ligand XCL1 (otherwise referred to as ATAC, lymphotactin, or SCM-1) remained elusive to date. In the present report we demonstrated that XCR1 is exclusively expressed on murine CD8(+) dendritic cells (DCs) and showed that XCL1 is a potent and highly specific chemoattractant for this DC subset. CD8(+) T cells abundantly secreted XCL1 8-36 hr after antigen recognition on CD8(+) DCs in vivo, in a period in which stable T cell-DC interactions are known to occur. Functionally, XCL1 increased the pool of antigen-specific CD8(+) T cells and their capacity to secrete IFN-gamma. Absence of XCL1 impaired the development of cytotoxicity to antigens cross-presented by CD8(+) DCs. The XCL1-XCR1 axis thus emerges as an integral component in the development of efficient cytotoxic immunity in vivo.

Simultaneous quantification of five bacterial and plant toxins from complex matrices using a multiplexed fluorescent magnetic suspension assay

Proteotoxins such as ricin, abrin, botulinum neurotoxins type A and B (BoNT/A, BoNT/B) and staphylococcal enterotoxin B (SEB) are regarded as potential biological warfare agents which could be used for bioterrorism attacks on the food chain. In this study we used a novel immunisation strategy to generate high-affinity monoclonal and polyclonal antibodies against native ricin, BoNT/A, and BoNT/B. The antibodies were used along with antibodies against SEB and abrin to establish a highly sensitive magnetic and fluorescent multiplex bead array with excellent sensitivities between 2 ng/L and 546 ng/L from a minimal sample volume of 50 microL. The assay was validated using 20 different related analytes and the assay precision was determined. Advancing the existing bead array technology, the novel magnetic and fluorescent microbeads proved amenable to enrichment procedures, by further increasing sensitivity to 0.3-85 ng/L, starting from a sample volume of 500 microL. Furthermore, the method was successfully applied for the simultaneous identification of the target toxins spiked into complex food matrices like milk, baby food and yoghurt. On the basis of our results, the assay appears to be a good tool for large-scale screening of samples from the food supply chain.

Ricinus communis Intoxications in Human and Veterinary Medicine—A Summary of Real Cases

Accidental and intended Ricinus communis intoxications in humans and animals have been known for centuries but the causative agent remained elusive until 1888 when Stillmark attributed the toxicity to the lectin ricin. Ricinus communis is grown worldwide on an industrial scale for the production of castor oil. As by-product in castor oil production ricin is mass produced above 1 million tons per year. On the basis of its availability, toxicity, ease of preparation and the current lack of medical countermeasures, ricin has gained attention as potential biological warfare agent. The seeds also contain the less toxic, but highly homologous Ricinus communis agglutinin and the alkaloid ricinine, and especially the latter can be used to track intoxications. After oil extraction and detoxification, the defatted press cake is used as organic fertilizer and as low-value feed. In this context there have been sporadic reports from different countries describing animal intoxications after uptake of obviously insufficiently detoxified fertilizer. Observations in Germany over several years, however, have led us to speculate that the detoxification process is not always performed thoroughly and controlled, calling for international regulations which clearly state a ricin threshold in fertilizer. In this review we summarize knowledge on intended and unintended poisoning with ricin or castor seeds both in humans and animals, with a particular emphasis on intoxications due to improperly detoxified castor bean meal and forensic analysis.

Monitoring of laying capacity, immunoglobulin Y concentration, and antibody titer development in chickens immunized with ricin and botulinum toxins over a two-year period

Abstract One of the key benefits in using chickens for immunization is the high yield of antibodies obtainable. It is known that egg production decreases over time, while animal maintenance costs remain stable. It would, however, be desirable to keep hens as long as possible to obtain maximal amounts of antibodies. To identify a suitable length of time that animals can be kept and to optimize the cost:yield ratio, we monitored the number of eggs laid, the total amount of chicken IgY, and the specific antibody titer from individually prepared eggs over a 2-yr period. The plant toxin ricin and the Clostridium botulinum neurotoxins type A and B were used to immunize 4 chickens. The number of eggs laid in 2 yr was approximately 600 per hen (about 80% of the maximum egg number), yielding about 20 to 40 g of total IgY per hen. A stable antibody titer of 1:100,000 to 1:1,000,000, as measured by ELISA, was obtained following up to 11 injections of 10 to 20 μg of immobilized native toxin. Laying capacities were found to decrease, on average, from 7 eggs/wk at the point of first immunization to 2 eggs/wk after more than 2 yr. In parallel, the yield of total and specific IgY increased over time, so that the antibody recovery remained high, even after prolonged immunization times. Using purified IgY preparations, classical immunological assays such as ELISA and Western blotting were performed. Furthermore, the IgY showed neutralizing capacity when used to block the functional activity of the toxins both in vitro and in vivo. Analysis of the total IgY content over time demonstrated a complex biological oscillation (and the antigen-specific titer), with a shorter time period of around 7 d (circaseptan rhythm). In summary, we successfully immunized chickens with ricin and botulinum neurotoxins and monitored laying capacity, IgY concentration, and specific antibody titer over an extended period of 2 yr.

Purification, Structural Analysis, and Function of Natural ATAC, a Cytokine Secreted by CD8+ T Cells

Recently, we identified a novel putative human cytokine expressed by activated CD8+ T cells, which was designated ATAC (ctivation-induced, cell-derived, nd hemokine-related; the same molecule has been identified independently as lymphotactin and single cysteine motif-1). In this report, we provide evidence that ATAC is a secreted 93-amino acid protein that is generated from its precursor by proteolytic cleavage between Gly21 and Val22. An estimated 60% of ATAC (Val22-Gly114) is secreted as an unmodified protein with a molecular mass of 10,271.72 Da (apparent molecular mass of 12 kDa in SDS-polyacrylamide gel electrophoresis) and in which Cys32 and Cys69 are linked by a disulfide bridge. Unmodified ATAC is a cationic protein with a pI of 11.35 and is capable of binding to heparin. Some 40% of ATAC is O-glycosylated within 25 min of synthesis, giving rise to the appearance of a homogeneous 15-kDa (minor fraction) and a heterogeneous, terminally sialylated 17-19-kDa (major fraction) protein species in SDS-polyacrylamide gel electrophoresis. The secretion of all ATAC protein variants is completed within 30-40 min of synthesis. In terms of function, various ATAC protein forms were consistently ineffective in chemotaxis assays. In contrast, both purified natural ATAC and a chemically synthesized aglycosyl analog induced locomotion (chemokinesis) in purified CD4+ and CD8+ T cell populations at 400 ng/ml.

Historical Perspectives and Guidelines for Botulinum Neurotoxin Subtype Nomenclature

Botulinum neurotoxins are diverse proteins. They are currently represented by at least seven serotypes and more than 40 subtypes. New clostridial strains that produce novel neurotoxin variants are being identified with increasing frequency, which presents challenges when organizing the nomenclature surrounding these neurotoxins. Worldwide, researchers are faced with the possibility that toxins having identical sequences may be given different designations or novel toxins having unique sequences may be given the same designations on publication. In order to minimize these problems, an ad hoc committee consisting of over 20 researchers in the field of botulinum neurotoxin research was convened to discuss the clarification of the issues involved in botulinum neurotoxin nomenclature. This publication presents a historical overview of the issues and provides guidelines for botulinum neurotoxin subtype nomenclature in the future.

Multiplex Detection of Microbial and Plant Toxins by Immunoaffinity Enrichment and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Plant and microbial toxins such as ricin, staphylococcal enterotoxin B (SEB), and the botulinum neurotoxins (BoNT) are considered as potential biological warfare agents. Specific screening methods are, therefore, required that enable unambiguous and sensitive identification of these biohazards, particularly for the occurrence of the toxins in complex sample matrixes. The present study describes a combination of a multiplex-immunoaffinity purification approach, followed by matrix-assisted laser desorption/ionization (MALDI)-based detection for the simultaneous identification of ricin, SEB, BoNT/A, and BoNT/B. The method comprises an affinity enrichment step, using specific monoclonal antibodies for each of the four toxins which have been selected from a pool of antibodies. The selected antibodies allow for specific and simultaneous capture of ricin, SEB, BoNT/A, BoNT/B, and the corresponding BoNT complexes. These were subsequently identified by MALDI time-of-flight (TOF) mass spectrometry (MS), following tryptic digest. The sensitivity of the technique was approximately 500 fmol for each of the toxins. These toxins were detectable within 8 h, even when present in complex matrixes such as milk or juice. Furthermore, the MALDI-based multiplex assay allowed for the discrimination of closely related BoNT sero- and subtypes, including a real case of food-borne botulism in Germany.

Isolation and functional characterization of the novel Clostridium botulinum neurotoxin A8 subtype.

Botulism is a severe neurological disease caused by the complex family of botulinum neurotoxins (BoNT). Based on the different serotypes known today, a classification of serotype variants termed subtypes has been proposed according to sequence diversity and immunological properties. However, the relevance of BoNT subtypes is currently not well understood. Here we describe the isolation of a novel Clostridium botulinum strain from a food-borne botulism outbreak near Chemnitz, Germany. Comparison of its botulinum neurotoxin gene sequence with published sequences identified it to be a novel subtype within the BoNT/A serotype designated BoNT/A8. The neurotoxin gene is located within an ha-orfX+ cluster and showed highest homology to BoNT/A1, A2, A5, and A6. Unexpectedly, we found an arginine insertion located in the HC domain of the heavy chain, which is unique compared to all other BoNT/A subtypes known so far. Functional characterization revealed that the binding characteristics to its main neuronal protein receptor SV2C seemed unaffected, whereas binding to membrane-incorporated gangliosides was reduced in comparison to BoNT/A1. Moreover, we found significantly lower enzymatic activity of the natural, full-length neurotoxin and the recombinant light chain of BoNT/A8 compared to BoNT/A1 in different endopeptidase assays. Both reduced ganglioside binding and enzymatic activity may contribute to the considerably lower biological activity of BoNT/A8 as measured in a mouse phrenic nerve hemidiaphragm assay. Despite its reduced activity the novel BoNT/A8 subtype caused severe botulism in a 63-year-old male. To our knowledge, this is the first description and a comprehensive characterization of a novel BoNT/A subtype which combines genetic information on the neurotoxin gene cluster with an in-depth functional analysis using different technical approaches. Our results show that subtyping of BoNT is highly relevant and that understanding of the detailed toxin function might pave the way for the development of novel therapeutics and tailor-made antitoxins.

Pentaplexed Quantitative Real-Time PCR Assay for the Simultaneous Detection and Quantification of Botulinum Neurotoxin-Producing Clostridia in Food and Clinical Samples

ABSTRACT Botulinum neurotoxins are produced by the anaerobic bacterium Clostridium botulinum and are divided into seven distinct serotypes (A to G) known to cause botulism in animals and humans. In this study, a multiplexed quantitative real-time PCR assay for the simultaneous detection of the human pathogenic C. botulinum serotypes A, B, E, and F was developed. Based on the TaqMan chemistry, we used five individual primer-probe sets within one PCR, combining both minor groove binder- and locked nucleic acid-containing probes. Each hydrolysis probe was individually labeled with distinguishable fluorochromes, thus enabling discrimination between the serotypes A, B, E, and F. To avoid false-negative results, we designed an internal amplification control, which was simultaneously amplified with the four target genes, thus yielding a pentaplexed PCR approach with 95% detection probabilities between 7 and 287 genome equivalents per PCR. In addition, we developed six individual singleplex real-time PCR assays based on the TaqMan chemistry for the detection of the C. botulinum serotypes A, B, C, D, E, and F. Upon analysis of 42 C. botulinum and 57 non-C. botulinum strains, the singleplex and multiplex PCR assays showed an excellent specificity. Using spiked food samples we were able to detect between 103 and 105 CFU/ml, respectively. Furthermore, we were able to detect C. botulinum in samples from several cases of botulism in Germany. Overall, the pentaplexed assay showed high sensitivity and specificity and allowed for the simultaneous screening and differentiation of specimens for C. botulinum A, B, E, and F.