A case report of a 25-year-old man who committed suicide by intravenous injection himself of an aqueous home-made castor bean extract is presented. The patient was hospitalized and treated symptomatically and was released at its own request fourth day after intoxication. The next day, the patient\'s condition deteriorated, and he died 6 days after intoxication even though he was given medical care. Case history, autopsy, and toxicological investigation of ante- and post-mortem collected materials are described. Blood and urine collected from the patient ante-mortem and other several biological materials (namely blood from the upper and lower limb, blood from the right and left ventricle, pericardial fluid, vitreous humour, liver, kidney, and spleen) were collected post-mortem during autopsy. Liquid-liquid extraction procedure followed by high-performance liquid chromatography tandem mass spectrometry analysis for identification and determination of ricinine as a biomarker of ricin/castor seed intoxication was developed and validated. The method was applied on analysis of collected ante- and post-mortem biological materials. The post-mortem contents of ricinine in organs (namely the liver, kidney, and spleen) are firstly reported. The obtained results indicated approximately uniform distribution of ricinine (concentration level about 1 ng mL-1) in the body after death. In addition, the GC-MS method was also applied for the analysis of extract of castor seed and the patient\'s urine, to demonstrate alternative possibility for identification of ricinine for clinical and forensic purposes.

Ricin is a potent toxin and potential bioterrorism weapon for which no specific licensed countermeasures are available. We report the safety and immunogenicity of the ricin vaccine RVEc™ in a Phase 1 (N=30) multiple-dose, open-label, non-placebo-controlled, dose-escalating (20, 50, and 100μg), single-center study. Each subject in the 20- and 50-μg dose groups (n=10 for each group) received three injections at 4-week intervals and was observed carefully for untoward effects of the vaccine; blood was drawn at predetermined intervals after each dose for up to 1 year. RVEc™ was safe and well tolerated at the 20- and 50-μg doses. The most common adverse events were pain at the injection site and headache. Of the 10 subjects who received a single 100-μg dose, two developed elevated creatine phosphokinase levels, which resolved without sequelae. No additional doses were administered to subjects in the 100-μg group. Immunogenicity of the vaccine was evaluated by measuring antibody response using the well standardized enzyme-linked immunosorbent assay (ELISA) and toxin neutralization assay (TNA). Of the subjects in the 20- and 50-μg dose groups, 100% achieved ELISA anti-ricin IgG titers of 1:500 to 1:121,500 and 50% produced neutralizing anti-ricin antibodies measurable by TNA. Four subjects in the 50-μg group received a single booster dose of RVEc™ 20-21 months after the initial dose. The single booster was safe and well tolerated, resulting in no serious adverse events, and significantly enhanced immunogenicity of the vaccine in human subjects. Each booster recipient developed a robust anamnestic response with ELISA anti-ricin IgG titers of 1:13,500 to 1:121,500 and neutralizing antibody titers of 1:400 to 1:3200. Future studies will attempt to optimize dose, scheduling, and route of administration. This study is registered at clinicaltrials.gov (NCT01317667 and NCT01846104).

Ricin, a large, water soluble toxic glycoprotein, is distributed majorly in the kernels of castor beans (the seeds of Ricinus communis L.) and has been used in traditional Chinese medicine (TCM) or other folk remedies throughout the world. The toxicity of crude ricin (CR) from castor bean kernels was investigated for the first time using an NMR-based metabolomic approach complemented with histopathological inspection and clinical chemistry. The chronic administration of CR could cause kidney and lung impairment, spleen and thymus dysfunction and diminished nutrient intake in rats. An orthogonal signal correction partial least-squares discriminant analysis (OSC-PLSDA) of metabolomic profiles of rat biofluids highlighted a number of metabolic disturbances induced by CR. Long-term CR treatment produced perturbations on energy metabolism, nitrogen metabolism, amino acid metabolism and kynurenine pathway, and evoked oxidative stress. These findings could explain well the CR induced nephrotoxicity and pulmonary toxicity, and provided several potential biomarkers for diagnostics of these toxicities. Such a (1)H NMR based metabolomics approach showed its ability to give a systematic and holistic view of the response of an organism to drugs and is suitable for dynamic studies on the toxicological effects of TCM.

Ricin is one of the most poisonous natural toxins from plants and is classified as a Class B biological threat pathogen by the Centers for Disease Control and Prevention (CDC) of U.S.A. Ricin exposure can occur through oral or aerosol routes. Ricin poisoning has a rapid onset and a short incubation period. There is no effective treatment for ricin poisoning. In this study, an aerosolized ricin-exposed mouse model was developed and the pathology was investigated. The protein expression profile in the ricin-poisoned mouse lung tissue was analyzed using proteomic techniques to determine the proteins that were closely related to the toxicity of ricin. 2D gel electrophoresis, mass spectrometry and subsequent biological functional analysis revealed that six proteins including Apoa1 apolipoprotein, Ywhaz 14-3-3 protein, Prdx6 Uncharacterized Protein, Selenium-binding protein 1, HMGB1, and DPYL-2, were highly related to ricin poisoning.

BACKGROUND: The modelling framework is proposed to study protection properties of antibodies to neutralize the effects of the plant toxin (ricin). The present study extends our previous work by including (i) the model of intracellular transport of toxin to the Endoplasmic Reticulum and (ii) the model of the internalised antibodies (when antibody is delivered directly into the cytosol).
METHODS: Simulation of the receptor-toxin-antibody interaction is implemented by solving the systems of PDEs (advection-diffusion models) or ODEs (rate models) for the underlying transport coupled with mass-action kinetics.
RESULTS: As the main application of the enhanced framework we present a comparative study of two kinds (external and internalised) of antibodies. This comparison is based on calculation of the non-dimensional protection factor using the same set of parameters (geometry, binding constants, initial concentrations of species, and total initial amount of the antibody).
CONCLUSIONS: This research will provide a framework for consistent evaluation and comparison of different types of antibodies for toxicological applications.

Ricin, Ricinus communis agglutinin 60 - RCA 60, is a deadly phytotoxic protein which inhibits ribosomes (class II), and there is no known effective antidote in living organisms. Ricin is composed of two polypeptide chains, A and B, linked covalently by a single disulfide bond. The analytical methods for the detection of RCA 60 are commonly laborious, expensive, require skilled labor, and involve sophisticated equipment. Aimed at the development of electroanalytical methods for RCA 60 detection, here we studied the electrochemical oxidation of RCA 60 on a glassy carbon (GC) electrode over a wide pH range, using cyclic voltammetry, differential pulse voltammetry (DPV) and square wave voltammetry (SWV). Two quasi-reversible electrochemical RCA 60 oxidation peaks were identified on the GC electrode by SWV. For values of 2.2 ≤ pH ≤ 10.2, DPV studies revealed that the peak potentials, EP1 and EP2, display a linear dependence with pH and the reaction mechanism involves the transfer of 2H⁺/2e⁻ (peak 1) and 1H⁺/1e⁻ (peak 2). The first and second RCA 60 oxidation steps may correspond to the oxidation of cysteine and tyrosine-tryptophan residues, respectively. The oxidation product of the second RCA 60 oxidation step appears at 7.0 ≤ pH ≤ 11.8. For pH ≥ 10.2, both processes are pH independent, resulting in a pKa of ca. 10.2. A third RCA 60 oxidation peak only appears at acidic pH. RCA 60 samples extracted from different castor seed cultivars showed similar electrochemical behavior, enabling the implementation of an analytical voltammetric method.

Fluorescent silica nanoparticles are reported to be highly stable and biocompatible materials with high water solubility, which make them ideal candidates for biological applications. These nanoparticles can also be modified with biocompatible and targeting moieties and can be used for a variety of in vitro and in vivo applications, such as targeting, particle tracking, cargo carrier, and as contrast agents. In this study, fluorescent dye-doped silica nanoparticles were prepared by a modified Stöber method. The nanoparticles produced were surface functionalized with amine moieties for their conjugation with glucose-derived and galactose-based residues. The amine, glucose-derived, and galactose-based functionalized fluorescent silica nanoparticles were analyzed for their physiochemical properties such as sizes, polydispersities, organic layer content, and surface chemistries. The nanoparticles produced were then studied for their interactions with carbohydrate-specific lectins. These lectin bioconjugates have helped in understanding their interactions with cell-surface receptors. As expected, galactose-functionalized nanoparticles were found to specifically interact with RCA120 , as compared to other nanoparticles. These specific interactions of galactose-lectin conjugates were further studied on the hepatocytes cell surface in vitro. The aggregation of galactose-lectins conjugates on the plasma membrane was possibly due to the specific interactions of carbohydrates with cell-surface glycoproteins, hence preventing the uptake of these nanoparticles. The study has provided an interesting approach to mark the cell-surface glycoproteins with fluorescent probes using a combination of lectin-carbohydrate conjugates.

There is no FDA-approved vaccine for the potent plant toxin ricin. We have developed a recombinant ricin vaccine, RiVax. Without adjuvant it is safe and immunogenic in mice, rabbits, and humans. Based on our studies in mice, we now report the results of a small clinical trial with Alhydrogel-adsorbed RiVax.

The plant A/B toxin ricin represents a heterodimeric glycoprotein belonging to the family of ribosome inactivating proteins, RIPs. Its toxicity towards eukaryotic cells results from the depurination of 28S rRNA due to the N-glycosidic activity of ricin toxin A chain, RTA. Since the extention of RTA by a mammalian-specific endoplasmic reticulum (ER) retention signal (KDEL) significantly increases RTA in vivo toxicity against mammalian cells, we here analyzed the phenotypic effect of RTA carrying the yeast-specific ER retention motif HDEL. Interestingly, such a toxin (RTA(HDEL)) showed a similar cytotoxic effect on yeast as a corresponding RTA(KDEL) variant on HeLa cells. Furthermore, we established a powerful yeast bioassay for RTA in vivo uptake and trafficking which is based on the measurement of dissolved oxygen in toxin-treated spheroplast cultures of S. cerevisiae. We show that yeast spheroplasts are highly sensitive against external applied RTA and further demonstrate that its toxicity is greatly enhanced by replacing the C-terminal KDEL motif by HDEL. Based on the RTA resistant phenotype seen in yeast knock-out mutants defective in early steps of endocytosis (∆end3) and/or in RTA depurination activity on 28S rRNA (∆rpl12B) we feel that the yeast-based bioassay described in this study is a powerful tool to dissect intracellular A/B toxin transport from the plasma membrane through the endosomal compartment to the ER.

Novel agents are needed for patients with refractory and relapsed acute lymphoblastic leukaemia (ALL). Combotox is a 1:1 mixture of two immunotoxins (ITs), prepared by coupling deglycosylated ricin A chain (dgRTA) to monoclonal antibodies directed against CD22 (RFB4-dgRTA) and CD19 (HD37-dgRTA). Pre-clinical data demonstrated that Combotox was effective in killing both pre-B-ALL cell lines and cells from patients with pre-B ALL. A clinical study of paediatric patients in which 3 of 17 patients with ALL experienced complete remission, supported the preclinical work and motivated this study. This study was a Phase I, dose-escalation trial using Combotox in adults with refractory or relapsed B-lineage-ALL. A cycle consisted of three doses, with one dose given every other day. Dose levels were 3, 5, 6, 7 and 8 mg/m(2) per dose. Seventeen patients, aged 19-72 years, were enrolled in this multi-institution study. The maximum tolerated dose was 7 mg/m(2) /dose (21 mg/m(2) /cycle) and vascular leak syndrome was the dose-limiting toxicity. Two patients developed reversible grade 3 elevations in liver function tests. One patient achieved partial remission and proceeded to allogeneic stem cell transplantation. All patients with peripheral blasts experienced decreased blast counts following the administration of Combotox. Thus, Combotox can be safely administered to adults with refractory leukaemia.