Gareth D. Griffiths

Morphology of ricin and abrin exposed endothelial cells is consistent with apoptotic cell death

Cultures of bovine pulmonary endothelial (BPE) cells were exposed to LC70 doses of ricin or abrin (15.5 and 4.5 pM respectively) over a period of up to 40 h. The viability of the cultures (as determined by the neutral red (NR) dye retention assay) declined after 6 h exposure to the toxins. From 15 h onwards, cellular material in toxin exposed cultures became detached from the substra tum of the culture vessels. Hoffman modulation contrast photomicrography showed that this process was due to ricin and abrin exposed cells collapsing into membrane bound vesicles which retained the NR dye, became detached and floated into the medium. These apoptotic-like structural changes were further investi gated by transmission electron microscopy (TEM) and by agarose gel electrophoresis of DNA from control and exposed cultures. Many of the characteristic changes associated with apoptotic cell death were seen using TEM, including heterochromatin condensation at the nuclear periphery, crenulation of the nuclear mem brane and progressive degeneration of residual nuclear and cytoplasmic structures. The plasma membrane of many cells remained intact, and contained nuclear and cytoplasmic debris. Agarose gel electrophoresis of DNA extracted from toxin-treated cells revealed oligonucleosome sized DNA fragments, characteristic of apoptosis, from adherent cells at 7 h and both adherent and floating populations when harvested from 15 h; DNA from unexposed control cells did not show this fragmentation. The identification of apopto sis as being a significant additional mechanism of toxicity following exposure to ricin and abrin holotox ins raises the possibility of developing new therapeutic strategies against poisoning by these phytotoxins.

Inhalation Toxicology and Histopathology of Ricin and Abrin Toxins

AbstractThe inhalation toxicology of ricin (supplied by Sigma) from the seed variety “Hale Queen” and abrin was examined following head-only exposure of rats to a range of concentrations of each toxin generated as an aerosol from solution using a constant-output nebulizer. The inhalation toxicity of an in-house preparation of ricin from a different seed type, Ricinus communis var. zanzibariensis (R. zanzibariensis), was also assessed for comparison. The approximate LCt50 values determined were very similar for the Sigma ricin and abrin (4.54–5.96 and 4.54 mg min m-3, respectively). However, the LCt50 of ricin toxin prepared in-house from seeds of the R. zanzibariensis variety was assessed to be 12.7 mg min m-3. Ricin prepared from this seed variety was therefore less toxic than Sigma ricin by a factor of almost threefold. Given that both ricin preparations were pure by silver-stained, sodium dodecyl sulfate polyacrylamide electrophoresis gels, the data must reflect differences in specific toxicity between...

Protection against inhalation toxicity of ricin and abrin by immunisation.

1 Abrin and ricin are highly toxic plant proteins which are very similar in structure and function and inhibit protein synthesis in eukaryotes. 2 Rats have been immunised against either toxin using formaldehyde-toxoids by three subcutaneous injections at intervals of 3 weeks. For abrin, serum titres in 14 out of 15 rats were raised to between 1 : 12800 and 1 : 51200 after two injections, 6 weeks from the start of the experiment. Titres of between 1 : 256 and 1 : 1024 were also measured in lung washes after challenge with active abrin toxin. 3 The three major antibody classes, IgG, IgM and IgA were present in the immune sera but IgG and IgA only were detected in lung washes. The proportion of IgA to IgG was higher in the lung fluid than in sera. Rats immunised by abrin toxoid were protected against 5 LCt50s of abrin by inhalation but others exposed to ricin were not. 4 For ricin, serum titres ranged from 1 : 800 to 1 : 25600 after two injections and after a third injection the titre range was the same but population samples were weighted towards the higher titres. All rats immunised with ricin toxoid survived the challenge of 5 LCt50s of ricin toxin by inhalation over the observation period of 28 days post-challenge. 5 Representative immunised rats (abrin toxoid) were taken at various times post-exposure, humanely killed and tissues were examined for pathological changes. It was concluded that an apparently severe lung lesion occurred at a later time than in non-immunised, toxin challenged rats. This damage was not lethal over the experimental observation periods. 6 Immunisation by the sub-cutaneous route therefore protects against lethality from challenge by inhalation of ricin or abrin toxins but does not prevent significant lung damage.

Liposomally-encapsulated ricin toxoid vaccine delivered intratracheally elicits a good immune response and protects against a lethal pulmonary dose of ricin toxin

A small study was performed to examine whether the instillation of ricin toxoid vaccine into the lungs of Porton rats offered protection from lethal effects of subsequent intratracheal challenge with ricin toxin. Further the immune response to liposomally-encapsulated vaccine and the protection offered was compared with vaccine either adsorbed to Alhydrogel adjuvant or as a simple aqueous solution. The formaldehyde-treated ricin toxin vaccine (RTV) was administered at two dose levels, 500 and 100 micrograms kg-1 body weight to groups of rats, on two occasions by intratracheal instillation. Liposomally-encapsulated vaccine (LRTV) produced a higher titre of ricin-specific antibodies than Alhydrogel-vaccine (ARTV) and vaccine solution. When challenged with 3 LD50 of ricin by intratracheal instillation 7 weeks after the second vaccine instillation, all rats in both LRTV dose groups survived with minimal signs of incapacitation. Analysis of antibody secretion by spleen cells, 14 days post challenge, showed that the IgG isotype in the LRTV group was significantly higher than that in the ARTV and RTV groups and also that the proportion of specific IgA in lung fluid was higher in the LRTV group than in the ARTV and RTV groups. The results of this study indicate that effective vaccinations against inhaled ricin could be achieved with liposomally-encapsulated ricin toxoid, via the lung and should be investigated further.

Comparison of the quality of protection elicited by toxoid and peptide liposomal vaccine formulations against ricin as assessed by markers of inflammation

Ricin is a very toxic substance which inhibits protein synthesis and produces severe tissue damage and inflammation. It is very potent when inhaled as an aerosol and protection has been examined in a series of studies using vaccine candidates including a formaldehyde inactivated ricin toxoid and the A chain of ricin, a polypeptide equivalent to half of the toxin molecule. Initially, subcutaneous injections of both compounds were found to protect against inhaled ricin but not without some subsequent adverse signs. Intra-pulmonary vaccination using liposomal formulations of these compounds was investigated with a view to improving lung condition following challenge. Using the humoral and local pulmonary immune responses as indices of vaccine performance, no significant difference between toxoid or peptide vaccines was found. In the third and current study, the quality of lung protection by vaccines was assessed using markers of inflammation. Thus, the profiles of inflammatory cell and protein influx into the lung were determined following intratracheal (i.t.) challenge with ricin of rats treated with liposomal vaccine formulations. Results showed that liposomal ricin toxoid offered a better quality of protection with a significantly lower influx of polymorphonuclear leucocytes (neutrophils) and little pulmonary oedema compared with the A chain/liposome formulation. Further, there was no significant difference between the quality of protection offered by the A chain when administered subcutaneously or locally into the lung by i.t. instillation. Liposomal ricin toxoid is a good candidate vaccine and optimised pulmonary delivery by inhalation should be further examined.

An extensive monoclonal antibody panel for the phenotyping of leukocyte subsets in the common marmoset and the cotton-top tamarin

New World monkeys are valuable animal models to study human diseases. To determine the phenotype of cells involved in immune responses, we used flow cytometry to screen a large panel of anti-human monoclonal antibodies (mAb) for cross-reactivity with cells of the common marmoset and the cotton-top tamarin. Certain antigens (e.g., CD2, CD8, CD20) are well conserved. However, CD10, CD23, and CD33 showed a clear discrepancy in their reaction patterns in both species, indicating that significant differences on the epitope level occurred during evolution. Epstein-Barr virus-transformed B-cell lines were shown to be a valuable tool for screening B-cell-specific reagents. In some cases, fluorescein isothiocyanate (FITC) and phycoerythrin (PE) modification of mAbs had a negative effect on the binding capacity, which stressed the importance of choosing the right label. Despite the fact that some CD antigens were not detected, adequate numbers of cross-reactive mAbs were identified to perform extensive studies on immunological functions in both the common marmoset and the cotton-top tamarin.

Examination of the toxicity of several protein toxins of plant origin using bovine pulmonary endothelial cells.

The bovine pulmonary endothelial (BPE) cell line was examined as a model to study the toxicity of ricin and abrin toxins currently under investigation. The BPE cell line was examined because ricin has been shown to bind to endothelial cells. Cell viability was assessed using several different biochemical parameters including growth (DNA by binding of gentian violet stain), mitochondrial function (succinate dehydrogenase activity) using MTT and lysosomal integrity (neutral red retention assay). In order to compare toxicities and investigate potential protective compounds, concentrations of toxins causing death of 50% and 70% of the (control) cell population (LC50 and LC70, respectively) were determined. It is concluded that while ricin and abrin share a common mechanism of action ricin is slightly less toxic than abrin. BPE cells are a good model for future mechanistic studies and particularly for initial phase screening of potentially therapeutic compounds. Carbohydrates were used in an attempt to examine which receptor types were involved in the binding and uptake of ricin and abrin by the cell line. It was found that only high concentrations of galactose prevented lethality while mannose apparently had no effect. Furthermore, the molar excess of carbohydrate to toxin required in order to achieve protection indicated that this would be an impractical approach to adopt in vivo.

Local and systemic responses against ricin toxin promoted by toxoid or peptide vaccines alone or in liposomal formulations.

The objective of this study was to develop a vaccine which would ultimately protect man from the lethal effects of inhaled ricin toxin. Porton rats have previously been protected from lethal quantities of inhaled ricin by subcutaneous (s.c.) ricin toxoid vaccine, but not without lung damage. This situation might be improved by an alternative vaccine such as the A chain of ricin, already known to protect against inhaled ricin. Another option would be to improve respiratory tract immunity by local vaccination in conjunction with liposomal formulation with a view to enhancing lung secretion of immune IgA. While boosted s.c. doses of ricin toxoid or A chain produced indistinguishable systemic immune responses 3 weeks later, when delivered by the intratracheal (i.t.) route, the A chain failed to elicit a specific immune response, unlike ricin toxoid. This situation was overcome by liposomal formulations and although ricin toxoid was readily encapsulated in liposomes, A chain was not. However, by simply mixing A chain and liposomes in the same weight ratio determined for liposomal toxoid, systemic immune responses for each formulation were indistinguishable 1 week after boosting. Ricin antibody responses in lung fluid monitored 1, 3, 7 and 14 days after i.t. challenge with ricin were statistically indistinguishable, but the group vaccinated with liposomal toxoid secreted 28.7% IgA compared with 0.9-14.9% for the A chain liposomal group. From this, it might be anticipated that the lungs would be better protected by liposomally-encapsulated ricin toxoid than by the A chain-liposome mixture.

A Histochemical Study of Changes Observed in the Mouse Diaphragm after Organophosphate Poisoning

A sublethal dose of sarin (GB, isopropyl methylphosphonofluoridate) was administered to mice. The animals were killed up to 28 d after dosing and frozen sections were made of the excised diaghragms which were stained using haematoxylin and eosin and a modified Gomori trichrome method. Muscle fibre degeneration and mononuclear infiltration were seen, notably at 24 h and 3 d. A number of histochemical procedures were carried out, including the GBHA procedure for ionized calcium. Calcium accumulation, seen at 4 h, was the earliest abnormality observed. All changes were rapidly regressing by 5 d and histological appearances were normal by 14 d. It was concluded that sarin produced myopathic changes preceded by calcium accumulation.

Broad recognition of ricin toxins prepared from a range of Ricinus cultivars using immunochromatographic tests.

Introduction. It is extremely important that diagnosis of persons exposed to ricin be achieved in a timely fashion for triage and appropriate treatment. A sensitive and specific immunochromatographic test (ICT) for ricin has been developed for this purpose and reported recently. This ICT detected ricin of a single cultivar, by naked eye, at concentrations as low as 1 ng/mL. However, there are many cultivars of the Ricinus communis plant, each producing ricin. Further, significant differences in ricin toxicity between two cultivars have been formerly demonstrated. Correspondingly, ricins extracted from different cultivars might exhibit variations in primary sequence or glycosylation, leading to differences in the limits of detectability. This was the purpose of this study. Methods. The ICT was used against solutions of ricin samples extracted from a wide range of cultivars to determine whether it could detect all the samples and to determine whether there were differences between the limits of visible detection using the ICT and also in the detection limits using a densitometer. Results. The ICT successfully identified ricins prepared from 19 Ricinus cultivars, all with a limit of visible detection between 1 and 2.5 ng/mL in buffer. This depended to some extent on whether the operator was experienced or not in reading the test. Discussion. This rapid and sensitive test provides a platform for the rapid diagnosis of ricin poisoning using relevant biological samples and will now be evaluated in animal models of inhalational exposure to ricin. Conclusion. This sensitive and rapid test may provide an early diagnosis to inform the administration of a ricin antitoxin currently in development at Defence Science and Technology Laboratory in the United Kingdom.