Toxicity Of Toxin Research Articles

Aedes aegypti alkaline phosphatase ALP1 is a functional receptor of Bacillus thuringiensis Cry4Ba and Cry11Aa toxins

Bacillus thuringiensis subs. israelensis produces at least three Cry toxins (Cry4Aa, Cry4Ba, and Cry11Aa) that are active against Aedes aegypti larvae. Previous work characterized a GPI-anchored alkaline phosphatase (ALP1) as a Cry11Aa binding molecule from the gut of A. aegypti larvae. We show here that Cry4Ba binds ALP1, and that the binding and toxicity of Cry4Ba mutants located in loop 2 of domain II is correlated. Also, we analyzed the contribution of ALP1 toward the toxicity of Cry4Ba and Cry11Aa toxins by silencing the expression of this protein though RNAi. Efficient silencing of ALP1 was demonstrated by real-time quantitative PCR (qPCR) and Western blot. ALP1 silenced larvae showed tolerance to both Cry4Ba and Cry11Aa although the silenced larvae were more tolerant to Cry11Aa in comparison to Cry4Ba. Our results demonstrate that ALP1 is a functional receptor that plays an important role in the toxicity of the Cry4Ba and Cry11Aa proteins.

T-2 toxin induces apoptosis in differentiated murine embryonic stem cells through reactive oxygen species-mediated mitochondrial pathway

T-2 toxin, a member of the trichothecene mycotoxin family produced by the Fusarium fungi, has been shown to exert a variety of toxic effects on multiple targets in vivo. However, the embryonic toxicity of T-2 toxin in vitro remains unclear. In the present study, two permanent cell lines, embryonic stem cells (ES cells D3) and fibroblast 3T3 cells, were used to evaluate T-2 toxin toxicity. Differentiated mouse ES cells were cultivated as embryoid bodies along with T-2 toxin at different concentrations (0.5, 1, and 2 ng/ml) for 24 h. The increases in cellular reactive oxygen species (ROS), lipid and DNA oxidative damage, and loss of mitochondrial transmembrane potential were observed at 1 and 2 ng/ml concentrations. Flow cytometry showed that T-2 toxin induced cell cycle arrest and apoptosis. Furthermore, T-2 toxin opened the mitochondrial permeability transition pore, caused the release of cytochrome c from mitochondria and induced the upregulation of p53, caspase-9, caspase-3 expression and increased the ratio of Bax/Bcl-2. However, T-2 toxin-induced oxidative damage and apoptosis in differentiated ES cells decreased significantly in the presence of the antioxidant Trolox. Taken together, these results demonstrate that T-2 toxin induces oxidative stress and apoptosis in differentiated murine ES cells, and ROS-mediated mitochondrial pathway plays an important role in T-2 toxin induced apoptosis.

Role of chondroitin sulfate C in the action of anthrax toxin

Anthrax toxin is produced by Bacillus anthracis, the causative agent of anthrax, and is responsible for the majority of disease symptoms. The toxin consists of 3 proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF), which combine to form lethal and edema toxin. Glycosaminoglycans, which are present on the surface of cells, were investigated with regard to their role in toxicity resulting from anthrax toxin exposure. Lethal toxin-induced cytotoxicity of the RAW 264.7 cells was significantly inhibited by the addition of chondroitin sulfate C as determined by the MTT assay. By contrast, several other glycosaminoglycans, including heparin, heparan sulfate, and dermatan sulfate did not show significant levels of inhibition. Studies utilizing fluorescence-labeled PA demonstrated decreased PA binding to RAW 264.7 cells with the addition of chondroitin sulfate C. Formation of PA oligomers at the surface of cells after binding was also inhibited by chondroitin sulfate C. Interestingly, enzymatic degradation of endogenous chondroitin sulfate C from the cell surface with chondroitinase ABC was accompanied by increased sensitivity to the toxin. These findings were further confirmed by pretreating cells with sodium chlorate to reduce the degree of cell surface glycosaminoglycans sulfation. In addition, chondroitin sulfate C effectively inhibits edema toxin-induced cAMP accumulation in cells. Our results indicate that chondroitin sulfate C may play an important role in the toxicity of anthrax toxin.

Clostridium difficile Toxin A Inhibits the Kinase Activity of Extracellular Signal-Related Kinases 1 and 2 Through Direct Binding

Clostridium difficile toxin A glucosylates Rho family proteins, resulting in actin filament disaggregation and cell rounding in cultured colonocytes. Given that the cellular toxicity of toxin A is dependent on its receptor binding and subsequent entry into the cell, we herein sought to identify additional colonocyte proteins that might bind to toxin A following its internalization. Our results revealed that toxin A interacted with ERK1 and ERK2 in two human colonocyte cell lines (NCM460 and HT29). A GST-pulldown assay also showed that toxin A can directly bind to ERK1 and ERK2. In NCM460 cells exposed to PMA (an ERK1/2 activator), the phosphorylation of ERK1/2 did not affect the interaction between toxin A and ERK1/2. However, an in vitro kinase assay showed that the direct binding of toxin A to ERK1 or ERK2 inhibited their kinase activities. These results suggest a new molecular mechanism for the cellular toxicity seen in cells exposed to toxin A.

Co-expression and Synergism Analysis of Vip3Aa29 and Cyt2Aa3 Insecticidal Proteins from Bacillus thuringiensis

Vegetative insecticidal protein (Vip3) from Bacillus thuringiensis shows high activity against lepidopteran insects. Cytolytic δ-endotoxin (Cyt) also has high toxicity to dipteran larvae and synergism with other crystal proteins (Cry), but synergism between Cyt and Vip3 proteins has not been tested. We analyzed for synergism between Cyt2Aa3 and Vip3Aa29. Both cyt2Aa3 and vip3Aa29 genes were co-expressed in Escherichia coli strain BL21 carried on vector pCOLADuet-1. Vip3Aa29 showed insecticidal activity against Chilo suppressalis and Spodoptera exigua, with 50% lethal concentration (LC(50)) at 24.0 and 36.6 μg ml(-1), respectively. It could also inhibit Helicoverpa armigera growth, with 50% inhibition concentration at 22.6 μg ml(-1). While Cyt2Aa3 was toxic to Culex quinquefasciatus (LC(50): 0.53 μg ml(-1)) and Chironomus tepperi (LC(50): 36 μg ml(-1)), it did not inhibit C. suppressalis, S. exigua, and H. armigera. However, the co-expression of Cyt2Aa3 and Vip3Aa29 showed synergistic effect on C. suppressalis and S. exigua, and the individual activities were strengthened 3.35- and 4.34-fold, respectively. The co-expression had no synergism against C. tepperi and H. armigera, but exerted some antagonistic effect on Cx. quinquefasciatus. The synergism between Cyt2Aa and Vip3Aa was thus discovered for the first time, which confirmed that Cyt toxin can enhance the toxicity of other toxins against some non-target insects. By synergism analysis, the effectiveness of microbial insecticides can be verified.

Targeting dysregulation of brain iron homeostasis in ageing

Brain iron is an essential nutrient for multiple functions, including gene expression, DNA synthesis, neurotransmission, myelination, oxygen transport, storage and activation, mitochondrial electron transport, numerous important metabolic processes and cofactor for several key enzymes of neurotransmitter biosynthesis. On the other hand, many investigators, among them the late Mark Smith, who was a pioneer neuroscientist and prominent investigator with regards to brain oxidative stress (OS) and iron in Alzheimer’s disease, have identified iron as an highly reactive element that can promote OS processes within the brain and might increase the toxicity of environmental or endogenous toxins. It is suggested that iron accumulation in the brain is capable of initiating free-radical reactions, which subsequently induce progressive loss of neurons, followed by a decrement in neuronal function characteristic of the ageing process. Indeed, it has become apparent that iron progressively accumulates in the brain as a function of age and that iron-induced OS can cause neurodegeneration. Chelation therapy was previously introduced as a novel therapy concept and rationale for the development of metal-binding drugs for neurodegeneration. The present review will discuss the involvement of dysregulation of brain iron homeostasis in the ageing process, addressing the potential importance of iron chelating therapeutic approaches.

Endosulfan Poisoning Resulting from Skin Exposure

Sir, In the letter to the editor by Kamate M et al., [1] important aspects of neurotoxicity of endosulfan have been highlighted. We wish to share our experience in handling a child with endosulfan poisoning resulting from skin exposure. We also want to add a few specific points in the management. A 2-y-old girl presented to the emergency with history of continuous generalized tonic clonic seizure for the past 1 h. There was a history of endosulfan application on head 2 h prior to the symptoms. The mother had applied endosulfan to remove head lice. There was no other significant history or any significant systemic findings. The seizure was immediately aborted by intravenous diazepam and child was loaded with intravenous phenytoin. The child’s head was shaved and surface decontamination was done with soap and water. The child persisted to be irritable for the next 12 h but there was no recurrence of seizures in the hospital. Her hemoglobin was 8.6 g/dL, and the total leukocyte count was 10,600/mm. Her serum electrolytes, blood gases, liver and renal functions were normal. She was asymptomatic and discharged on day 3 of presentation. Parents were counseled regarding the safe handling of endosulfan and other pesticides at home. Endosulfan and other organochlorines are central nervous system stimulants. They are thought to exert their toxic effects through inhibition of γ amino butyric acid (GABA) receptors. Toxicity commonly manifests as altered sensorium (81%), generalised seizures (75%) including status epilepticus (33%) [2]. Control of seizures is an important step in management of this toxicity. There are no trials investigating an optimal anti-convulsant or treatment algorithm for drug or toxin induced seizures. In general, benzodiazepines followed by barbiturates are the first and second-line therapies for drug or toxin induced seizures unless a specific antidote is available [3, 4]. Benzodiazepines and barbiturates are preferred over phenytoin because both these anticonvulsants are agonists at the GABA-A receptors, which facilitate chloride influx through the chloride ionophore, enhancing GABA inhibition [5]. Although phenytoin is the second line agent indicated in the treatment of most causes of status epilepticus, it is not as useful in the management of drug or toxin induced seizures. Moreover its use can potentially enhance cardiac toxicity of certain toxins. There is general lack of awareness to this fact, which is evident by the emergency room use of phenytoin in our patient and the one reported by authors. While using benzodiazapines and/or barbiturates it is important to watch for respiratory depression and hemodynamic status. If facilities for respiratory or cardiovascular support are not readily available then alternative antiepileptics like valproate or levetiracetam may be safer options.

Essential role of tryptophan residues in toxicity of binary toxin from Bacillus sphaericus

Bacillus sphaericus produces mosquito-larvicidal binary toxin composed of BinA and BinB. While BinB is expected to bind to a specific receptor on the cell membrane, BinA interacts to BinB or BinB receptor complex and translocates into the cytosol to exert its activity via unknown mechanism. To investigate functional roles of aromatic cluster in BinA, amino acids at positions Y213, Y214, Y215, W222 and W226 were substituted by leucine. All mutant proteins were highly produced and their secondary structures were not affected by these substitutions. All mutants are able to insert into lipid monolayers as observed by Langmuir-Blodgett trough and could permeabilize the liposomes in a similar manner as the wild type. However, mosquito-larvicidal activity was abolished for W222L and W226L mutants suggesting that tryptophan residues at both positions play an important role in the toxicity of BinA, possibly involved in the cytopathological process after toxin entry into the cells.

Toxin composition and toxicity dynamics of marine gastropod Nassarius spp. collected from Lianyungang, China

Consumption of nassariid gastropods often leads to poisoning incidents in some coastal provinces in China. To elucidate the pattern of toxicity dynamics and origin of toxins, samples of gastropod Nassarius spp. were collected from late May to early August 2007 from Lianyungang, Jiangsu province, where the poisoning incidents have been frequently reported. Toxicity was first screened with the mouse bioassay method, and tetrodotoxin and its analogues (TTXs) were analysed with high-performance liquid chromatography coupled with an ion-trap mass spectrometer (HPLC-MS n ). The toxicity of nassariid N. semiplicatus showed an ‘M’-shaped pattern of fluctuation during the sampling season. Two peaks of toxicity appeared in late May and late July. The maximum toxicity was recorded on 24 May, with the value of 846 mouse unit (MU) g−1 of tissue (wet weight). TTX and its analogues trideoxyTTX, 4-epiTTX, anhydroTTX and oxoTTX were detected in the nassariid samples. TrideoxyTTX but not TTX was the major toxin in all the samples. No paralytic shellfish poison (PSP) was detected in the sample with the maximum toxicity by HPLC-FLD analysis. Variation of TTX content in the tissue of nassariid gastropods correlates well with the dynamics of toxicity. It is suggested that TTXs are the major toxins corresponding to the toxicity of the nassariids, and May and July are the high-risk seasons for consumption of nassariids, which is critical for the management of poisoning incidents.

Structure-Function Map of the Receptor Site for β-Scorpion Toxins in Domain II of Voltage-gated Sodium Channels

Voltage-gated sodium (Na(v)) channels are the molecular targets of β-scorpion toxins, which shift the voltage dependence of activation to more negative membrane potentials by a voltage sensor-trapping mechanism. Molecular determinants of β-scorpion toxin (CssIV) binding and action on rat brain sodium channels are located in the S1-S2 (IIS1-S2) and S3-S4 (IIS3-S4) extracellular linkers of the voltage-sensing module in domain II. In IIS1-S2, mutations of two amino acid residues (Glu(779) and Pro(782)) significantly altered the toxin effect by reducing binding affinity. In IIS3-S4, six positions surrounding the key binding determinant, Gly(845), define a hot spot of high-impact residues. Two of these substitutions (A841N and L846A) reduced voltage sensor trapping. The other three substitutions (N842R, V843A, and E844N) increased voltage sensor trapping. These bidirectional effects suggest that the IIS3-S4 loop plays a primary role in determining both toxin affinity and efficacy. A high resolution molecular model constructed with the Rosetta-Membrane modeling system reveals interactions of amino acid residues in sodium channels that are crucial for toxin action with residues in CssIV that are required for its effects. In this model, the wedge-shaped CssIV inserts between the IIS1-S2 and IIS3-S4 loops of the voltage sensor, placing key amino acid residues in position to interact with binding partners in these extracellular loops. These results provide new molecular insights into the voltage sensor-trapping model of toxin action and further define the molecular requirements for the development of antagonists that can prevent or reverse toxicity of scorpion toxins.

Recombinant Shiga toxin B subunit elicits protection against Shiga toxin via mixed Th type immune response in mice

Shigella dysenteriae is the causative agent of the third commonest bacterial disease for childhood diarrhoea and responsible for millions of deaths per year. It produces potent toxin termed Shiga toxin which is listed in category B biological warfare agent of CDC, USA. Earlier we have reported production of recombinant Shiga toxin B subunit that produced antibodies which neutralized Shiga toxin toxicity in HeLa cells. In the present study, we have evaluated the immunomodulatory potential of rStxB protein in Balb/c mice using Freunds adjuvants. Animal protection with recombinant StxB was conferred through both humoral and cellular immune responses as indicated by an increased antibody titre with predominance of IgG2a and IgG2b isotypes along with elevated levels of IgG1 subtype. Cytokine profile of the mice antiserum and splenocyte also indicates Th2 and Th1 type of immune responses where former dominates in early stage of immunization. Histopathology study of kidneys of vaccinated mice showed remarkable differences when compared to the animals infected with Shigella dysenteriae type1. The present study indicates that recombinant StxB is a promising vaccine candidate and can be used for production of therapeutic antibodies to counter Shiga intoxication.

Ongoing Development of Monoclonal Antibodies and Antibody Drug-Conjugates in Lymphoma

Rituximab, a monoclonal antibody (mAb) directed against CD20, has changed practices in the treatment of patients with B-cell lymphoma. The large success of rituximab has contributed to validate immunotherapy with monoclonal antibodies as a valuable strategy in lymphoma. Recently, better-engineered anti-CD20-mAbs have been designed to improve efficacy and safety. Also, new antibodies targeting other lymphoma subtypes including T-cell lymphoma and Hodgkin's disease have been developed. Ongoing pharmacology development is concentrating on immuno-drug-conjugates combining the toxicity of chemotherapy, isotopes, or toxins with the specificity of mAbs. This article offers an overview on the development of new antibodies and immuno conjugates in lymphoma.

Dominant Negative Phenotype of Bacillus thuringiensis Cry1Ab, Cry11Aa and Cry4Ba Mutants Suggest Hetero-Oligomer Formation among Different Cry Toxins

Background Bacillus thuringiensis Cry toxins are used worldwide in the control of different insect pests important in agriculture or in human health. The Cry proteins are pore-forming toxins that affect the midgut cell of target insects. It was shown that non-toxic Cry1Ab helix α-4 mutants had a dominant negative (DN) phenotype inhibiting the toxicity of wildtype Cry1Ab when used in equimolar or sub-stoichiometric ratios (1∶1, 0.5∶1, mutant∶wt) indicating that oligomer formation is a key step in toxicity of Cry toxins.Methodology/Principal FindingsThe DN Cry1Ab-D136N/T143D mutant that is able to block toxicity of Cry1Ab toxin, was used to analyze its capacity to block the activity against Manduca sexta larvae of other Cry1 toxins, such as Cry1Aa, Cry1Ac, Cry1Ca, Cry1Da, Cry1Ea and Cry1Fa. Cry1Ab-DN mutant inhibited toxicity of Cry1Aa, Cry1Ac and Cry1Fa. In addition, we isolated mutants in helix α-4 of Cry4Ba and Cry11Aa, and demonstrate that Cry4Ba-E159K and Cry11Aa-V142D are inactive and completely block the toxicity against Aedes aegypti of both wildtype toxins, when used at sub-stoichiometric ratios, confirming a DN phenotype. As controls we analyzed Cry1Ab-R99A or Cry11Aa-E97A mutants that are located in helix α-3 and are affected in toxin oligomerization. These mutants do not show a DN phenotype but were able to block toxicity when used in 10∶1 or 100∶1 ratios (mutant∶wt) probably by competition of binding with toxin receptors.Conclusions/SignificanceWe show that DN phenotype can be observed among different Cry toxins suggesting that may interact in vivo forming hetero-oligomers. The DN phenotype cannot be observed in mutants affected in oligomerization, suggesting that this step is important to inhibit toxicity of other toxins.

The Role of Endothelial Cell Injury in Thrombotic Microangiopathy

Thrombotic microangiopathy (TMA) refers to a clinical and pathologic syndrome in which endothelial injury results in the manifestations of thrombocytopenia, microangiopathic hemolytic anemia, and kidney injury. A host of causes may induce endothelial injury and TMA, including enteric bacterial toxins, deficiency or dysfunction of complement regulatory proteins, deficiency or inhibition of von Willebrand factor-cleaving proteases, and factors that inhibit endothelial cell proliferation and turnover. This has led specialists to concentrate on these specific inciting factors in terms of designing treatment and management. However, a key and less recognized factor is the underlying level of endothelial health. Many persons with hereditary causes may remain disease free for years or may never develop disease. Others with acute inciting events, such as Escherichia coli O157 enteritis, never manifest TMA. Experimental studies document the importance of specific factors, such as endothelial nitric oxide levels, in helping protect animals from TMA. This suggests that one might approach the management of TMA not simply with specific treatments aimed at the underlying hereditary cause or inciting event, but rather at general measures that may improve overall endothelial health. We propose studies to determine whether interventions that improve endothelial health, such as the administration of angiotensin-converting enzyme inhibitors, statins, vitamin C, allopurinol, or nitric oxide-producing drugs, may be able to prevent TMA, even in persons with underlying hereditary conditions that otherwise would predispose them to these diseases.

Scientific Opinion on marine biotoxins in shellfish – Emerging toxins: Ciguatoxin group

The EFSA Panel on Contaminants in the Food Chain (CONTAM Panel) assessed the risks to human health related to the consumption of ciguatoxin (CTX)-group toxins in fish. CTX-group toxins occur in fish as a result of biotransformation of precursor gambiertoxins produced by the benthic dinoflagellate Gambierdiscus toxicus. CTX-group toxins cause ciguatera fish poisoning. They are mainly found in Pacific, Caribbean and Indian Ocean regions and are classified as Pacific (P), Caribbean (C) and Indian Ocean (I) CTX-group toxins. Recently CTX-group toxins were identified for the first time in fish in Europe. Currently there are no regulatory limits for CTX-group toxins in fish in Europe, but the regulation requires that no fish products containing CTX-group toxins are placed on the market. The toxicological database for CTX-group toxins is limited, comprising mostly acute toxicity studies. In view of the acute toxicity of CTX-group toxins the CONTAM Panel considered establishing an acute reference dose (ARfD). However, due to the very limited quantitative data both in experimental animals as well as related to human intoxications, the CONTAM Panel concluded that the establishment of an oral ARfD was not possible. Based on case reports on human intoxications it appears that a concentration of 0.01 μg P CTX-1 equivalents/kg fish is expected not to exert effects in sensitive individuals when consuming a single fish meal. The mouse bioassay (MBA) has been widely used to detect CTX-group toxins. However, due to insufficient detection capability and ethical concerns the MBA is not considered an appropriate method. In vitro (cytotoxicity and receptor binding) assays have been developed as alternative, but they need further development. Liquid chromatography-tandem mass spectrometry methods can be of value for the quantification of CTX-group toxins, but certified reference standards and reference materials need to be provided to allow method development and (inter-laboratory) validation.

Washing mineral oil reduces contaminants and embryotoxicity

To determine if washing improves the quality of mineral oil used for embryo culture. A 2 × 3 factorial experimental study. University hospital-based infertility center. Mice. The chemical nature of contaminants present in two lots of mineral oil was determined. Effect of washing on toxicity and amount of toxin present in media was determined. The effect of washing was determined by a quality control bioassay or by directly determining the level of contaminant in oil-conditioned culture media. Water, culture media, and media plus albumin were equally effective in reducing toxicity and concentration of toxin. Temperature did not affect washing results. Peroxide, aldehydes, and alkenals were present in one lot of oil, and Triton X-100 was identified in the other lot. Washed oil containing peroxide passed the one-cell mouse embryo bioassay, and washing reduced the amount of Triton X-100 by 25%. Mineral oil is the least defined component used for in vitro fertilization and embryo culture; therefore, it is important to determine if washing oil is beneficial. This study provides clear evidence that washing reduces toxicity of mineral oil.

Genetic transformation and pyramiding of aprotinin-expressing sugarcane with cry1Ab for shoot borer (Chilo infuscatellus) resistance

We evaluated the insecticidal toxicity of Cry1Aa, Cry1Ab and Cry1Ac toxins against neonate larvae of sugarcane shoot borer Chilo infuscatellus Snellen (Lepidoptera: Crambidae) in vitro on diet surface. With the lowest LC(50) value, Cry1Ab emerged as the most effective among the three toxins. Sugarcane cultivars Co 86032 and CoJ 64 were transformed with cry1Ab gene driven by maize ubiquitin promoter through particle bombardment and Agrobacterium-mediated transformation systems. Gene pyramiding was also attempted by retransforming sugarcane plants carrying bovine pancreatic trypsin inhibitor (aprotinin) gene, with cry1Ab. Southern analysis confirmed multiple integration of the transgene in case of particle bombardment and single site integration in Agrobacterium-mediated transformants. The expression of cry1Ab was demonstrated through Western analysis and the toxin was quantified using ELISA. The amount of Cry1Ab protein in different events varied from 0.007 to 1.73% of the total soluble leaf protein; the events transformed by Agrobacterium method showed significantly higher values. In in vivo bioassay with neonate larvae of shoot borer, transgenics produced considerably lower percentage of deadhearts despite suffering feeding damage by the borer compared with the untransformed control plants. Expressed Cry1Ab content was negatively related to deadheart damage. Aprotinin-expressing sugarcane pyramided with cry1Ab also showed reduction in damage. The potential of producing sugarcane transgenics with cry1Ab and aprotinin genes resistant to early shoot borer was discussed in the light of the results obtained.

Pore-forming properties of the Bacillus thuringiensis toxin Cry9Ca in Manduca sexta brush border membrane vesicles

The toxicity and pore-forming ability of the Bacillus thuringiensis Cry9Ca insecticidal toxin, its single-site mutants, R164A and R164K, and the 55-kDa fragment resulting from its proteolytic cleavage at residue 164 were investigated using Manduca sexta neonate larvae and fifth-instar larval midgut brush border membrane vesicles, respectively. Neither the mutations nor the proteolytic cleavage altered Cry9Ca toxicity. Compared with Cry1Ac, Cry9Ca and its mutants formed large poorly selective pores in the vesicles. Pore formation was highly dependent on pH, however, especially for wild-type Cry9Ca and both mutants. Increasing pH from 6.5 to 10.5 resulted in an irregular step-wise decrease in membrane permeabilization that was not related to a change in the ionic selectivity of the pores. Pore formation was much slower with Cry9Ca and its derivatives, including the 55-kDa fragment, than with Cry1Ac and its rate was not influenced by the presence of protease inhibitors or a reducing agent.

Active immunization induces toxicity of diphtheria toxin in diphtheria resistant mice — Implications for neuroinflammatory models

Cell-type specific expression of the human diphtheria toxin receptor in generally toxin resistant mice represents an innovative approach for the selective depletion of pre-defined cell populations. We demonstrate that in wildtype mice diphtheria toxin – in concentrations otherwise well tolerated – is highly toxic and lethal together with active immunization irrespective of the immunogenic peptide applied. We found increased lung cellularity as only pathological abnormality. Animal models of inflammatory diseases requiring active immunization including experimental autoimmune encephalomyelitis may thus not be applicable in diphtheria receptor transgenic mice pointing to a major limitation of this otherwise technically interesting approach.

Interplay between Toxin Transport and Flotillin Localization

The flotillin proteins are localized in lipid domains at the plasma membrane as well as in intracellular compartments. In the present study, we examined the importance of flotillin-1 and flotillin-2 for the uptake and transport of the bacterial Shiga toxin (Stx) and the plant toxin ricin and we investigated whether toxin binding and uptake were associated with flotillin relocalization. We observed a toxin-induced redistribution of the flotillins, which seemed to be regulated in a p38-dependent manner. Our experiments provide no evidence for a changed endocytic uptake of Stx or ricin in cells silenced for flotillin-1 or -2. However, the Golgi-dependent sulfation of both toxins was significantly reduced in flotillin knockdown cells. Interestingly, when the transport of ricin to the ER was investigated, we obtained an increased mannosylation of ricin in flotillin-1 and flotillin-2 knockdown cells. The toxicity of both toxins was twofold increased in flotillin-depleted cells. Since BFA (Brefeldin A) inhibits the toxicity even in flotillin knockdown cells, the retrograde toxin transport is apparently still Golgi-dependent. Thus, flotillin proteins regulate and facilitate the retrograde transport of Stx and ricin.