Mucoricin is a ricin-like toxin that is critical for the pathogenesis of mucormycosis.

Fungi of the order Mucorales cause mucormycosis, a lethal infection with an incompletely understood pathogenesis. We demonstrate that Mucorales fungi produce a toxin, which plays a central role in virulence. Polyclonal antibodies against this toxin inhibit its ability to damage human cells in vitro and prevent hypovolemic shock, organ necrosis and death in mice with mucormycosis. Inhibition of the toxin in Rhizopus delemar through RNA interference compromises the ability of the fungus to damage host cells and attenuates virulence in mice. This 17 kDa toxin has structural and functional features of the plant toxin ricin, including the ability to inhibit protein synthesis through its N-glycosylase activity, the existence of a motif that mediates vascular leak and a lectin sequence. Antibodies against the toxin inhibit R. delemar- or toxin-mediated vascular permeability in vitro and cross react with ricin. A monoclonal anti-ricin B chain antibody binds to the toxin and also inhibits its ability to cause vascular permeability. Therefore, we propose the name 'mucoricin' for this toxin. Not only is mucoricin important in the pathogenesis of mucormycosis but our data suggest that a ricin-like toxin is produced by organisms beyond the plant and bacterial kingdoms. Importantly, mucoricin should be a promising therapeutic target.

Pretreatment with Retro-2 protects cells from death caused by ricin toxin by retaining the capacity of protein synthesis.

The current study explores the detoxification effect of Retro-2 on ricin toxin (RT) cytotoxicity, as well as the mechanisms underlying such effects, to provide a basis for follow-up clinical applications of Retro-2. The mouse-derived mononuclear/macrophage cell line, RAW264.7, was used to evaluate the detoxification effect of Retro-2 on RT by detecting cell viability, capacity for protein synthesis and the expression of cytokines, as well as endoplasmic reticulum stress (ERS)-related mRNA. The results indicated that many cells died when challenged with concentrations of RT ≥50ng/mL. The protein synthesis capacity of cells decreased when challenged with 200ng/mL RT for 2hours. Furthermore, the synthesis and release of many cytokines decreased, while the expression of cytokines or ERS-related mRNA increased when challenged with 200ng/mL of RT for 12 or more hours. However, cell viability, capacity for protein synthesis and release levels of many cytokines were higher, while the expression levels of cytokine, or ERS-related mRNA, were lower in cells pretreated with 20µm Retro-2 and challenged with RT, compared with those that had not been pretreated with Retro-2. In conclusion, Retro-2 retained the capacity for protein synthesis inhibited by RT, alleviated ERS induced by RT and increased the viability of cells challenged with RT. Retro-2 shows the potential for clinical applications.

Adaptogens in chemobrain (Part III): Antitoxic effects of plant extracts towards cancer chemotherapy-induced toxicity - transcriptome-wide microarray analysis of neuroglia cells.

BACKGROUND: Toxicity of chemotherapeutics is a serious problem in cancer therapy. Adaptogens are known to increase adaptability and survival organisms. AIM: The aim of this study was to assess the effects of selected adaptogenic herbal extracts on FEC (fixed combination of 5-fluorouracil, epirubicin and cyclophosphamide) induced changes in transcriptome-wide microarray profiles of neuroglia cells. Another task of the study was to identify those genes, which are associated with FEC-induced hepato-, cardio- and nephrotoxicity to predict potential effects of andrographolide (AND), Andrographis herb, Eleutherococcus roots genuine extracts (ES), their fixed combination (AE) and the combination of Rhodiola roots, Schisandra berries and Eleutherococcus roots (RSE) on the organismal level. METHODS: Gene expression profiling was performed by transcriptome-wide mRNA microarray in the human T98G neuroglia cells after treatment with adaptogens. Interactive pathways downstream analysis was performed with data sets of significantly up- or down-regulated genes and predicted effects on cellular functions and diseases were identified by Ingenuity IPA database software. RESULT: Significant differences of transcriptome-wide microarray profiles were observed after treatment of T98G cells with FEC and after co-incubation with adaptogens. FEC induced deregulation of certain genes with suggested toxicity associated with liver fibroses, necrosis and congenital heart diseases. Co-incubation of AE with FEC prevented FEC-induced deregulation of 66 genes increasing organismal death, 37 genes decreasing cell survival, 37 genes decreasing DNA repair, 37 genes decreasing viral infection and some other functions, indicating on potential beneficial effects of AE. Furthermore, FEC-induced hepato-, nephro- and cardiotoxicity related to deregulation of genes was predictably attenuated by AE. Moreover, co-incubation of AE with FEC caused differential expression of genes, which presumably are beneficial for an organism during chemotherapy. They include predicted activation of DNA repair, activation of movement of antigen presenting cells and inhibition of muscle cells death. The main active constituent of AE is AND. Co-incubation of FEC only with AND results in deregulation of 10 genes causing death of breast cancer cells, decrease of liver toxicity and attenuation of organismal death. Co-incubation of ES extract with FEC showed that ES suppressed FEC-induced deregulation of genes, which inhibit organismal death and fertility. Co-incubation of FEC with RSE indicated potential hepatoprotective effect against FEC-induced apoptosis of liver cells presumably due to suppression of FEC-induced expressions of genes, which increased liver cell apoptosis. Simultaneously, RSE activated expression of genes inhibiting tumor growth. Though, microarray analysis did not provide final proof that the genes induced by the AE, AP and ES are responsible for the physiological effects observed in human patients following their oral administration, it provided insights into putative genes and directions for future research and possible implementation into practice. CONCLUSION: Application of cytostatic drugs in combination with adaptogenic plant extracts induced significant changes in transcriptome-wide microarray profiles of neuroglial cells. These changes indicate on potential beneficial effects of adaptogens on FEC induced adverse events in cancer chemotherapy.

Natural Compounds and Their Analogues as Potent Antidotes against the Most Poisonous Bacterial Toxin.

Botulinum neurotoxins (BoNTs), the most poisonous proteins known to humankind, are a family of seven (serotype A to G) immunologically distinct proteins synthesized primarily by different strains of the anaerobic bacterium Clostridium botulinum Being the causative agents of botulism, the toxins block neurotransmitter release by specifically cleaving one of the three soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins, thereby inducing flaccid paralysis. The development of countermeasures and therapeutics against BoNTs is a high-priority research area for public health because of their extreme toxicity and potential for use as biowarfare agents. Extensive research has focused on designing antagonists that block the catalytic activity of BoNTs. In this study, we screened 300 small natural compounds and their analogues extracted from Indian plants for their activity against BoNT serotype A (BoNT/A) as well as its light chain (LCA) using biochemical and cellular assays. One natural compound, a nitrophenyl psoralen (NPP), was identified to be a specific inhibitor of LCA with an in vitro 50% inhibitory concentration (IC50) value of 4.74 ± 0.03 µM. NPP was able to rescue endogenous synaptosome-associated protein 25 (SNAP-25) from cleavage by BoNT/A in human neuroblastoma cells with an IC50 of 12.2 ± 1.7 µM, as well as to prolong the time to the blocking of neutrally elicited twitch tensions in isolated mouse phrenic nerve-hemidiaphragm preparations.IMPORTANCE The long-lasting endopeptidase activity of BoNT is a critical biological activity inside the nerve cell, as it prompts proteolysis of the SNARE proteins, involved in the exocytosis of the neurotransmitter acetylcholine. Thus, the BoNT endopeptidase activity is an appropriate clinical target for designing new small-molecule antidotes against BoNT with the potential to reverse the paralysis syndrome of botulism. In principle, small-molecule inhibitors (SMIs) can gain entry into BoNT-intoxicated cells if they have a suitable octanol-water partition coefficient (log P) value and other favorable characteristics (P. Leeson, Nature 481:455-456, 2012, https://doi.org/10.1038/481455a). Several efforts have been made in the past to develop SMIs, but inhibitors effective under in vitro conditions have not in general been effective in vivo or in cellular models (L. M. Eubanks, M. S. Hixon, W. Jin, S. Hong, et al., Proc Natl Acad Sci U S A 104:2602-2607, 2007, https://doi.org/10.1073/pnas.0611213104). The difference between the in vitro and cellular efficacy presumably results from difficulties experienced by the compounds in crossing the cell membrane, in conjunction with poor bioavailability and high cytotoxicity. The screened nitrophenyl psoralen (NPP) effectively antagonized BoNT/A in both in vitro and ex vivo assays. Importantly, NPP inhibited the BoNT/A light chain but not other general zinc endopeptidases, such as thermolysin, suggesting high selectivity for its target. Small-molecule (nonpeptidic) inhibitors have better oral bioavailability, better stability, and better tissue and cell permeation than antitoxins or peptide inhibitors.

Combined therapy with gas gangrene antitoxin and recombinant human soluble thrombomodulin for Clostridium perfringens sepsis in a rat model.

Cases of Clostridium perfringens septicemia, such as liver abscess, often develop a rapidly progressive intravascular hemolysis and coagulation; the mortality rate with current standard care including antibiotics and surgery is high. Herein, we firstly investigated the effects of gas gangrene antitoxin (GGA) (antitoxin against C. perfringens) and recombinant human soluble thrombomodulin (rTM) on the hemolysis, coagulation status, inflammatory process, and mortality in α-toxin-treated rats. Male 11-week-old Sprague Dawley rats were randomly divided into five groups: control group, α-toxin group, GGA group, rTM group, and combined GGA and rTM (combination group). After α-toxin injection, mortality and platelet counts, and hemolysis were observed for 6 h. The fibrin/fibrinogen degradation products (FDP), and plasma high-mobility group box 1 (HMGB1) were also measured at 6 h. The combination group demonstrated 100% survival compared with 50% survival in the α-toxin group and demonstrated significantly improved hemolysis, platelet counts, and lactate levels compared with those in the α-toxin group (p < .01). The FDP and HMGB1 levels in the combination therapy group were significantly lower than those in the α-toxin group (p < .05). Combination therapy with GGA and rTM administration is applicable as adjunct therapy for fatal C. perfringens sepsis.

Toxin ζ reversible induces dormancy and reduces the UDP-N-acetylglucosamine pool as one of the protective responses to cope with stress.

Toxins of the ζ/PezT family, found in the genome of major human pathogens, phosphorylate the peptidoglycan precursor uridine diphosphate-N-acetylglucosamine (UNAG) leading to unreactive UNAG-3P. Transient over-expression of a PezT variant impairs cell wall biosynthesis and triggers autolysis in Escherichia coli. Conversely, physiological levels of ζ reversibly induce dormancy produce a sub-fraction of membrane-compromised cells, and a minor subpopulation of Bacillus subtilis cells become tolerant of toxin action. We report here that purified ζ is a strong UNAG-dependent ATPase, being GTP a lower competitor. In vitro, ζ toxin phosphorylates a fraction of UNAG. In vivo, ζ-mediated inactivation of UNAG by phosphorylation does not deplete the active UNAG pool, because expression of the toxin enhances the efficacy of genuine cell wall inhibitors (fosfomycin, vancomycin or ampicillin). Transient ζ expression together with fosfomycin treatment halt cell proliferation, but ε2 antitoxin expression facilitates the exit of ζ-induced dormancy, suggesting that there is sufficient UNAG for growth. We propose that ζ induces diverse cellular responses to cope with stress, being the reduction of the UNAG pool one among them. If the action of ζ is not inhibited, e.g., by de novo ε2 antitoxin synthesis, the toxin markedly enhances the efficacy of antimicrobial treatment without massive autolysis in Firmicutes.

Toxic shock syndrome toxin-1-mediated toxicity inhibited by neutralizing antibodies late in the course of continual in vivo and in vitro exposure.

Toxic shock syndrome (TSS) results from the host's overwhelming inflammatory response and cytokine storm mainly due to superantigens (SAgs). There is no effective specific therapy. Application of immunoglobulins has been shown to improve the outcome of the disease and to neutralize SAgs both in vivo and in vitro. However, in most experiments that have been performed, antiserum was either pre-incubated with SAg, or both were applied simultaneously. To mirror more closely the clinical situation, we applied a multiple dose (over five days) lethal challenge in a rabbit model. Treatment with toxic shock syndrome toxin 1 (TSST-1) neutralizing antibody was fully protective, even when administered late in the course of the challenge. Kinetic studies on the effect of superantigen toxins are scarce. We performed in vitro kinetic studies by neutralizing the toxin with antibodies at well-defined time points. T-cell activation was determined by assessing T-cell proliferation (3H-thymidine incorporation), determination of IL-2 release in the cell supernatant (ELISA), and IL-2 gene activation (real-time PCR (RT-PCR)). Here we show that T-cell activation occurs continuously. The application of TSST-1 neutralizing antiserum reduced IL-2 and TNFα release into the cell supernatant, even if added at later time points. Interference with the prolonged stimulation of proinflammatory cytokines is likely to be in vivo relevant, as postexposure treatment protected rabbits against the multiple dose lethal SAg challenge. Our results shed new light on the treatment of TSS by specific antibodies even at late stages of exposure.

Review of the inhibition of biological activities of food-related selected toxins by natural compounds.

There is a need to develop food-compatible conditions to alter the structures of fungal, bacterial, and plant toxins, thus transforming toxins to nontoxic molecules. The term 'chemical genetics' has been used to describe this approach. This overview attempts to survey and consolidate the widely scattered literature on the inhibition by natural compounds and plant extracts of the biological (toxicological) activity of the following food-related toxins: aflatoxin B1, fumonisins, and ochratoxin A produced by fungi; cholera toxin produced by Vibrio cholerae bacteria; Shiga toxins produced by E. coli bacteria; staphylococcal enterotoxins produced by Staphylococcus aureus bacteria; ricin produced by seeds of the castor plant Ricinus communis; and the glycoalkaloid α-chaconine synthesized in potato tubers and leaves. The reduction of biological activity has been achieved by one or more of the following approaches: inhibition of the release of the toxin into the environment, especially food; an alteration of the structural integrity of the toxin molecules; changes in the optimum microenvironment, especially pH, for toxin activity; and protection against adverse effects of the toxins in cells, animals, and humans (chemoprevention). The results show that food-compatible and safe compounds with anti-toxin properties can be used to reduce the toxic potential of these toxins. Practical applications and research needs are suggested that may further facilitate reducing the toxic burden of the diet. Researchers are challenged to (a) apply the available methods without adversely affecting the nutritional quality, safety, and sensory attributes of animal feed and human food and (b) educate food producers and processors and the public about available approaches to mitigating the undesirable effects of natural toxins that may present in the diet.

Nitrosylation: an adverse factor in Uremic Hemolytic Syndrome. Antitoxin effect of Ziziphus mistol Griseb.

Toxins of Escherichia coli (STEC) causing Uremic Hemolytic Syndrome (UHS) generate oxidative stress in human blood with more production of nitric oxide (NO) than reactive oxygen species (ROS). Shiga toxin (Stx) together with the hemolysin (Hly) increased lipid oxidation, as evaluated by malondialdehyde MDA and oxidation of proteins. The addition of Ziziphus mistol Griseb extracts decreased NO, ROS, MDA and simultaneously caused an increase in the degradation of oxidized proteins to advanced oxidation protein products (AOPPs) in controls and samples with toxins. Furthermore, the nitrosylated proteins/AOPP ratio was reduced, due to the increase of AOPP. Z. mistol Griseb extracts exhibited a high proportion of polyphenols and flavonoids, with evident correlation with ferrous reduction antioxidant potential (FRAP). The plasma of eight children with UHS showed oxidative stress and NO stimulus, comparable to the effect of toxins during the assays in vitro. UHS children presented high levels of nitrosylated proteins respect to control children of similar age. Although the degradation of oxidized proteins to AOPP rose in UHS children, the nitrosylated proteins/AOPP rate increased as a consequence of the elevated nitrosative stress observed in these patients.

Antioxidant, antimicrobial and antiverotoxic potentials of extracts of Curtisia dentata.

ETHNOPHARMACOLOGICAL RELEVANCE: There is an increase in antimicrobial resistance and complexities arising from verotoxic related bacterial infections as well as rise in demand for application of natural antioxidants to combat oxidative damage by free radicals in many oxidative stress-mediated disease conditions such as cancer. Thus the potential of Curtisia dentata as antimicrobial, antioxidant and antiverotoxin against environmental isolates of Escherichia coli and Acinetobacter spp. as well as the presence of phytochemicals and some organic compounds, was determined. MATERIALS AND METHODS: Phytochemical analysis was carried out using standard methods and antioxidant activity was determined using the DPPH radical scavenging activity. Effect of extracts on bacterial cell wall was also determined. RESULTS: Extracts contained anthraquinones, alkaloids, essential oils, glycosides, phenols, steroids, saponins, tannins, quinones, anthocyanins, amines and carboxylic acids as phytochemicals. Extracts demonstrated high antimicrobial activity and low minimum inhibitory concentrations as well as inhibitory action against the expression of both Vtx1 and Vtx2 genes in Escherichia coli, Acinetobacter haemolyticus and Acinetobacter lwoffii. Ethanol root bark extracts consistently showed the highest DPPH radical scavenging activity (62.43%), total phenol content (TPH) (57.62 26 mg GAE/g) and reducing power (RP) (41.32%), followed by those of the stem bark and leaf extracts with the respective values of 54.68%, 37.77 mg GAE/g and 21.83%. The extracts induced the leakage of Na(+) and K(+) ions from both test bacteria. CONCLUSION: Curtisia dentata is a very effective source of antioxidant and a possible alternative to sourcing antiverotoxic antibiotics with novel mechanism of action.

Identification of anti-alpha toxin monoclonal antibodies that reduce the severity of Staphylococcus aureus dermonecrosis and exhibit a correlation between affinity and potency.

Staphylococcus aureus alpha toxin (AT) is an important virulence determinant and may be a valid target for immunoprophylaxis against staphylococcal disease. Here we report the identification of potent inhibitory anti-AT monoclonal antibodies (MAbs) derived using B-cell hybridoma technology from VelocImmune mice engineered to produce IgG with a human variable domain. A small panel of inhibitory MAbs blocked AT-mediated lysis of rabbit red blood cells, A549 human lung epithelial cells, and THP-1 human monocytic cells, in a dose-dependent manner. Binding studies indicated that these MAbs recognize a similar epitope on AT and exhibit dissociation constants (K(D)) ranging from 0.50 to 15 nM. In an S. aureus dermonecrosis model, mice passively immunized with anti-AT inhibitory MAbs exhibited significant reductions of lesion size relative to mice treated with an irrelevant IgG control. Interestingly, there was a correlation between MAb affinity for a single epitope, the 50% inhibitory concentration (IC(50)) in the AT hemolytic assay, and lesion size reduction in the dermonecrosis model. A representative high-affinity MAb, 2A3.1, was demonstrated to significantly reduce lesion size following infection with three different clinical isolates (USA300, CC30, and CC5). Taken together, these results indicate that in vitro potency of anti-AT MAbs predicts in vivo potency in this model, supporting their continued preclinical evaluation as molecules for immunoprophylaxis against staphylococcal skin and soft tissue infections caused by diverse clinical isolates.

[Sophoridine inhibits NF-kappaB signaling pathway activation in kidney tissue of endotoxemia mice].

This study is to investigate the effects of sophoridine on NF-kappaB signaling pathway in kidney tissue of endotoxemia mice and the mechanism involved. BALB/c mice were challenged with lipopolysaccharide (LPS) caudal vein injection, then sophoridine was administered by intraperitoneal injection. Totally 50 mice were divided into 5 groups: control group, LPS model group, sophoridine treatment 12 mg x kg(-1) group, 6 mg x kg(-1) group and 3 mg x kg(-1) group. All animals were sacrificed at 6 hours after treatment. Kidney and blood samples were harvested. IKKbeta mRNA and TNF-alpha mRNA expression of renal tissue was measured by the reverse transcription polymerase chain reaction (RT-PCR), and phosphorylation IKKbeta protein (pIKKbeta) was detected by immunohistochemistry. NF-kappaB P65 protein expression and distribution of renal tissue were observed by Western blotting and immunofluorescence laser confocal microscopy. Serum TNF-alpha level was detected by radioimmunoassay. The results showed that the sophoridine significantly reduced the expression of IKKbeta mRNA and pIKKbeta protein, and inhibited the expression of NF-kappaB P65 protein and decreased the entry nuclear rate of NF-kappaB P65 in the renal tissue of endotoxemia mice. Thereby the renal TNF-alpha mRNA expression and serum TNF-alpha level were significantly reduced. These results suggest that sophoridine could inhibit inflammatory reaction induced by LPS through inhibiting activation of NF-kappaB signaling pathway.

Evaluation of the ability of LL-37 to neutralise LPS in vitro and ex vivo.

BACKGROUND: Human cathelicidin LL-37 is a cationic antimicrobial peptide (AMP) which possesses a variety of activities including the ability to neutralise endotoxin. In this study, we investigated the role of LPS neutralisation in mediating LL-37's ability to inhibit Pseudomonas aeruginosa LPS signalling in human monocytic cells. METHODOLOGY/PRINCIPAL FINDINGS: Pre-treatment of monocytes with LL-37 significantly inhibited LPS-induced IL-8 production and the signalling pathway of associated transcription factors such as NF-κB. However, upon removal of LL-37 from the media prior to LPS stimulation, these inhibitory effects were abolished. These findings suggest that the ability of LL-37 to inhibit LPS signalling is largely dependent on extracellular LPS neutralisation. In addition, LL-37 potently inhibited cytokine production induced by LPS extracted from P. aeruginosa isolated from the lungs of cystic fibrosis (CF) patients. In the CF lung, polyanionic molecules such as glycosaminoglycans (GAGs) and DNA bind LL-37 and impact negatively on its antibacterial activity. In order to determine whether such interactions interfere with the LPS neutralising ability of LL-37, the status of LL-37 and its ability to bind LPS in CF sputum were investigated. Overall our findings suggest that in the CF lung, the ability of LL-37 to bind LPS and inhibit LPS-induced IL-8 production is attenuated as a result of binding to DNA and GAGs. However, LL-37 levels and its concomitant LPS-binding activity can be increased with a combination of DNase and GAG lyase (heparinase II) treatment. CONCLUSIONS/SIGNIFICANCE: Overall, these findings suggest that a deficiency in available LL-37 in the CF lung may contribute to greater LPS-induced inflammation during CF lung disease.

Tumor endothelium marker-8 based decoys exhibit superiority over capillary morphogenesis protein-2 based decoys as anthrax toxin inhibitors.

Anthrax toxin is the major virulence factor produced by Bacillus anthracis. The toxin consists of three protein subunits: protective antigen (PA), lethal factor, and edema factor. Inhibition of PA binding to its receptors, tumor endothelium marker-8 (TEM8) and capillary morphogenesis protein-2 (CMG2) can effectively block anthrax intoxication, which is particularly valuable when the toxin has already been overproduced at the late stage of anthrax infection, thus rendering antibiotics ineffectual. Receptor-like agonists, such as the mammalian cell-expressed von Willebrand factor type A (vWA) domain of CMG2 (sCMG2), have demonstrated potency against the anthrax toxin. However, the soluble vWA domain of TEM8 (sTEM8) was ruled out as an anthrax toxin inhibitor candidate due to its inferior affinity to PA. In the present study, we report that L56A, a PA-binding-affinity-elevated mutant of sTEM8, could inhibit anthrax intoxication as effectively as sCMG2 in Fisher 344 rats. Additionally, pharmacokinetics showed that L56A and sTEM8 exhibit advantages over sCMG2 with better lung-targeting and longer plasma retention time, which may contribute to their enhanced protective ability in vivo. Our results suggest that receptor decoys based on TEM8 are promising anthrax toxin inhibitors and, together with the pharmacokinetic studies in this report, may contribute to the development of novel anthrax drugs.

Protective effects of Mucuna pruriens seed extract pretreatment against cardiovascular and respiratory depressant effects of Calloselasma rhodostoma (Malayan pit viper) venom in rats.

The protective effects of Mucuna pruriens seed extract (MPE) against the cardio-respiratory depressant and neuromuscular paralytic effects induced by injection of Calloselasma rhodostoma (Malayan pit viper) venom in anaesthetized rats were investigated. While MPE pretreatment did not reverse the inhibitory effect of the venom on the gastrocnemius muscle excitability, it significantly attenuated the venom-induced cardio-respiratory depressant effects (p < 0.05). The protection effects may have an immunological mechanism, as indicated by the presence of several proteins in the venom that are immunoreactive against anti-MPE. However, we cannot rule out the possibility that the pretreatment may exert a direct, non-immunological protective action against the venom.

Antibody protection against botulinum neurotoxin intoxication in mice.

Adulteration of food or feed with any of the seven serotypes of botulinum neurotoxin (BoNT) is a potential bioterrorism concern. Currently, there is strong interest in the development of detection reagents, vaccines, therapeutics, and other countermeasures. A sensitive immunoassay for detecting BoNT serotype A (BoNT/A), based on monoclonal antibodies (MAbs) F1-2 and F1-40, has been developed and used in complex matrices. The epitope for F1-2 has been mapped to the heavy chain of BoNT/A, and the epitope of F1-40 has been mapped to the light chain. The ability of these MAbs to provide therapeutic protection against BoNT/A intoxication in mouse intravenous and oral intoxication models was tested. High dosages of individual MAbs protected mice well both pre- and postexposure to BoNT/A holotoxin. A combination therapy consisting of antibodies against both the light and heavy chains of the toxin, however, significantly increased protection, even at a lower MAb dosage. An in vitro peptide assay for measuring toxin activity showed that pretreatment of toxin with these MAbs did not block catalytic activity but instead blocked toxin entry into primary and cultured neuronal cells. The timing of antibody rescue in the mouse intoxication models revealed windows of opportunity for antibody therapeutic treatment that correlated well with the biologic half-life of the toxin in the serum. Knowledge of BoNT intoxication and antibody clearance in these mouse models and understanding of the pharmacokinetics of BoNT are invaluable for future development of antibodies and therapeutics against intoxication by BoNT.

Investigation of new dominant-negative inhibitors of anthrax protective antigen mutants for use in therapy and vaccination.

The lethal toxin (LeTx) of Bacillus anthracis plays a key role in the pathogenesis of anthrax. The protective antigen (PA) is a primary part of the anthrax toxin and forms LeTx by combination with lethal factor (LF). Phenylalanine-427 (F427) is crucial for PA function. This study was designed to discover potential novel therapeutic agents and vaccines for anthrax. This was done by screening PA mutants that were mutated at the F427 residue for a dominant-negative inhibitory (DNI) phenotype which was nontoxic but inhibited the toxicity of the wild-type LeTx. For this, PA residue F427 was first mutated to each of the other 19 naturally occurring amino acids. The cytotoxicity and DNI phenotypes of the mutated PA proteins were tested in the presence of 1 microg/ml LF in RAW264.7 cells and were shown to be dependent on the individual amino acid replacements. A total of 16 nontoxic mutants with various levels of DNI activity were identified in vitro. Among them, F427D and F427N mutants had the highest DNI activities in RAW264.7 cells. Both mutants inhibited LeTx intoxication in mice in a dose-dependent way. Furthermore, they induced a Th2-predominant immune response and protected mice against a challenge with five 50% lethal doses of LeTx. The protection was correlated mainly with a low level of interleukin-1 beta (IL-1 beta) and with high levels of PA-specific immunoglobulin G1, IL-6, and tumor necrosis factor alpha. Thus, PA DNI mutants, such as F427D and F427N mutants, may serve in the development of novel therapeutic agents and vaccines to fight B. anthracis infections.

Identification and biochemical characterization of small-molecule inhibitors of Clostridium botulinum neurotoxin serotype A.

An integrated strategy that combined in silico screening and tiered biochemical assays (enzymatic, in vitro, and ex vivo) was used to identify and characterize effective small-molecule inhibitors of Clostridium botulinum neurotoxin serotype A (BoNT/A). Virtual screening was initially performed by computationally docking compounds of the National Cancer Institute (NCI) database into the active site of BoNT/A light chain (LC). A total of 100 high-scoring compounds were evaluated in a high-performance liquid chromatography (HPLC)-based protease assay using recombinant full-length BoNT/A LC. Seven compounds that significantly inhibited the BoNT/A protease activity were selected. Database search queries of the best candidate hit [7-((4-nitro-anilino)(phenyl)methyl)-8-quinolinol (NSC 1010)] were performed to mine its nontoxic analogs. Fifty-five analogs of NSC 1010 were synthesized and examined by the HPLC-based assay. Of these, five quinolinol derivatives that potently inhibited both full-length BoNT/A LC and truncated BoNT/A LC (residues 1 to 425) were selected for further inhibition studies in neuroblastoma (N2a) cell-based and tissue-based mouse phrenic nerve hemidiaphragm assays. Consistent with enzymatic assays, in vitro and ex vivo studies revealed that these five quinolinol-based analogs effectively neutralized BoNT/A toxicity, with CB 7969312 exhibiting ex vivo protection at 0.5 microM. To date, this is the most potent BoNT/A small-molecule inhibitor that showed activity in an ex vivo assay. The reduced toxicity and high potency demonstrated by these five compounds at the biochemical, cellular, and tissue levels are distinctive among the BoNT/A small-molecule inhibitors reported thus far. This study demonstrates the utility of a multidisciplinary approach (in silico screening coupled with biochemical testing) for identifying promising small-molecule BoNT/A inhibitors.

The effects of Panax ginseng and Spirulina platensis on hepatotoxicity induced by cadmium in rats.

Cadmium is an environmental and industrial cumulative pollutant that affects many organs, specially the liver. The protective effect of Spirulina platensis and Panax ginseng on cadmium-induced oxidative stress and hepatotoxicity was evaluated in adult female Wistar albino rats. At the end of the 1-month experimental period, all animals were fasted for 12h and liver samples were taken for the determination of malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD) and nitric oxide (NO) levels. S. platensis and P. ginseng treatments showed marked decrease lipid peroxidation and increase of the endogenous antioxidants levels. The cadmium-induced histopathological changes were also minimized with the tested extracts. These results suggest that S. platensis and P. ginseng might play a role in reducing the toxic effect of cadmium and its antioxidant properties seem to mediate such a protective effect.

Inhibitors of anthrax lethal factor based upon N-oleoyldopamine.

The structural features of an anthrax lethal factor inhibitor, N-oleoyldopamine (OLDA, 1) have been probed. The oleic acid moiety is critical, but, more interestingly, the presence of the double bond and its geometry were found to play an essential role. One compound, 5, was found to be an uncompetitive inhibitor of lethal factor (LF) with a K(i) value of 2.2microM and a cell-based IC(50) value of 4.3microM.