Increasing the Antitumor Effect of an EpCAM-Targeting Fusion Toxin by Facile Click PEGylation

Fusion toxins used for cancer-related therapy have demonstrated short circulation half-lives, which impairs tumor localization and, hence, efficacy. Here, we demonstrate that the pharmacokinetics of a fusion toxin composed of a designed ankyrin repeat protein (DARPin) and domain I–truncated Pseudomonas Exotoxin A (PE40/ETA″) can be significantly improved by facile bioorthogonal conjugation with a polyethylene glycol (PEG) polymer at a unique position. Fusion of the anti-EpCAM DARPin Ec1 to ETA″ and expression in methionine-auxotrophic E. coli enabled introduction of the nonnatural amino acid azidohomoalanine (Aha) at position 1 for strain-promoted click PEGylation. PEGylated Ec1-ETA″ was characterized by detailed biochemical analysis, and its potential for tumor targeting was assessed using carcinoma cell lines of various histotypes in vitro, and subcutaneous and orthotopic tumor xenografts in vivo. The mild click reaction resulted in a well-defined mono-PEGylated product, which could be readily purified to homogeneity. Despite an increased hydrodynamic radius resulting from the polymer, the fusion toxin demonstrated high EpCAM-binding activity and retained cytotoxicity in the femtomolar range. Pharmacologic analysis in mice unveiled an almost 6-fold increase in the elimination half-life (14 vs. 82 minutes) and a more than 7-fold increase in the area under the curve (AUC) compared with non-PEGylated Ec1-ETA″, which directly translated in increased and longer-lasting effects on established tumor xenografts. Our data underline the great potential of combining the inherent advantages of the DARPin format with bioorthogonal click chemistry to overcome the limitations of engineering fusion toxins with enhanced efficacy for cancer-related therapy.

Studying the fate of non-volatile organic compounds in a commercial plasma air purifier

Degradation of non-volatile organic compounds-environmental toxins (methyltriclosane and phenanthrene), bovine serum albumin, as well as bioparticles (Legionella pneumophila, Bacillus subtilis, and Bacillus anthracis)-in a commercially available plasma air purifier based on a cold plasma was studied in detail, focusing on its efficiency and on the resulting degradation products. This system is capable of handling air flow velocities of up to 3.0m s(-1) (3200Lmin(-1)), much higher than other plasma-based reactors described in the literature, which generally are limited to air flow rates below 10Lmin(-1). Mass balance studies consistently indicated a reduction in concentration of the compounds/particles after passage through the plasma air purifier, 31% for phenanthrene, 17% for methyltriclosane, and 80% for bovine serum albumin. L. pneumophila did not survive passage through the plasma air purifier, and cell counts of aerosolized spores of B. subtilis and B. anthracis were reduced by 26- and 15-fold, depending on whether it was run at 10Hz or 50Hz, respectively. However rather than chemical degradation, deposition on the inner surfaces of the plasma air purifier occured. Our interpretation is that putative "degradation" efficiencies were largely due to electrostatic precipitation rather than to decomposition into smaller molecules.

Evaluation of the Hepa Wash® treatment in pigs with acute liver failure

BACKGROUND Mortality of patients with acute liver failure (ALF) is still unacceptably high. Available liver support systems are still of limited success at improving survival. A new type of albumin dialysis, the Hepa Wash® system, was newly introduced. We evaluated the new liver support system as well as the Molecular Adsorbent Recycling System (MARS) in an ischemic porcine model of ALF. METHODS In the first study animals were randomly allocated to control (n=5) and Hepa Wash (n=6) groups. In a further pilot study, two animals were treated with the MARS-system. All animals received the same medical and surgical procedures. An intraparenchymal intracranial pressure was inserted. Hemodynamic monitoring and goal-directed fluid therapy using the PiCCO system was done. Animals underwent functional end-to-side portacaval shunt and ligation of hepatic arteries. Treatment with albumin dialysis was started after fall of cerebral perfusion pressure to 45 mmHg and continued for 8 h. RESULTS All animals in the Hepa Wash group survived the 13-hour observation period, except for one that died after stopping treatment. Four of the control animals died within this period (p=0.03). Hepa Wash significantly reduced impairment of cerebral perfusion pressure (23±2 vs. 10±3 mmHg, p=0.006) and mean arterial pressure (37±1 vs. 24±2 mmHg, p=0.006) but had no effect on intracranial pressure (14±1 vs. 15±1 mmHg, p=0.72). Hepa Wash also enhanced cardiac index (4.94±0.32 vs. 3.36±0.25 l/min/m2, p=0.006) and renal function (urine production, 1850 ± 570 vs. 420 ± 180 ml, p=0.045) and eliminated water soluble (creatinine, 1.3±0.2 vs. 3.2±0.3 mg/dl, p=0.01; ammonia 562±124 vs. 1382±92 μg/dl, p=0.006) and protein-bound toxins (nitrate/nitrite 5.54±1.57 vs. 49.82±13.27 μmol/l, p=0.01). No adverse events that could be attributed to the Hepa Wash treatment were observed. CONCLUSIONS Hepa Wash was a safe procedure and improved multiorgan system failure in pigs with ALF. The survival benefit could be the result of ameliorating different organ functions in association with the detoxification capacity of water soluble and protein-bound toxins.

The Aeromonas salmonicida subsp. salmonicida exoproteome: determination of the complete repertoire of Type-Three Secretion System effectors and identification of other virulence factors

BACKGROUND Aeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis, is a major pathogen of fisheries worldwide. Several virulence factors have been described, but the type-three secretion system (T3SS) is recognized as having a major effect on virulence by injecting effectors directly into fish cells. In this study we used high-throughput proteomics to display the differences between in vitro secretome of A. salmonicida wild-type (wt, hypervirulent, JF2267) and T3SS-deficient (isogenic ΔascV, extremely low-virulent, JF2747) strains in exponential and stationary phases of growth. RESULTS Results confirmed the secretion of effectors AopH, AexT, AopP and AopO via T3SS, and for the first time demonstrated the impact of T3SS in secretion of Ati2, AopN and ExsE that are known as effectors in other pathogens. Translocators, needle subunits, Ati1, and AscX were also secreted in supernatants (SNs) dependent on T3SS. AopH, Ati2, AexT, AopB and AopD were in the top seven most abundant excreted proteins. EF-G, EF-Tu, DnaK, HtpG, PNPase, PepN and MdeA were moderately secreted in wt SNs and predicted to be putative T3 effectors by bioinformatics. Pta and ASA_P5G088 were increased in wt SNs and T3-associated in other bacteria. Ten conserved cytoplasmic proteins were more abundant in wt SNs than in the ΔascV mutant, but without any clear association to a secretion system. T1-secreted proteins were predominantly found in wt SNs: OmpAI, OmpK40, DegQ, insulinase ASA_0716, hypothetical ASA_0852 and ASA_3619. Presence of T3SS components in pellets was clearly decreased by ascV deletion, while no impact was observed on T1- and T2SS. Our results demonstrated that the ΔascV mutant strain excreted well-described (VapA, AerA, AerB, GCAT, Pla1, PlaC, TagA, Ahe2, GbpA and enolase) and yet uncharacterized potential toxins, adhesins and enzymes as much as or even more than the wt strain. Other putative important virulence factors were not detected. CONCLUSIONS We demonstrated the whole in vitro secretome and T3SS repertoire of hypervirulent A. salmonicida. Several toxins, adhesins and enzymes that are not part of the T3SS secretome were secreted to a higher extent in the extremely low-virulent ΔascV mutant. All together, our results show the high importance of an intact T3SS to initiate the furunculosis and offer new information about the pathogenesis.

P2X7 receptors mediate resistance to toxin-induced cell lysis

In the majority of cells, the integrity of the plasmalemma is recurrently compromised by mechanical or chemical stress. Serum complement or bacterial pore-forming toxins can perforate the plasma membrane provoking uncontrolled Ca(2+) influx, loss of cytoplasmic constituents and cell lysis. Plasmalemmal blebbing has previously been shown to protect cells against bacterial pore-forming toxins. The activation of the P2X7 receptor (P2X7R), an ATP-gated trimeric membrane cation channel, triggers Ca(2+) influx and induces blebbing. We have investigated the role of the P2X7R as a regulator of plasmalemmal protection after toxin-induced membrane perforation caused by bacterial streptolysin O (SLO). Our results show that the expression and activation of the P2X7R furnishes cells with an increased chance of surviving attacks by SLO. This protective effect can be demonstrated not only in human embryonic kidney 293 (HEK) cells transfected with the P2X7R, but also in human mast cells (HMC-1), which express the receptor endogenously. In addition, this effect is abolished by treatment with blebbistatin or A-438079, a selective P2X7R antagonist. Thus blebbing, which is elicited by the ATP-mediated, paracrine activation of the P2X7R, is part of a cellular non-immune defense mechanism. It pre-empts plasmalemmal damage and promotes cellular survival. This mechanism is of considerable importance for cells of the immune system which carry the P2X7R and which are specifically exposed to toxin attacks.

Microvesicle Shedding and Lysosomal Repair Fulfill Divergent Cellular Needs during the Repair of Streptolysin O-Induced Plasmalemmal Damage

Pathogenic bacteria secrete pore-forming toxins that permeabilize the plasma membrane of host cells. Nucleated cells possess protective mechanisms that repair toxin-damaged plasmalemma. Currently two putative repair scenarios are debated: either the isolation of the damaged membrane regions and their subsequent expulsion as microvesicles (shedding) or lysosome-dependent repair might allow the cell to rid itself of its toxic cargo and prevent lysis. Here we provide evidence that both mechanisms operate in tandem but fulfill diverse cellular needs. The prevalence of the repair strategy varies between cell types and is guided by the severity and the localization of the initial toxin-induced damage, by the morphology of a cell and, most important, by the incidence of the secondary mechanical damage. The surgically precise action of microvesicle shedding is best suited for the instant elimination of individual toxin pores, whereas lysosomal repair is indispensable for mending of self-inflicted mechanical injuries following initial plasmalemmal permeabilization by bacterial toxins. Our study provides new insights into the functioning of non-immune cellular defenses against bacterial pathogens.

Ceramide in Plasma Membrane Repair

The perforation of the plasmalemma by pore-forming toxins causes an influx of Ca2+ and an efflux of cytoplasmic proteins. In order to ensure cellular survival, lesions have to be identified, plugged and removed from the membrane. The Ca2+-driven fusion of lysosomes with the plasma membrane leads to hydrolysis of sphingomyelin by acid sphingomyelinase and a formation of ceramide platforms in the outer leaflet of the lipid bilayer. We propose that the negative curvature, promoted by tighter packing of lipids in the outer layer, leads to an inward vesiculation of the damaged area for its endocytotic uptake and internal degradation. In contrast, the activation of neutral sphingomyelinase triggers the production of ceramide within the inner leaflet of the lipid bilayer, thereby promoting an outward curvature, which enables the cell to shed the membrane-containing toxin pore into the extracellular space. In this process, ceramide is supported by members of the annexin protein family which act as Ca2+ sensors and as membrane fusion agents.

Veterinary hemodialysis: advances in management and technology.

Hemodialysis (HD) is a renal replacement therapy that can enable recovery of patients in acute kidney failure and prolong survival for patients with end-stage kidney failure. HD is also uniquely suited for management of refractory volume overload and removal of certain toxins from the bloodstream. Over the last decade, veterinary experience with HD has deepened and refined and its geographic availability has increased. As awareness of the usefulness and availability of dialytic therapy increases among veterinarians and pet owners and the number of veterinary dialysis facilities increases, dialytic management will become the standard of advanced care for animals with severe intractable uremia.

Detection of RTX toxin genes in gram-negative bacteria with a set of specific probes

The family of RTX (RTX representing repeats in the structural toxin) toxins is composed of several protein toxins with a characteristic nonapeptide glycine-rich repeat motif. Most of its members were shown to have cytolytic activity. By comparing the genetic relationships of the RTX toxin genes we established a set of 10 gene probes to be used for screening as-yet-unknown RTX toxin genes in bacterial species. The probes include parts of apxIA, apxIIA, and apxIIIA from Actinobacillus pleuropneumoniae, cyaA from Bordetella pertusis, frpA from Neisseria meningitidis, prtC from Erwinia chrysanthemi, hlyA and elyA from Escherichia coli, aaltA from Actinobacillus actinomycetemcomitans and lktA from Pasteurella haemolytica. A panel of pathogenic and nonpathogenic gram-negative bacteria were investigated for the presence of RTX toxin genes. The probes detected all known genes for RTX toxins. Moreover, we found potential RTX toxin genes in several pathogenic bacterial species for which no such toxins are known yet. This indicates that RTX or RTX-like toxins are widely distributed among pathogenic gram-negative bacteria. The probes generated by PCR and the hybridization method were optimized to allow broad-range screening for RTX toxin genes in one step. This included the binding of unlabelled probes to a nylon filter and subsequent hybridization of the filter with labelled genomic DNA of the strain to be tested. The method constitutes a powerful tool for the assessment of the potential pathogenicity of poorly characterized strains intended to be used in biotechnological applications. Moreover, it is useful for the detection of already-known or new RTX toxin genes in bacteria of medical importance.

Transient Fanconi syndrome with severe polyuria and polydipsia in a 4-year old Shih Tzu fed chicken jerky treats.

Acquired Fanconi syndrome is characterized by inappropriate urinary loss of amino acids, bicarbonate, electrolytes, and water. It has recently been described in dogs fed chicken jerky treats from China, a new differential diagnosis to the classical inciting infectious diseases (e.g. leptospirosis, pyelonephritis) and toxins. A dog fed exclusively chicken jerky treats purchased in Switzerland was presented to our clinic with severe polyuria, polydipsia and profound electrolyte and acid base disturbances. Other inciting causes of Fanconi syndrome were ruled out. The requirement of a very intensive supportive treatment in this dog stands in contrast to treatment of chronic forms of Fanconi syndrome as described in the Basenji. This intensive therapy and the associated monitoring can be a real challenge and a limiting factor for the prognosis of acquired Fanconi syndrome. Veterinarians should be aware of the risk of excessive feeding of chicken jerky treats.

Engineered liposomes sequester bacterial exotoxins and protect from severe invasive infections in mice

Gram-positive bacterial pathogens that secrete cytotoxic pore-forming toxins, such as Staphylococcus aureus and Streptococcus pneumoniae, cause a substantial burden of disease. Inspired by the principles that govern natural toxin-host interactions, we have engineered artificial liposomes that are tailored to effectively compete with host cells for toxin binding. Liposome-bound toxins are unable to lyse mammalian cells in vitro. We use these artificial liposomes as decoy targets to sequester bacterial toxins that are produced during active infection in vivo. Administration of artificial liposomes within 10 h after infection rescues mice from septicemia caused by S. aureus and S. pneumoniae, whereas untreated mice die within 24-33 h. Furthermore, liposomes protect mice against invasive pneumococcal pneumonia. Composed exclusively of naturally occurring lipids, tailored liposomes are not bactericidal and could be used therapeutically either alone or in conjunction with antibiotics to combat bacterial infections and to minimize toxin-induced tissue damage that occurs during bacterial clearance

Novel Prodrug-Like Fusion Toxin with Protease-Sensitive Bioorthogonal PEGylation for Tumor Targeting

Highly potent biotoxins like Pseudomonas exotoxin A (ETA) are attractive payloads for tumor targeting. However, despite replacement of the natural cell-binding domain of ETA by tumor-selective antibodies or alternative binding proteins like designed ankyrin repeat proteins (DARPins) the therapeutic window of such fusion toxins is still limited by target-independent cellular uptake, resulting in toxicity in normal tissues. Furthermore, the strong immunogenicity of the bacterial toxin precludes repeated administration in most patients. Site-specific modification to convert ETA into a prodrug-like toxin which is reactivated specifically in the tumor, and at the same time has a longer circulation half-life and is less immunogenic, is therefore appealing. To engineer a prodrug-like fusion toxin consisting of the anti-EpCAM DARPin Ec1 and a domain I-deleted variant of ETA (ETA″), we used strain-promoted azide alkyne cycloaddition for bioorthogonal conjugation of linear or branched polyethylene glycol (PEG) polymers at defined positions within the toxin moiety. Reversibility of the shielding was provided by a designed peptide linker containing the cleavage site for the rhinovirus 3C model protease. We identified two distinct sites, one within the catalytic domain and one close to the C-terminal KDEL sequence of Ec1-ETA″, simultaneous PEGylation of which resulted in up to 1000-fold lower cytotoxicity in EpCAM-positive tumor cells. Importantly, the potency of the fusion toxin was fully restored by proteolytic unveiling. Upon systemic administration in mice, PEGylated Ec1-ETA″ was much better tolerated than Ec1-ETA″; it showed a longer circulation half-life and an almost 10-fold increased area under the curve (AUC). Our strategy of engineering prodrug-like fusion toxins by bioorthogonal veiling opens new possibilities for targeting tumors with more specificity and efficacy.

Patho-genetics of Clostridium chauvoei

The genomic sequence of Clostridium chauvoei, the etiological agent of blackleg, a severe disease of ruminants with high mortality specified by a myonecrosis reveals a chromosome of 2.8 million base-pairs and a cryptic plasmid of 5.5 kilo base-pairs. The chromosome contains the main pathways like glycolysis/gluconeogenesis, sugar metabolism, purine and pyrimidine metabolisms, but the notable absence of genes of the citric acid cycle and deficient or partially deficient amino acid metabolism for Histidine, Tyrosine, Phenylalanine, and Tryptophan. These essential amino acids might be acquired from host tissue damage caused by various toxins and by protein metabolism that includes 57 genes for peptidases, and several ABC transporters for amino acids import.

Defensive weapons and defense signals in plants: Some metabolites serve both roles

The defense of plants against herbivores and pathogens involves the participation of an enormous range of different metabolites, some of which act directly as defensive weapons against enemies (toxins or deterrents) and some of which act as components of the complex internal signaling network that insures that defense is timed to enemy attack. Recent work reveals a surprising trend: The same compounds may act as both weapons and signals of defense. For example, two groups of well-studied defensive weapons, glucosinolates and benzoxazinoids, trigger the accumulation of the protective polysaccharide callose as a barrier against aphids and pathogens. In the other direction, several hormones acting in defense signaling (and their precursors and products) exhibit activity as weapons against pathogens. Knowing which compounds are defensive weapons, which are defensive signals and which are both is vital for understanding the functioning of plant defense systems.

Metabolomics reveals herbivore-induced metabolites of resistance and susceptibility in maize leaves and roots

Plants respond to herbivory by reprogramming their metabolism. Most research in this context has focused on locally induced compounds that function as toxins or feeding deterrents. We developed an ultra-high-pressure liquid chromatography time-of-flight mass spectrometry (UHPLC-TOF-MS)-based metabolomics approach to evaluate local and systemic herbivore-induced changes in maize leaves, sap, roots and root exudates without any prior assumptions about their function. Thirty-two differentially regulated compounds were identified from Spodoptera littoralis-infested maize seedlings and isolated for structure assignment by microflow nuclear magnetic resonance (CapNMR). Nine compounds were quantified by a high throughput direct nano-infusion tandem mass spectrometry/mass spectrometry (MS/MS) method. Leaf infestation led to a marked local increase of 1,3-benzoxazin-4-ones, phospholipids, N-hydroxycinnamoyltyramines, azealic acid and tryptophan. Only few changes were found in the root metabolome, but 1,3-benzoxazin-4-ones increased in the vascular sap and root exudates. The role of N-hydroxycinnamoyltyramines in plant–herbivore interactions is unknown, and we therefore tested the effect of the dominating p-coumaroyltyramine on S. littoralis. Unexpectedly, p-coumaroyltyramine was metabolized by the larvae and increased larval growth, possibly by providing additional nitrogen to the insect. Taken together, this study illustrates that herbivore attack leads to the induction of metabolites that can have contrasting effects on herbivore resistance in the leaves and roots.