Anthrax toxins regulate pain signaling and can deliver molecular cargoes into ANTXR2+ DRG sensory neurons.

Bacterial products can act on neurons to alter signaling and function. In the present study, we found that dorsal root ganglion (DRG) sensory neurons are enriched for ANTXR2, the high-affinity receptor for anthrax toxins. Anthrax toxins are composed of protective antigen (PA), which binds to ANTXR2, and the protein cargoes edema factor (EF) and lethal factor (LF). Intrathecal administration of edema toxin (ET (PA + EF)) targeted DRG neurons and induced analgesia in mice. ET inhibited mechanical and thermal sensation, and pain caused by formalin, carrageenan or nerve injury. Analgesia depended on ANTXR2 expressed by Nav1.8+ or Advillin+ neurons. ET modulated protein kinase A signaling in mouse sensory and human induced pluripotent stem cell-derived sensory neurons, and attenuated spinal cord neurotransmission. We further engineered anthrax toxins to introduce exogenous protein cargoes, including botulinum toxin, into DRG neurons to silence pain. Our study highlights interactions between a bacterial toxin and nociceptors, which may lead to the development of new pain therapeutics.

A comparison of biological activity of commercially available purified native botulinum neurotoxin serotypes A1 to F1 in vitro, ex vivo, and in vivo.

Botulinum neurotoxin (BoNT) is a major therapeutic agent. Of seven native BoNT serotypes (A to G), only A and B are currently used in the clinic. Here we compared the potency of commercially available purified native serotypes A1 to F1 across in vitro, ex vivo, and in vivo assays. BoNT potency in vitro was assessed in rat primary cells (target protein cleavage and neurotransmitter release assays) in supraspinal, spinal, and sensory systems. BoNT potency ex vivo was measured in the mouse phrenic nerve hemidiaphragm (PNHD) assay, measuring muscle contractility. In vivo, BoNT-induced muscle relaxation in mice and rats was assessed in the Digit Abduction Score (DAS) test, while effects on body weight (BW) gain were used to assess tolerability. In all assays, all BoNT serotypes were potent toxins, except serotype D1 in vivo which failed to produce significant muscle flaccidity in mice and rats. In rats, all serotypes were well-tolerated, whereas in mice, reductions in BW were detected at high doses. Serotype A1 was the most potent serotype across in vitro, ex vivo, and in vivo assays. The rank order of potency of the serotypes revealed differences among assays. For example, species-specificity was seen for serotype B1, and to a lesser extent for serotype C1. Serotypes F1 and C1, not currently in the clinic, showed preference for sensory over motor models and therefore could be considered for development in conditions involving the somatosensory system.

Augmentation of VAMP-catalytic activity of botulinum neurotoxin serotype B does not result in increased potency in physiological systems.

Botulinum neurotoxins (BoNTs) are used extensively as therapeutic agents. Serotypes A and B are available as marketed products. Higher doses of BoNT/B are required to reach an efficacy similar to that of products containing BoNT/A. Advances in our understanding of BoNT/B mechanism of action have afforded the opportunity to make rational modifications to the toxin aimed at increasing its activity. Recently, a mutation in the light chain of BoNT/B (S201P) was described that increases the catalytic activity of the isolated BoNT/B light chain in biochemical assays. In this study, we have produced two full-length recombinant BoNT/B toxins in E.coli-one wild type (rBoNT/B1) and one incorporating the S201P mutation (rBoNT/B1(S201P)). We have compared the activity of these two molecules along with a native BoNT/B1 in biochemical cell-free assays and in several biological systems. In the cell-free assay, which measured light-chain activity alone, rBoNT/B1(S201P) cleaved VAMP-2 and VAMP-1 substrate with an activity 3-4-fold higher than rBoNT/B1. However, despite the enhanced catalytic activity of rBoNT/B1(S201P), there was no significant difference in potency between the two molecules in any of the in vitro cell-based assays, using either rodent spinal cord neurons or cortical neurons. Similarly in ex vivo tissue preparations rBoNT/B1(S201P) was not significantly more potent than rBoNT/B1 at inhibiting either diaphragm or detrusor (bladder) muscle activity in C57BL/6N and CD1 mice. Finally, no differences between rBoNT/B1 and rBoNT/B1(S201P) were observed in an in vivo digit abduction score (DAS) assay in C57BL/6N mice, either in efficacy or safety parameters. The lack of translation from the enhanced BoNT/B1(S201P) catalytic activity to potency in complex biological systems suggests that the catalytic step is not the rate-limiting factor for BoNT/B to reach maximum efficacy. In order to augment the efficacy of BoNT/B in humans, strategies other than enhancing light chain activity may need to be considered.

Monoclonal antibody-based time-resolved fluorescence immunoassays for daidzein, genistein, and equol in blood and urine: application to the Isoheart intervention study.

BACKGROUND: Time-resolved fluorescence immunoassays (TR-FIAs) for phytoestrogens in biological samples are an alternative to mass spectrometric methods. These immunoassays were used to test urine and plasma samples from individuals in a dietary intervention trial aimed at determining the efficacy of dietary isoflavones in reducing the risk of coronary heart disease in postmenopausal women. METHODS: We established murine monoclonal TR-FIA methods for daidzein, genistein, and equol. These assays could be performed manually or adapted to an automated analyzer for high throughput and increased accuracy. Analysis of urine was conducted on nonextracted samples. Blood analysis was performed on nonextracted samples for daidzein, whereas genistein and equol required diethyl-ether extraction. RESULTS: Comparison of monoclonal TR-FIA, commercial polyclonal antibody-based TR-FIA, and gas chromatography-mass spectrometry showed correlations (r, 0.911-0.994) across the concentration range observed in the Isoheart study (50 mg/day isoflavones). The concentrations of urinary daidzein and genistein observed during intervention demonstrated good compliance, and a corresponding increase in serum daidzein and genistein confirmed bioavailability of the isoflavone-rich foods; 33 of the 117 volunteers (28.2%) were classified as equol producers on the basis of their urinary equol concentration (>936 nmol/L), and significant differences in the numbers of equol producers were observed between Berlin and the 3 other European cohorts studied. CONCLUSIONS: The validated monoclonal TR-FIA methods are applicable for use in large-scale human phytoestrogen intervention studies and can be used to monitor compliance, demonstrate bioavailability, and assess equol producer status.