Botulinum toxins produced by the anaerobic bacterium Clostridium botulinum are the most potent biological toxins in nature. Traditionally, people at risk are immunized with a formaldehyde-inactivated toxin complex. Second generation vaccines are based on the recombinant carboxy-terminal heavy-chain (Hc) fragment of the neurotoxin. However, the materialization of this approach is challenging, mainly due to the high AT content of clostridial genes. Herein, we present an alternative strategy in which the native genes encoding Hc proteins of botulinum toxins A, B, and E were used to express the recombinant Hc fragments in a cell-free expression system. We used the unique property of this open system to introduce different combinations of chaperone systems, protein disulfide isomerase (PDI), and reducing/oxidizing environments directly to the expression reaction. Optimized expression conditions led to increased production of soluble Hc protein, which was successfully scaled up using a continuous exchange (CE) cell-free system. Hc proteins were produced at a concentration of more than 1 mg/ml and purified by one-step Ni ؉ affinity chromatography. Mice immunized with three injections containing 5 g of any of the in vitro-expressed, alum-absorbed, Hc vaccines generated a serum enzyme-linked immunosorbent assay (ELISA) titer of 10 5 against the native toxin complex, which enabled protection against a high-dose toxin challenge (10 3 to 10 6 mouse 50% lethal dose [MsLD 50 ]). Finally, immunization with a trivalent HcA, HcB, and HcE vaccine protected mice against the corresponding trivalent 10 5 MsLD 50 toxin challenge. Our results together with the latest developments in scalability of the in vitro protein expression systems offer alternative routes for the preparation of botulinum vaccine.

A preadmission social work intervention was evaluated for impact on length of hospital stay (LOS) and patient satisfaction. Psychosocial issues related to function and post-discharge needs were assessed at an exploratory level. A modified post-test only control group design was used. Study group patients were screened before hospitalization and offered services on admission. Control group patients received standard care. Study group patients were significantly more satisfied with services but impact on length of stay was not demonstrated with one possible exception. Post-operative complications were significantly related to longer LOS; however, unlike control group patients, study group patients with complications did not have significantly longer LOS. Women and those limited in preadmission physical function were most likely to report insufficient help after discharge. A more intensive preadmission intervention is recommended to improve impact on LOS and informal support system involvement, while future outcome studies would clarify the nature of service gaps and high risk groups.

Botulinum toxin was detected in patient serum using Endopeptidase-mass-spectrometry assay, although all conventional tests provided negative results. Antitoxin was administered, resulting in patient improvement. Implementing this highly sensitive and rapid assay will improve preparedness for foodborne botulism and deliberate exposure.

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