CBMTS III: A Summary
by Dr. Rudolf Portmann
AC Laboratorium Spiez, Switzerland

At CBMTS III ninety-eight scientists from 29 countries participated and presented papers in the areas of defense against chemical and biological warfare and chemical and bioterrorism. The presentations took place in a total of 16 sessions, including the three poster sessions.

Symposium Chair: Dr. Brian Davey
Symposium Co-chair: Dr. Barbara Price

Sector Chemical (Sessions: 1,2, 4, 5, 8, 9, 10, 11, 13, 15)
Chair: Dr. Rudolf Portmann
Co-Chair: Dr. David Moore

Sector Biological (Sessions: 3, 6, 7, 12, 14, 16)
Chair: Dr. Barbara Price
Co-chair: Prof. Ji-Sheng Chen

Session 1 - OP Treatment: State of the Art
Chair: Peter Eyer; Co-chair: Jiri Bajgar
This session was devoted to the practical question of treatment of OP intoxication as well as the effects of low dose exposure to OP compounds. Dr Elsa Reiner gave an introductory overview of the current knowledge about research topics concerned with treatment of OP intoxication. Dr. Balali-Mood presented the results of a study aimed at investigation the effects of extra sodium bicarbonate infusion (alkalinization to pH 7.47) in OP-poisoned patients which all received atropine. There was the suggestion of some benefits, but this was not statistically significant. Due to the potential low cost of bicarbonate therapy if it is successful, the study will be extended with even higher alkalinization. Dr. Bajgar presented a study using the erythrocyte-AChE inhibition to identify the underlying nerve agent. Dr. Worek presented results indicating that if an oxime substantially reactivates the BuChE (serumcholinesterase), this will lead to a re-inhibition of the AChE (acetylcholinesterase). Dr. Scott presented a highly advanced approach to follow subtle behavioral and electrophysiological changes upon low-dose exposure towards OP in marmosets. It appears that a single sign-free dose exposure of non-human primates to sarin was without any lasting significant effect.

Session 2 - Posters Animal Treatment/ChE Measurement
Chair: Elsa Reiner; Co-chair: Atilla Hincal
This first poster session included treatment studies of OP intoxicated animals (Dr. Antonijevic, Dr. Dorandeu). The authors gave a brief platform presentation, followed by an extremely lively discussion in the poster area. This format seems to be very stimulating for further discussions and collaborations.

Session 3 - Bioterrorism: National Approach
Chair: Murray Hamilton; Co-chair: Lotfali Haghighi
This session was dedicated to the national approach against chemical and biological terrorism. Here overviews of national readiness and response planning were presented Dr. Erasmus, South Africa, Dr. Harwood, Canada, Dr. Treadwell, USA, Dr. Spies, USA and Dr. Robertson, Australia. Col. Khan from Pakistan opened the Session with a presentation detailing characteristics of modern terrorists as they have moved away from large organizations towards smaller but more focussed groups.

Session 4 - OP Treatment Studies
Chair: Leah Scott; Co-chair: Florin Paul
This session's theme concerned the studies of treatment of OP-intoxications. Dr. Phillipens presented a summary of guinea pig studies investigating whether stressors are influencing the effects of pretreatment using physostigmine and the effectiveness of such pretreatment in protecting against soman. Stressors appear to induce adverse behavioral effects in pretreated animals and that protective efficacy was compromised. Dr. Zhou described in vitro studies involving cholinolytic drugs and oximes. Unlike the oximes, toxogonin and Hl-6, these compounds did not by themselves reactivate AChE. But the results confirmed that, if added at the start of ageing, reversible ChE inhibitors increased the portion of AChE reactivatible by the oximes. Dr. Tonkopii discussed the effectiveness of a series of familiar and less familiar carbamates as pretreatment for nerve agent poisoning in vivo. Interestingly, some of the carbamates were more toxic than the nerve agents and others made the nerve agent mixture more toxic.

Session 5 - Medical Treatment
Chair: Mahdi Balali-Mood; Co-chair: Cornelis Erasmus
Questions on the treatment of convulsion, mustard and heavy metal intoxication were discussed. Dr. Rump first pointed out the side effects of benzodiazepines, and in particular, diazepam as anti-convulsant, and then introduced imidazenil as the ideal drug for the management of OP-induced convulsions. He concluded that imidazenil, in a dose of 2 mg/kg, blocked soman induced convulsions and increased the anti-lethal effectiveness of atropine and HI-6 in soman poisoned mice. Dr Szinicz pointed out that the role of inflammatory cytokines released after sulfur mustard (SM) injury is not well known. The data indicate HaCaT cells to be an appropriate model for the study of SM effects in keratinocytes compared to SCL II cells. According to Dr. Jaiswal no satisfactory prophylactic for the treatment regimen has evolved for sulfur mustard. He introduced Amifostine and its analogs as prophylactic agents for SM poisoning. Amifostine and one of its analogs, DRDE-07, gave significant protection. Dr. Wormser carried out a prolonged study on the therapeutic effects of iodine at 15, 30, 45, and 60 minutes after SM exposure on guinea pig skin. Gross pathology analysis showed a strong protective effect at intervals of 15 and 30 minutes, and to a lesser extent at 45 and 60 minutes. Dr. Mircioiu presented a paper on using chelators or "decorporators" as a possible medical treatment for poisoning by radioactive metals. Using pharmacokinetic theory he explored a bicompartmental pharmacokinetic model, that appeared to work, in spite of the fact that the ion cannot leave the central compartment. Ca, Zn and Cu complexes of EDTA were among the "decorporators" studied.

Session 6 - Toxins
Chair: Leo Laughlin; Co-chair: Liaquat Ali Khan
Toxins were the theme. Dr. Laughlin discussed selected US BW Agent detection technologies from an historical perspective. The initial problems identified in the methods discussed still persist: sensitivity, time for detection / identification, and specificity of the methodology. Dr. Chen from China provided an assessment of toxins that clearly indicated that that there are far more toxins of concern than are currently listed in the Chemical Weapons or Biological & Toxins Weapons Conventions. He described a number of factors to assess the threat from toxins. Dr. Hamilton explained how botulinum toxins inhibited acetylcholine release by cleaving presynaptic membrane proteins necessary for exocytosis of the neurotransmitter acetylcholine. A specific library was prepared by combinatorial synthesis that contained a repeating sequence (probably the binding recognition site) that did inhibit botulinum toxin.

Session 7 - Bio/Toxin: Threat and Detection
Chair: Paul Russel; Co-chair: Sven-Åke Pearson
Main theme: identifying infectious diseases and toxins as candidate biological warfare agents and techniques to assist diagnosis of both natural disease outbreaks and biological warfare agent outbreaks. In the first instance criteria based on biological and physical properties of pathogens and toxins such as inactivity, lethality, transmissibility, disseminability and weaponisation were discussed by Dr Bokan as tools for identifying candidate biological warfare agents against human, animal and plant targets. A scoring mechanism was used to create a threat list which largely concurred with agents listed in the BTWC. The use of rapid PCR and light cycler were mentioned by Dr Schütz as a rapid means of diagnosing a disease outbreak. The use of genetic techniques for rapid identification and epidemiology was discussed using Swiss studies of army training areas as an example. The dual use of these techniques was highlighted and the fact that recombinant gene research had increased dramatically over the last 15 years. The best means of preventing less ethical research would be through the BTWC and honesty in such fora as the CBMTS meetings. Dr Taleski discussed direct epidemiological studies as a means of identifying a disease outbreak due to the dissemination of a biological weapon. Dr Paul, as an example, used anthrax's capabilities as a biological weapon to show how the epidemiology would apply. The epidemiological curve, with casualty numbers and fatalities were presented and the current medical countermeasures to anthrax were shown. The role of macrophages in viral hemorrhagic fevers (VHFs) was presented by Dr. Ryabchikova, indicating that most viruses causing VHF are able to multiply within these cells. Hemorrhagic rash, and bleeding were seen in the baboon model.

Session 8 - Risk Assessment/Data Bank/Mass Casualty
Chair: Gurayten Ozyurt; Co-chair: Julie Tremblay-Lutter
Risk assessment, data banks and mass casualty management were the overall theme of this session. Dr. Hincal pointed out, that Drug and Poison information Centers may well provide the informational links between medical institutions, military groups, police forces, civil defense personnel and public transportation systems in mass casualty management. Dr. Hughart introduced the developing database, consisting of information about a chemical disaster, likely to be encountered by first responders (military men or women, disaster assessment teams, police officers, firemen and emergency medical technicians). It has key words as DISASTER, ASSESSMENT and ASSISTANCE. The understanding of the long-term toxic effects of environmental toxicants and example data sets from DYNCORP Project covering civilian and military risk management were presented by Dr. Rao. Dr. Kosnett presented the key services Poison Centers can provide in the event of CB incident: a) centralized communication to hospital emergency department b) assistance of antidote and related emergency medical supplies, c) communication of information to the general public either through the media or coming telephone calls. As pointed out by Dr. Mathenge, industrial accidents are becoming a common feature in the process of industrial development. The proactive action that would prevent the hazards of these factories brought by natural and human induction, was introduced. Dr. Hincal introduced the Mass Casualty Management programs and preparations for Pharmaceutical Industry, which is mainly located in Istanbul, Turkey, and their risk management approaches.

Session 9 - Riot control and Detection
Chair: Richard Gordon; Co-chair: Urs Brodbeck
Questions on detection, direct or retrospective, and riot control agents were discussed by Dr. Davey, Dr. Noort, Dr. Juruli, Dr. Surovtsev and Dr. Wicki. The overall message from this Session was that both innocent control of civilians (such as pepper spray or the events that occurred at Tbilisi), as well as more threatening events (such as the Japan Tokyo incident that used impure sarin), require techniques to identify the toxic chemical agents used. Furthermore, a better understanding of the mechanism of action of the agents would provide a means to detect the agents better, and determine those agents that are indeed toxic. Most important, the standardization of the methodology to identify post-exposure to agents (biomedical samples) would provide the means to better enforce the CWC. A result of the discussions was that the estimation of cholinesterase activity is a useful indication of pesticide intoxication in occupational health and therapeutic monitoring of various neurological diseases.

Session 10 - Cholinesterase Determination
Chair: Uri Wormser; Co-chair: Valerii Tonkopii
As discussed, determination of ChE, is a crucial factor in detection and monitoring exposure of humans to organophosphates and carbamates. Much effort has been invested in developing appropriate analysis that can simply and reliably determine ChE. The papers presented in this Session discussed the recent advances in this area. Dr. Eyer presented an improved method that, in addition of being easily performed, gives reliable results down to 3% residual activity. Dr. Feaster demonstrated a new and accurate method for simultaneous determination of blood concentrations of both AChE and BuChE. This method was successfully employed in screening 320 blood samples in one four-hour time interval. Dr. Portmann discussed the reasons for the relatively high variations in BuChE levels obtained in different laboratories. He discussed several important technical improvements, such as the use of detergent and the loss of hydrocyanic acid in the reagent mixture. Dr. Simeon-Rudolf described a simple method for AChE and BuChE using a colorimetric method and DTNB for thiol groups determination. Dr. Brodbeck compared AChE and BuChE levels in blood taken from capillary (finger) and from venous blood. He also tested the effect of different detergent on these enzymatic activities. The Session was an important methodological step towards a commonly accepted methods that may be applied in practice.

Session 11 - Decontamination
Chair: Devendra Jaiswal; Co-chair: Slavomir Rump
Decontamination plays a vital role in defence against toxic chemicals including chemical warfare agents. It is a process of removal and conversion of the toxic chemicals into non-toxic or harmless substances by physical, chemical or biochemical methods, depending on the nature and type of contamination and surface on which the decontaminant is dispersed. The research & development in NBC defence obviously includes decontamination as an important area of work. The Session gave a wide spectrum of information on new developments on materials, methods and technologies for decontamination. The decontamination of mass casualties after exposure to harmful liquid chemicals can be quite important in several situations particularly in an industrial accident as was highlighted by Dr.Persson. The casualties with traumatic injuries and exposure to toxic chemicals will create problems with regard to transportation, decontamination and medical treatment. The polyurethane foam linked mammalian cholinesterase for decontamination and detection of OP nerve agents was an interesting development reported by Dr. Gordon. It involves a bioscavenger approach to the protection against OP toxicity. A sponge product containing ChEs, oximes and polyurethane was developed for removal and decontamination of OP compounds from medically important surfaces such as skin and wounds. The bioscavengers can also act as a biosensor for detection of OPs. Maj. Eaton discussed large-scale mass decontamination and special event planning criteria for special international events based on Canadian Aqueous System for Chemical, Biological and Radiological Decontamination (CASCAD) system and Canadian model. Recent and planned research to extend and further validate RSDL as a broad spectrum personal decontamination system for BCW agent decontamination had been studied by Dr. Philip O'Dell. The Reactive Skin Decontamination Lotion (RSDL) has been fielded in a number of countries for C agent protection of personnel. It is also useful for BW agent decontamination and for riot agent removal. Mr. Earl Laurie presented the useful development of new Chemical and Biological (CB) moulded gloves with far better tear resistance and physical properties He highlighted the general composition of the material used in such gloves and the advantages of the new product.

Session 12 - B-Agent Protection and Treatment
Chair: Elena Ryabchikova; Co-chair: Kim Lindsay
This session was devoted to discussion of B-agent protection and treatment. Only a small part of this huge field may be discussed in any meeting, and the reports presented in this Session were related to several events. Dr. Moore presented new promising biocidal resin Triosyn, characterized by high potential of microorganisms destruction and simplicity and convenience in practical application. The resin represents new approach to usage of iodine abilities to kill various microorganisms. Dr. Ryabchikova presented current situation of smallpox researches, and proposed plan of variola virus studies in SRC VB "Vector" (Russia). Dr. Haghighi's topic was related to analysis of the infections among Iranian troops during Iran-Iraq war. Isolated agents were identified and their biological properties were examined. Resistance of the selected microorganisms to antibiotics was evaluated. Dr. Russel's presentation gave an overview of plague vaccine history, current situation and main directions for future. Plague is still dangerous disease requiring attention of epidemiologists and development of new modern vaccines. Dr. Melling, who presented common principles of biodefense vaccinology, completed the Session. Biodefense vaccines are a special kind of vaccines, however, they should correspond to requirements for vaccines destined for public health. The report summarized general problems of biodefense vaccinology. Taken together, the Session paid attention to the most significant problems of B-agent protection and treatment.

Session 13 - Poster 2: Chem Poisoning, Treatment, Decon
Chair: Keith Vesely; Co-chair: Michael McMillan
This poster session was devoted to chemical poisoning, treatment, warning and decontamination. Topics included: antidote delivery systems; effects of low-level inhaled sarin; development of adequate warning systems and risk assessment procedures in and around chemical demilitarization sites; present and future treatments for chemical warfare agents; and examination of mycotoxin levels in feed and animal livers. Dr. Pita explained the functioning, manufacturing process and technical characteristics of Spanish Armed Forces autoinjectors. New directions will include: replacing 1 mL syringes with 2 mL syringes; introducing HI-6; research and development of combination atropine, oxime and anticonvulsant; and possibility of formulating autoinjectors with other drugs. Dr. Bajgar reported on a study examining the effects of sublethal concentrations of inhaled sarin as measured by AChE activity in rats. Results suggested that at low concentrations, AChE in blood is "titrated" by sarin. AChE in the blood appears to be is a very sensitive marker for diagnosis of inhalation exposure to low concentrations of sarin. Dr. Kassa described the long-term effects of low-level sarin inhalation in rats. Non-convulsive symptomatic levels of sarin can cause subtle long-term signs of neurotoxicity and immunotoxicity. These sarin-induced long-term signs of neurotoxicity may be caused by mechanism(s) separate from the cholinergic nervous system. Dr. Petrov described the need to replace obsolete warning systems to properly warn personnel at chemical warfare storage facilities, as well as surrounding populations in Russia. Dr. Juruli evaluated the health risk of the "old military waste" in Georgia. To prevent damage to human health, the national legal instruments related to chemical management must be improved. Dr. Saxena presented work establishing a relationship between ligand-induced acceleration of AChE reactivation and phosphoryloxime inhibition of the reactivated enzyme. Results suggest that ChE, oximes and organophosphorus hydrolases can work in tandem to hydrolyze or inactivate organophosphorus compounds in vitro and in vivo. Dr. Kalantari presented a study designed to determine the extent of contamination of mixed feed by mycotoxin and the subsequent presence of the toxin in livers of poultry fed the contaminated feed. Results showed aflatoxin contamination but at levels that were safe. Dr. Ozyurt described the role of serum ChE and the Namba classification in diagnosing and managing organophosphorus intoxication in humans. Clinical appearance was found to be more important than only BuChE levels in determining when to cease antidotal therapy.

Session 14 - Poster 3: B-Agents and Terrorism
Chair: Lisa Rotz; Co-chair: Maarten Nieuwenhuizen
This poster session addressed a variety of subjects from policy approaches to combating chemical terrorism to specific methods for detection and early warning for chemical and biological agents. In addition, research projects that evaluated countermeasures for botulinum toxin B and T-2 mycotoxin were presented. Dr. Huber described the Swiss approach to protect from chemical terrorism. The observation was made that the problems and approaches for responding to chemical terrorism seem to be similar for various countries. Discussion occurred regarding the necessity of each country developing their own solutions, or could a common response template be developed that may then be used by several countries. Dr. Schürch discussed research regarding the development of a detection method for biological agents and toxins using monoclonal antibodies. The authors used a semisynthetic method to develop antibody libraries against F. tulariensis. The antibody libraries created using the animal model and the semisynthetic method were compared and appeared to be similar in their specificity for F. tularensis antigens. Buforin I (a peptide from the stomach of Asian frog) was found to be a competitive inhibitor of botulinum toxin B by Dr. Gordon who evaluated several peptides that contained the QF cleavage site. Buforin I functioned as a competitive, dose-dependent inhibitor for botulinum B toxin activity but not as a substrate. A method for assessing the risk of different agrochemicals for use as a terrorist weapon utilizing an analysis of their availability, volatility, and human and animal toxicity was presented by Dr. Yousefi. Ratings of high, medium, or low for each category were assigned to the agrochemicals currently available for industry use in South Africa and this scoring system was used to determine which chemicals present the greatest risk. The efficacy of multiple treatment regimens utilizing corticosteroids in rats exposed to T-2 mycotoxin was evaluated by Dr. Jovanovic. The dose of corticosteroid but not the route of administration produced significant differences in the protective index. A novel strategy using synthetic nucleic acids combine with conventional immunoassays for biological agent detection was described by Dr. Filer. The poster presentation was a description of a Multiplexed Detection System that would serve as a bridge between two distinct assay technologies in an attempt to ameliorate the limits endemic to current immuno-diagnostic systems.

Session 15 - Bioscavenger
Chair: Bhupendra Doctor; Co-chair: Yongxiang Zhang
The session addressed bioscavengers, research on the development of stoichiometric bioscavengers such as cholinesterases (ChEs) and catalytic bioscavengers such as OPAA and OPH was presented. Dr. Doctor demonstrated the use of three different types of ChEs (HuS BChE, Eq BChE, and FBS AchE) in protection against OP intoxication in four animal species (rats, mice, guinea pigs, and rhesus monkeys). He was followed by Dr. Broomfield, who described the kinetics of nerve agent hydrolysis by human OPAA hydrolase and demonstrated that despite the high Km, this enzyme can catalyze the rapid hydrolysis of soman and sarin, and to a lesser extent VX. Dr. Cheng described his studies on the isolation of an OPAA hydrolase from Alteromonas, its characterization as a prolidase, and the cloning and large-scale production of recombinant enzyme. He also described the formulation of recombinant enzyme in different matrices for possible use in chemical agent decontamination, personnel protection, and detoxification. Dr. Saxena discussed her attempt at improving the bioscavenging property of Mo AChE by site directed mutagenesis studies and demonstrated that the E202Q AChE was 2- to 3-fold more efficient than wild-type AChE in detoxifying soman and sarin compared to wild-type AChE. Dr. Way demonstrated the use of recombinant OPH encapsulated into sterically stabilized liposomes (SL) in the presence of 2-PAM and atropine, for the protection of mice against a 1000 LD50 of paraoxon. In similar studies, Dr. Petrikovics demonstrated the use of recombinant OPAA encapsulated into sterically stabilized liposomes (SL) in the presence of 2-PAM and atropine, for the protection of mice against DFP.

Session 16 - Terror
Chair: Ladislaw Szinicz; Co-chair: Andrew Robertson
Session 16 was devoted to terrorism. Various presentations from other Sessions (3, 7, 14) already stressed the disastrous results that may arise from terrorist or non-state use of chemical and biological warfare (CBW) agents. Mitigating the impact of a CBW attack requires timely planning, properly trained personnel, rapid detection methods, protection and medical equipment, pharmaceuticals, dedicated facilities and a rapid, and well trained, emergency response organization. Any reaction to a CBW terrorist attack will be infused with high levels of anxiety, emotion and psychological pressure. Dr. Santana noted that intensive communication at different levels is inevitable in any incident response and stressed the importance of the early "hardening" of any disaster communication system. Intensive education and training, coupled with appropriate equipment, is also important. Col. Centonze outlined the particular CBW protection and detection training which is provided by the Swiss Government and emphasized the benefits of cooperation between civil and military authorities. Dr. Lisa Rotz addressed the evaluation and prioritization of potential biological agents against objective criteria to ensure appropriate allocation of the limited funding by public health authorities. By identifying priority agents, public health programs can be targeted appropriately. Dr. Lindsey stressed the importance of the availability of standardized laboratory protocols during a CBW attack and outlined the approach CDC had taken to make this information available using the internet. Dr. Treadwell, using the Seattle World Trade Organization meeting as an example, emphasized the need for surveillance programs for early recognition of characteristic symptom clusters, which may be the first indication of terrorist use of biological agents. The daunting task of improving protection against CBW terrorist attack will be more cost-effective with continued national and international cooperation.