Harry Salem

Good cell culture practice for stem cells and stem-cell-derived models

The first guidance on Good Cell Culture Practice (GCCP) dates back to 2005. This document expands this to include aspects of quality assurance for in vitro cell culture focusing on the increasingly diverse cell types and culture formats used in research, product development, testing and manufacture of biotechnology products and cell-based medicines. It provides a set of basic principles of best practice that can be used in training new personnel, reviewing and improving local procedures, and helping to assure standard practices and conditions for the comparison of data between laboratories and experimentation performed at different times. This includes recommendations for the documentation and reporting of culture conditions. It is intended as guidance to facilitate the generation of reliable data from cell culture systems, and is not intended to conflict with local or higher level legislation or regulatory requirements. It may not be possible to meet all recommendations in this guidance for practical, legal or other reasons. However, when it is necessary to divert from the principles of GCCP, the risk of decreasing the quality of work and the safety of laboratory staff should be addressed and any conclusions or alternative approaches justified. This workshop report is considered a first step toward a revised GCCP 2.0.

Issues in Chemical and Biological Terrorism

This manuscript describes the overview presented at the 23rd Annual Meeting of the American College of Toxicology in 2002. Although it is recognized that weapons of mass destruction that can be used against our military and civilian populations include chemical, biological, radiological, and nuclear (CBRN) agents, this overview is limited primarily to chemical and biological (CB) agents. The issues of CB terrorism are discussed in terms of When, What, How, and Who. The US Army has been providing chemical and biological solutions since 1917, and has since 1996 applied these solutions to homeland defense and domestic preparedness. The use of chemical and biological agents as terrorist weapons both in the United States and elsewhere in the world is reviewed. The CB threat spectrum is presented, as is the further categorization of biological threat agents by the Centers for Disease Control and Prevention (CDC). In addition, the CB agents considered to be a potential threat to our water supply are also presented. These are agents that are water soluble, stable, and resistant to water treatment and/or disinfection. The overview concludes with the chronological accomplishments of ECBC since 1917.

Chemical warfare agent and biological toxin-induced pulmonary toxicity: could stem cells provide potential therapies?

Chemical warfare agents (CWAs) as well as biological toxins present a significant inhalation injury risk to both deployed warfighters and civilian targets of terrorist attacks. Inhalation of many CWAs and biological toxins can induce severe pulmonary toxicity leading to the development of acute lung injury (ALI) as well as acute respiratory distress syndrome (ARDS). The therapeutic options currently used to treat these conditions are very limited and mortality rates remain high. Recent evidence suggests that human stem cells may provide significant therapeutic options for ALI and ARDS in the near future. The threat posed by CWAs and biological toxins for both civilian populations and military personnel is growing, thus understanding the mechanisms of toxicity and potential therapies is critical. This review will outline the pulmonary toxic effects of some of the most common CWAs and biological toxins as well as the potential role of stem cells in treating these types of toxic lung injuries.

High Content Analysis of an In Vitro Model for Metabolic Toxicity Results with the Model Toxicants 4-Aminophenol and Cyclophosphamide

In vitro models that accurately and rapidly assess hepatotoxicity and the effects of hepatic metabolism on nonliver cell types are needed by the U.S. Department of Defense and the pharmaceutical industry to screen compound libraries. Here, we report the first use of high content analysis on the Integrated Discrete Multiple Organ Co-Culture (IdMOC) system, a high-throughput method for such studies. We cultured 3T3-L1 cells in the presence and absence of primary human hepatocytes, and exposed the cultures to 4-aminophenol and cyclophosphamide, model toxicants that are respectively detoxified and activated by the liver. Following staining with calcein-AM, ethidium homodimer-1, and Hoechst 33342, high content analysis of the cultures revealed four cytotoxic endpoints: fluorescence intensities of calcein-AM and ethidium homodimer-1, nuclear area, and cell density. Using these endpoints, we observed that the cytotoxicity of 4-aminophenol in 3T3-L1 cells in co-culture was less than that observed for 3T3-L1 monocultures, consistent with the known detoxification of 4-aminophenol by hepatocytes. Conversely, cyclophosphamide cytotoxicity for 3T3-L1 cells was enhanced by co-culturing with hepatocytes, consistent with the known metabolic activation of this toxicant. The use of IdMOC plates combined with high content analysis is therefore a multi-endpoint, high-throughput capability for measuring the effects of metabolism on toxicity.

The Pesticide Metabolites Paraoxon and Malaoxon Induce Cellular Death by Different Mechanisms in Cultured Human Pulmonary Cells.

Organophosphorus (OP) pesticides are known to induce pulmonary toxicity in both humans and experimental animals. To elucidate the mechanism of OP-induced cytotoxicity, we examined the effects of parathion and malathion and their respective metabolites, paraoxon and malaoxon, on primary cultured human large and small airway cells. Exposure to paraoxon and malaoxon produced a dose-dependent increase in cytotoxicity following a 24-hour exposure, while treatment with parathion or malathion produced no effects at clinically relevant concentrations. Exposure to paraoxon-induced caspase activation, but malaoxon failed to induce this response. Since caspases have a major role in the regulation of apoptosis and cell death, we evaluated OP-induced cell death in the presence of a caspase inhibitor. Pharmacological caspase inhibition protected against paraoxon-induced cell death but not malaoxon-induced cell death. These data suggest that caspase activation is a key signaling element in paraoxon-induced cell death, but not malaoxon-induced cellular death in the pulmonary epithelium.

Generalized Additive Mixed-Models for Pharmacology Using Integrated Discrete Multiple Organ Co-Culture.

Integrated Discrete Multiple Organ Co-culture (IDMOC) is emerging as an in-vitro alternative to in-vivo animal models for pharmacology studies. IDMOC allows dose-response relationships to be investigated at the tissue and organoid levels, yet, these relationships often exhibit responses that are far more complex than the binary responses often measured in whole animals. To accommodate departure from binary endpoints, IDMOC requires an expansion of analytic techniques beyond simple linear probit and logistic models familiar in toxicology. IDMOC dose-responses may be measured at continuous scales, exhibit significant non-linearity such as local maxima or minima, and may include non-independent measures. Generalized additive mixed-modeling (GAMM) provides an alternative description of dose-response that relaxes assumptions of independence and linearity. We compared GAMMs to traditional linear models for describing dose-response in IDMOC pharmacology studies.

CHAPTER 5:The Structural Biology and Biochemistry of the Ricin Toxin and the Military Use and Inhalation Toxicology of Ricin Aerosols

The US Government has classified the biological agents most commonly associated with military and terrorist use as biological select agents and toxins (BSATs) because of the severe threat that they pose to public health and safety. The agents have become particularly attractive to foreign states and terrorist groups because they are relatively inexpensive to produce and require minimal technical infrastructures. The most widely recognized BSAT is the ricin protein toxin found in the seeds of the castor bean plant Ricinus communis. This chapter reviews the structural biology and biochemistry of the ricin protein, especially in terms of its internalization into eukaryotic host cells and the details of its N-glycosidase activity responsible for inactivating ribosome function in protein synthesis. The chapter also reviews some of the more applied aspects of using the toxin as an offensive weapon, including an account the brief history of ricin weaponization programs during World Wars I and II. Also, because aerosol delivery of appropriately formulated ricin powders is the most effective means for its use as an offensive weapon, there is a sharp focus on the particle dynamics of aerosolized ricin, its aerodynamics within the respiratory tract and the signs, symptoms and inhalation toxicology associated with ricin exposure. Much of the respiratory dynamics and inhalation toxicology information derives from testing laboratory animals, especially nonhuman primates, because human exposures to BSAT aerosols are exceedingly rare. Finally, the expected difficulties and caveats of using aerosolized ricin as an agent of bioterrorism are addressed in the Conclusion section.