Ken Alibek

Lethal toxin of Bacillus anthracis causes apoptosis of macrophages

Lethal toxin is a major anthrax virulence factor, causing the rapid death of experimental animals. Lethal toxin can enter most cell types, but only certain macrophages and cell lines are susceptible to toxin-mediated cytolysis. We have shown that in murine RAW 264.7 cells, sublytic amounts of lethal toxin trigger intracellular signaling events typical for apoptosis, including changes in membrane permeability, loss of mitochondrial membrane potential, and DNA fragmentation. The cells were protected from the toxin by specific inhibitors of caspase-1, -2, -3, -4, -6, and -8. Phagocytic activity of macrophages was inhibited by sublytic concentrations of lethal toxin. Infection of cells with anthrax (Sterne) spores impaired their bactericidal capacity, which could be reversed by a lethal toxin inhibitor, bestatin. We suggest that apoptosis rather than direct lysis is biologically relevant to lethal toxin intracellular activity.

Effect of Bacillus anthracis lethal toxin on human peripheral blood mononuclear cells

Lethal toxin (LeTx) plays a central role in anthrax pathogenesis, however a cytotoxicity of LeTx has been difficult to demonstrate in vitro. No cytolytic effect has been reported for human cells, in contrast to murine cell lines, indicating that cell lysis can not be considered as a marker of LeTx activity. We have recently shown that murine macrophage‐like RAW 264.7 cells treated with LeTx or infected with anthrax spores underwent changes typical of apoptotic death. Here we demonstrate that cells from human peripheral blood display a proapoptotic behavior similar to murine cells. TUNEL assay detected a nucleosomal degradation typical of apoptosis in peripheral blood mononuclear cells (PBMC) treated with LeTx. Membrane staining with apoptotic dyes was detected in macrophages derived from monocytes in presence of LeTx. The toxin inhibited production of proinflammatory cytokines in PBMC stimulated with a preparation of Bacillus anthracis cell wall. Infection of PBMC with anthrax spores led to the appearance of a large population of cells stained positively for apoptosis, with a reduced capacity to eliminate spores and vegetative bacteria. The aminopeptidase inhibitor, bestatin, capable of protecting cells from LeTx, restored a bactericidal activity of infected cells. These findings may be explained by LeTx expression within phagocytes and support an important role of LeTx as an early intracellular virulence factor contributing to bacterial dissemination and disease progression.

Systemic cytokine response in murine anthrax

Systemic pro‐inflammatory cytokine release has been previously implicated as a major death‐causing factor in anthrax, however, direct data have been absent. We determined the levels of IL‐1β, IL‐6 and TNF‐α in serum of mice challenged with virulent (Ames) or attenuated (Sterne) strains of Bacillus anthracis. More than 10‐fold increase in the IL‐1β levels was detected in Ames‐challenged Balb/c mice, in contrast to more susceptible C57BL/6 mice, which showed no IL‐1β response. Balb/c mice have also responded with higher levels of IL‐6. The A/J mice demonstrated IL‐1β and IL‐6 systemic response to either Ames or Sterne strain of B. anthracis, whereas no increase in TNF‐α was detected in any murine strain. We used RT‐PCR for gene expression analyses in the liver which often is a major source of cytokines and one of the main targets in infectious diseases. A/J mice challenged with B. anthracis (Sterne) showed increased gene expression for Fas, FasL, Bax, IL‐1β, TNF‐α, TGF‐β, MIP‐1α, KC and RANTES. These data favour the hypothesis that apoptotic cell death during anthrax infection causes chemokine‐induced transmigration of inflammatory cells to vitally important organs such as liver. Administration of caspase inhibitors z‐VAD‐fmk and ac‐YVAD‐cmk improved survival in Sterne‐challenged mice indicating a pathogenic role of apoptosis in anthrax.

Anaerobic induction of Bacillus anthracis hemolytic activity.

A number of genes in Bacillus anthracis encode for proteins homologous to the membrane-damaging factors known as pathogenic determinants in different bacteria. B. anthracis, however, has been traditionally considered non-hemolytic, and the recently identified hemolytic genes have been suggested to be transcriptionally silent. We found that the hemolytic genes of B. anthracis, collectively designated as anthralysins (Anls), could be induced in strict anaerobic conditions. We also demonstrate that Anl genes are expressed at the early stages of infection within macrophages by vegetating bacilli after spore germination. Cooperative and synergistic enhancement of the pore-forming and phospholipase C (PLC) activities of the Anls was found in hemolytic tests on human, but not sheep, red blood cells (RBC). These findings imply Anls as B. anthracis pathogenic determinants and highlight oxygen limitation as environmental factor controlling their expression at both early and late stages of infection.

Treatment of anthrax infection with combination of ciprofloxacin and antibodies to protective antigen of Bacillus anthracis.

Currently there is no effective treatment for inhalational anthrax beyond administration of antibiotics shortly after exposure. There is need for new, safe and effective treatments to supplement traditional antibiotic therapy. Our study was based on the premise that simultaneous inhibition of lethal toxin action with antibodies and blocking of bacterial growth by antibiotics will be beneficial for the treatment of anthrax. In this study, we tested the effects of a combination treatment using purified rabbit or sheep anti-protective antigen (PA) antibodies and the antibiotic ciprofloxacin in a rodent anthrax model. In mice infected with a dose of Bacillus anthracis Sterne strain corresponding to 10 LD(50), antibiotic treatment with ciprofloxacin alone only cured 50% of infected animals. Administration of anti-PA IgG in combination with ciprofloxacin produced 90-100% survival. These data indicate that a combination of antibiotic/immunoglobulin therapy is more effective than antibiotic treatment alone in a rodent anthrax model.

Effective antiprotease-antibiotic treatment of experimental anthrax

BackgroundInhalation anthrax is characterized by a systemic spread of the challenge agent, Bacillus anthracis. It causes severe damage, including multiple hemorrhagic lesions, to host tissues and organs. It is widely believed that anthrax lethal toxin secreted by proliferating bacteria is a major cause of death, however, the pathology of intoxication in experimental animals is drastically different from that found during the infectious process. In order to close a gap between our understanding of anthrax molecular pathology and the most prominent clinical features of the infectious process we undertook bioinformatic and experimental analyses of potential proteolytic virulence factors of B. anthracis distinct from lethal toxin.MethodsSecreted proteins (other than lethal and edema toxins) produced by B. anthracis were tested for tissue-damaging activity and toxicity in mice. Chemical protease inhibitors and rabbit immune sera raised against B. anthracis proteases were used to treat mice challenged with B. anthracis (Sterne) spores.ResultsB. anthracis strain delta Ames (pXO1-, pXO2-) producing no lethal and edema toxins secrets a number of metalloprotease virulence factors upon cultivation under aerobic conditions, including those with hemorrhagic, caseinolytic and collagenolytic activities, belonging to M4 and M9 thermolysin and bacterial collagenase families, respectively.These factors are directly toxic to DBA/2 mice upon intratracheal administration at 0.5 mg/kg and higher doses. Chemical protease inhibitors (phosphoramidon and 1, 10-phenanthroline), as well as immune sera against M4 and M9 proteases of B. anthracis, were used to treat mice challenged with B. anthracis (Sterne) spores. These substances demonstrate a substantial protective efficacy in combination with ciprofloxacin therapy initiated as late as 48 h post spore challenge, compared to the antibiotic alone.ConclusionSecreted proteolytic enzymes are important pathogenic factors of B. anthrasis, which can be considered as effective therapeutic targets in the development of anthrax treatment and prophylactic approaches complementing anti-lethal toxin therapy.

Prevention of lethal respiratory vaccinia infections in mice with interferon‐α and interferon‐γ

The antiviral efficacy of interferons (IFNs) was evaluated using a vaccinia intranasal infection model in mice in this study. We provide evidence that intranasal administration of IFN-α and IFN-γ (days −1 to +3) resulted in 100 and 90% survival against a lethal respiratory vaccinia infection (8 LD50) in mice, respectively; whereas no animals in the placebo group survived through the study period (21 days). The IFN treatment consisted of a single daily dose of 5×103 U per mouse for 5 consecutive days. The efficacy of IFN-γ was evident even when the IFN-γ treatments started 1–2 days after infection and when a lower dose (2×103 U per mouse) was used. The treatment of IFN-α and IFN-γ reduced the virus titers in the lungs of infected mice by 1000–10,000-fold, when the administration started 1 day after infection. Our data suggest that IFN-α and IFN-γ are effective in protecting vaccinia-infected mice from viral replication in lungs and mortality, and may be beneficial in other human orthopoxvirus infections.

In vitro-generated respiratory mucosa: a new tool to study inhalational anthrax.

We generated a three-dimensional (3-D) model of human airway tissues in order to study initiation of inhalational form of anthrax infection. The system was designed to model the air-blood barrier of the respiratory tract represented by epithelial cells and macrophages. When grown on collagen/fibronectin gel support at an air-liquid interface, airway epithelial cells formed cell layers morphologically resembling those in vivo. These preformed epithelial cell cultures were further supplemented with monocytes/macrophages isolated from human blood. After 2-5 days of co-culture, monocytes differentiated into a phenotype of resident macrophages, which was evaluated by the expression of specific cell surface markers. This model allowed sorting out the role of each type of cell found at the air surface of the lung. The interdependence of macrophages and epithelial cells in the clearance of anthrax spores from airways and the capacity of the airway epithelial cells to protect from anthrax infection was demonstrated.

Biological and chemical terrorism : a guide for healthcare providers and first responders

A Quick Reference for Potential Biological Weapons (tear out sheet) Preface How to Use This Book Introduction Basic Bioterrorism (Section I) What is Bioterrorism? Flu-Like Illness What Makes an Effective Weapon Personal Precautions: Table 1 (Comparison of Symptoms of Potential Biological Weapons to Influenza) Table 2 (Signs that Suggest an Attack with a Biological Weapon) Syndromic Cross References (Section 2) Fever with Prominent Arthralgias Acute Hepatic Syndrome Hemorrhagic Diathesis Hilar Adenopathy or Widened Mediastinum Fever with Petechiae Cutaneuos Lesions of Ulcertations Acute Respiratory Syndrome With Fever Acute Respiratory Syndrome Without Fever Flu-Like Illness with Rash Fever with Lymphadenopathy Acute Neurologic Syndrome with Fever Acute Neurologic Syndrome without Fever Acute GI Syndrome with Fever Acute GI Syndrome without Fever Individual Biologic Weapon Detalied Quick Reference (Section 3) Aflatoxins Anthrax (Inhalational & gastrointestinal) Anthrax (Cutaneous) Blastomucosis Botulism (Botulism Toxins) Brucellosis Crimean-Congo Hemorrhagic Fever Chikungunya Cholera Dengue Fever (Dengue Hemorrhagic Fever) Domoic Acid (Amnesic Shellfish Poisining) Ebola and Marburg Viral Hemorrhagic Fevers Encephalis, Viral (Venezuelan, Eastern, Western, St. Louis, Japanese, and West Nile Virus) Imfluenza Glanders Hantavirus Kyansunur Forest Disease Lassa Virus and the South American Viral Hemorrhagic Fevers Legionellosis Leptospirosis Melioidosis Nipah Virus Omsk Hemorrhagic Fever Plague Psittacosis Q Fever Ricin and Abrin Rocky Mountain Spotted Fever Rift Valley Fever Salmonellosis (Typhoid, parathypoid and Non-Typhoidal) Saxitoxin (Paralytic Shellfish Poisoning) Staphylococcal Enterotoxin B Shigellosis Smallpox (information sheet) Smallpox (Color Atlas and Visual Guide) Trichothescene (T-2) Mycotoxins Tetrodotoxin Tularemia Typhus (Epedemic, Murine and Scrub) Yellow Fever Basic Chemical Terrorism (Section 4) What is Chemical Terrorism Physical Properties of Chemical Weapons Protecting Yourself Chemical Weapon Syndriomic Cross References (Section 5) Prominent Skin Rash, Blistering, and/or Burns Prominent Pulmonary Symptoms Seizures and/or Sudde Coma Acute Hemolytic Anemia Individual Chemical Weapon Detailed Quick References (Section 6) Nerve Agents Cyanides (Blood Agents) Hydrogen Sulfide Pulmonary Agents Mustard Lewisite hosgene Oxime (CX) Anhydrous Ammonia, Sulfur Dioxide, and Hydrogen Chloride Hydrogen Flouride Arsine Lacrimators (Tear Gas) References Resources on the Internet Glossary Index

Anthrax Lethal Toxin Inhibits the Production of Proinflammatory Cytokines

In previous studies, we have found that anthrax lethal toxin (LeTx) induces apoptosis in both murine macrophages and human peripheral blood mononuclear cells (PBMCs). In this study, we further report that bacterial cell wall (CW) components of Bacillus (B.) anthracis are powerful inducers of proinflammatory cytokines from the PBMCs. These effects are deprived when the LeTx is present. The major causative element for this suppression is lethal factor (LF) rather than protective antigen (PA). These results indicate that the roles of LeTx in anthrax pathogenesis, particularly its effects on cytokine production, should be reevaluated as our findings and other reports are controversial to the conventional concept.