Biochemistry, 1999 Aug 10, 38(32), 10432 - 41 Anthrax protective antigen: prepore-to-pore conversion; Miller CJ et al.; PA(63), the active 63 kDa form of anthrax protective antigen, forms a heptameric ring-shaped oligomer that is believed to represent a precursor of the membrane pore formed by this protein . When maintained at pH >/=8.0, this "prepore" dissociated to monomeric subunits upon treatment with SDS at room temperature, but treatment at pH </=7 (or with beta-octylglucoside at pH 8.0) caused it to convert to an SDS-resistant pore-like form . Transition to this form involved major changes in the conformation of loop 2 of domain 2 (D2L2), as evidenced by (i) occlusion of a chymotrypsin site within D2L2 and (ii) excimer formation by pyrene groups linked to N306C within this loop . The pore-like form retained the capacity to bind anthrax toxin A moieties and cell surface receptors, but was unable to form pores in membranes or mediate translocation . Mutant PA(63) in which D2L2 had been deleted was inactive in pore formation and translocation but, like the prepore, was capable of forming heptamers that converted to an SDS-resistant form under acidic conditions . Our findings support a model of pore formation in which the D2L2 loops move to the membrane-proximal face of the heptamer and interact to form a 14-strand transmembrane beta-barrel . Concomitantly, domain 2 undergoes a major conformational rearrangement, independent of D2L2, that renders the heptamer resistant to dissociation by SDS . These results provide a basis for further exploration of the role of PA(63) in translocation of anthrax toxin's enzymic moieties across membranes. Ann Emerg Med, 1999 Aug, 34(2), 177 - 82 Principles for emergency response to bioterrorism; Keim M et al.; The recent occurrence of a series of anthrax-related hoaxes illustrates the need to educate emergency services personnel about how to best ensure patient and worker safety in the case of suspected exposure to biological threat agents . There are very few data to support the methods being used or the variation in current care . Emergency physicians, first responders, and hazardous materials response teams need a standardized approach to the management of patients who may have been exposed to biological threat agents . Currently recommended hospital infection control procedures seem appropriate for the level of risk involved with aerosolized biological threat agents . Such recommendations include standard and transmission-based precautions . These groups need a working knowledge of the isolation and infection control measures recommended for the treatment of patients exposed to those biological threat agents at outlined in the Centers for Disease Control and Prevention Guideline for Isolation Precautions in Hospitals. Infect Immun, 1999 Jul, 67(7), 3290 - 6 Cytotoxic T-lymphocyte epitopes fused to anthrax toxin induce protective antiviral immunity; Doling AM et al.; We have investigated the use of the protective antigen (PA) and lethal factor (LF) components of anthrax toxin as a system for in vivo delivery of cytotoxic T-lymphocyte (CTL) epitopes . During intoxication, PA directs the translocation of LF into the cytoplasm of mammalian cells . Here we demonstrate that antiviral immunity can be induced in BALB/c mice immunized with PA plus a fusion protein containing the N-terminal 255 amino acids of LF (LFn) and an epitope from the nucleoprotein (NP) of lymphocytic choriomeningitis virus . We also demonstrate that BALB/c mice immunized with a single LFn fusion protein containing NP and listeriolysin O protein epitopes in tandem mount a CTL response against both pathogens . Furthermore, we show that NP-specific CTL are primed in both BALB/c and C57BL/6 mice when the mice are immunized with a single fusion containing two epitopes, one presented by Ld and one presented by Db . The data presented here demonstrate the versatility of the anthrax toxin delivery system and indicate that this system may be used as a general approach to vaccinate outbred populations against a variety of pathogens. Infect Immun, 1999 Jun, 67(6), 3055 - 60 Proteasome activity is required for anthrax lethal toxin to kill macrophages; Tang G et al.; Anthrax lethal toxin (LeTx), consisting of protective antigen (PA) and lethal factor (LF), rapidly kills primary mouse macrophages and macrophage-like cell lines such as RAW 264.7 . LF is translocated by PA into the cytosol of target cells, where it acts as a metalloprotease to cleave mitogen-activated protein kinase kinase 1 (MEK1) and possibly other proteins . In this study, we show that proteasome inhibitors such as acetyl-Leu-Leu-norleucinal, MG132, and lactacystin efficiently block LeTx cytotoxicity, whereas other protease inhibitors do not . The inhibitor concentrations that block LF cytotoxicity are similar to those that inhibit the proteasome-dependent IkappaB-alpha degradation induced by lipopolysaccharide . The inhibitors did not interfere with the proteolytic cleavage of MEK1 in LeTx-treated cells, indicating that they do not directly block the proteolytic activity of LF . However, the proteasome inhibitors did prevent ATP depletion, an early effect of LeTx . No overall activation of the proteasome by LeTx was detected, as shown by the cleavage of fluorogenic substrates of the proteasome . All of these results suggest that the proteasome mediates a toxic process initiated by LF in the cell cytosol . This process probably involves degradation of unidentified molecules that are essential for macrophage homeostasis . Moreover, this proteasome-dependent process is an early step in LeTx intoxication, but it is downstream of the cleavage by LF of MEK1 or other putative substrates. Infect Immun, 1999 Jun, 67(6), 3026 - 30 Anthrax toxin entry into polarized epithelial cells; Beauregard KE et al.; We examined the entry of anthrax edema toxin (EdTx) into polarized human T84 epithelial cells using cyclic AMP-regulated Cl- secretion as an index of toxin entry . EdTx is a binary A/B toxin which self assembles at the cell surface from anthrax edema factor and protective antigen (PA) . PA binds to cell surface receptors and delivers EF, an adenylate cyclase, to the cytosol . EdTx elicited a strong Cl- secretory response when it was applied to the basolateral surface of T84 cells but no response when it was applied to the apical surface . PA alone had no effect when it was applied to either surface . T84 cells exposed basolaterally bound at least 30-fold-more PA than did T84 cells exposed apically, indicating that the PA receptor is largely or completely restricted to the basolateral membrane of these cells . The PA receptor did not fractionate with detergent-insoluble caveola-like membranes as cholera toxin receptors do . These findings have implications regarding the nature of the PA receptor and confirm the view that EdTx and CT coopt fundamentally different subcellular systems to enter the cell and cause disease. Biochem J, 1999 May 1, 339 ( Pt 3), 639 - 47 Endoprotease PACE4 is Ca2+-dependent and temperature-sensitive and can partly rescue the phenotype of a furin-deficient cell strain; Sucic JF et al.; PACE4 is a member of the eukaryotic subtilisin-like endoprotease family . The expression of human PACE4 in RPE.40 cells (furin-null mutants derived from Chinese hamster ovary K1 cells) resulted in the rescue of a number of wild-type characteristics, including sensitivity to Sindbis virus and the ability to process the low-density-lipoprotein receptor-related protein . Expression of PACE4 in these cells failed to restore wild-type sensitivity to Pseudomonas exotoxin A . Co-expression of human PACE4 in these cells with either a secreted form of the human insulin pro-receptor or the precursor form of von Willebrand factor resulted in both proproteins being processed; RPE.40 cells were unable to process either precursor protein in the absence of co-expressed PACE4 . Northern analysis demonstrated that untransfected RPE.40 cells express mRNA species for four PACE4 isoforms, suggesting that any endogenous PACE4 proteins produced by these cells are either non-functional or sequestered in a compartment outside of the secretory pathway . In experiments in vitro, PACE4 processed diphtheria toxin and anthrax toxin protective antigen, but not Pseudomonas exotoxin A . The activity of PACE4 in vitro was Ca2+-dependent and, unlike furin, was sensitive to temperature changes between 22 and 37 degrees C . RPE.40 cells stably expressing human PACE4 secreted an endoprotease with the same Ca2+ dependence and temperature sensitivity as that observed in membrane fractions of these cells assayed in vitro . These results, in conjunction with other published work, demonstrate that PACE4 is an endoprotease with more stringent substrate specificity and more limited operating parameters than furin. Infect Dis Clin North Am, 1999 Mar, 13(1), 187 - 208, viii Anthrax vaccine . Model of a response to the biologic warfare threat; Nass M; Anthrax vaccine is being administered to all 2.4 million active duty, reserve, and National Guard troops, as prophylaxis against biologic warfare . The vaccine's effectiveness in this setting may be limited . This article discusses unresolved issues of safety, with an emphasis on the need for careful surveillance of vaccines used by the military, which has sidestepped the commercial process . Also considered are ethical issues related to the development and use of military biologics, as the United States Army advances its Joint Vaccine Acquisition Program, a plan to produce more than ten vaccines specifically for biologic warfare threat, and to administer them to all military servicemembers. Arch Dermatol, 1999 Mar, 135(3), 311 - 22 Cutaneous manifestations of biological warfare and related threat agents; McGovern TW et al.; The specter of biological warfare (BW) looms large in the minds of many Americans . The US government has required that emergency response teams in more than 100 American cities be trained by the year 2001 to recognize and contain a BW attack . The US military is requiring active duty soldiers to receive immunization against anthrax . Dermatologists need not feel helpless in the face of a potential BW attack . Many potential agents have cutaneous manifestations that the trained eye of a dermatologist can recognize . Through early recognition of a BW attack, dermatologists can aid public health authorities in diagnosing the cause so that preventive and containment measures can be instituted to mitigate morbidity and mortality . This article reviews bacterial, viral, and toxin threat agents and emphasizes those that would have cutaneous manifestations following an aerosol attack . We conclude with clues that can help one recognize a biological attack. Infect Immun, 1999 Apr, 67(4), 1853 - 9 Oligomerization of anthrax toxin protective antigen and binding of lethal factor during endocytic uptake into mammalian cells; Singh Y et al.; The protective antigen (PA) protein of anthrax toxin binds to a cellular receptor and is cleaved by cell surface furin to produce a 63-kDa fragment (PA63) . The receptor-bound PA63 oligomerizes to a heptamer and acts to translocate the catalytic moieties of the toxin, lethal factor (LF) and edema factor (EF), from endosomes to the cytosol . In this report, we used nondenaturing gel electrophoresis to show that each PA63 subunit in the heptamer can bind one LF molecule . Studies using PA immobilized on a plastic surface showed that monomeric PA63 is also able to bind LF . The internalization of PA and LF by cells was studied with radiolabeled and biotinylated proteins . Uptake was relatively slow, with a half-time of 30 min . The number of moles of LF internalized was nearly equal to the number of moles of PA subunit internalized . The essential role of PA oligomerization in LF translocation was shown with PA protein cleaved at residues 313-314 . The oligomers formed by these proteins during uptake into cells were not as stable when subjected to heat and detergent as were those formed by native PA . The results show that the structure of the toxin proteins and the kinetics of proteolytic activation, LF binding, and internalization are balanced in a way that allows each PA63 subunit to internalize an LF molecule . This set of proteins has evolved to achieve highly efficient internalization and membrane translocation of the catalytic components, LF and EF. Cell Signal, 1999 Feb, 11(2), 111 - 6 Activation of phospholipase C and protein kinase C is required for expression of anthrax lethal toxin cytotoxicity in J774A.1 cells; Bhatnagar R et al.; Anthrax lethal toxin (LT) comprises two proteins: the protective antigen (PA) and the lethal factor (LF) . The LT is cytotoxic to macrophage-like cell line J774A.1 . Pre-treatment of these cells with neomycin, a phospholipase C inhibitor, protected them against anthrax LT cytotoxicity . Protection obtained with neomycin indicated that LT stimulates phospholipase C in these cells . It was found that levels of inositol 1,4,5-triphosphate (IP3) dramatically increased in toxin-treated cells . The rise in IP3 levels was proportional to the dose of LF that was allowed to bind to receptor-bound PA . By using protein kinase C (PKC) inhibitors, we found that the activation of PKC is required for mediating anthrax LT cytotoxicity . Activation of phospholipase C or PKC is not required for the binding of PA to the cell surface receptors or for the uptake or internalisation of the toxin . In this study, we demonstrate that the IP3 signalling cascade is initiated by anthrax lethal toxin in J774A.1 cells . The second messengers generated during the cascade aid LF in mediating lethality only after its translocation into the cytosol. MMWR Morb Mortal Wkly Rep, 1999 Feb 5, 48(4), 69 - 74 Bioterrorism alleging use of anthrax and interim guidelines for management--United States, 1998; Protection against anthrax toxin by vaccination with a DNA plasmid encoding anthrax protective antigen; Section of Retroviral Immunology, Food and Drug Administration, Bethesda, MD, USAA DNA vaccine encoding the immunogenic and biologically active portion of anthrax protective antigen (PA) was constructed . Spleen cells from BALB/c mice immunized intramuscularly with this vaccine were stimulated to secrete IFN gamma and IL-4 when exposed to PA in vitro . Immunized mice also mounted a humoral immune response dominated by IgG1 anti-PA antibody production, the subclass previously shown to confer protection against anthrax toxin . A 1:100 dilution of serum from these animals protected cells in vitro against cytotoxic concentrations of PA . Moreover, 7/8 mice immunized three times with the PA DNA vaccine were protected against lethal challenge with a combination of anthrax protective antigen plus lethal factor. Adv Vet Med, 1999, 41, 7 - 24 Grease, anthraxgate, and kennel cough: a revisionist history of early veterinary vaccines; Tizard I; In conclusion, it is remarkable just how farsighted many of the early vaccine investigators were . Jenner was apparently very comfortable with contagion and even recognized that infectious agents could gradually change and adapt to a new species . Pasteur, long before his fowl cholera experiment, dreamed that attenuation could yield safe vaccines and it took him no time at all therefore to recognize the significance of that serendipitous experiment . The fact that two other investigators were also developing anthrax vaccines simultaneously is yet another example of how the times favor certain discoveries . Finally Ferry, while constrained by the fact that he had no idea that distemper was caused by a virus, recognized well the concept of secondary infection and rationalized, not unreasonably, that his vaccine might assist in controlling this . It is also clear that we must look skeptically at the accepted historical record . Thus, it is clear that Jenner used horse-derived material as a source of vaccine material and that vaccinia may in fact be the long-lost agent of horsepox . Certainly this would not be news to many nineteenth-century investigators and veterinarians . Individuals planning to use live vaccinia in recombinant vaccines may wish to keep this in mind . Who discovered anthrax vaccine? Burdon-Sanderson clearly recognized that he could attenuate the organism . Greenfield showed that this could protect against disease although he was far from developing an effective vaccine . Poor Henri Toussaint was probably the first to develop an effective product but did not publicize his results widely . It was left to Louis Pasteur to take the risks inherent in a high-profile public experiment and win . I believe that he richly deserves the prize . Finally, who deserves the credit for distemper vaccine? First, Carre deserves much more credit than hitherto for discovering that distemper was caused by a virus . Second, Ferry, although misled by his identification of B . bronchiseptica deserves credit for realizing that his vaccine could play a role in controlling secondary infections . The true discoverer of an effective distemper vaccine was the Italian, Puntoni, but once again the publicity went to others, Laidlaw and Dunkin . Thus a pattern emerges that prior discovery matters little in the face of aggressive publicity . If nobody knows you did the experiment you might as well have never done it in the first place . Publish or perish is by no means a new phenomenon. Biochemistry, 1998 Nov 10, 37(45), 15737 - 46 Characterization of membrane translocation by anthrax protective antigen; Wesche J et al.; Solving the crystallographic structure of the ring-shaped heptamer formed by protective antigen (PA), the B moiety of anthrax toxin, has focused attention on understanding how this oligomer mediates membrane translocation of the toxin's A moieties . We have developed an assay for translocation in which radiolabeled ligands are bound to proteolytically activated PA (PA63) at the surface of CHO or L6 cells, and translocation across the plasma membrane is induced by lowering the pH . The cells are then treated with Pronase E to degrade residual surface-bound material, and protected ligands are quantified after fractionation by SDS-PAGE . Translocation was most efficient (35%-50%) with LFN, the N-terminal PA binding domain of the anthrax lethal factor (LF) . Intact LF, edema factor (EF), or fusion proteins containing LFN fused to certain heterologous proteins {the diphtheria toxin A chain (DTA) or dihydrofolate reductase (DHFR)} were less efficiently translocated (15%-20%); and LFN fusions to several other proteins were not translocated at all . LFN with different N-terminal residues was found to be degraded according to the N-end rule by the proteasome, and translocation of LFN fused to a mutant form of DHFR with a low affinity for methotrexate (MTX) protected cells from the effects of MTX . Both results are consistent with a cytosolic location of protected proteins . Evidence that a protein must unfold to be translocated was obtained in experiments showing that (i) translocation of LFNDTA was blocked by introduction of an artificial disulfide into the DTA moiety, and (ii) translocation of LFNDHFR and LFNDTA was blocked by their ligands (MTX and adenine, respectively) . These results demonstrate that the acid-induced translocation by anthrax toxin closely resembles that of diphtheria toxin, despite the fact that these two toxins are unrelated and form pores by different mechanisms. J Immunol Methods, 1998 Sep 1, 218(1-2), 1 - 8 Comparative studies of magnetic particle-based solid phase fluorogenic and electrochemiluminescent immunoassay; Yu H; Two solid phase immunoassays, an electrochemiluminescent immunoassay (ECLIA) and a magnetic particle fluorogenic immunoassay (MPFIA) were evaluated and compared for bacterial detection . Briefly, the ECLIA is based on a redox reaction between ruthenium (II)-trisbipyridyl Ru{(bpy)3}2+ labeled antibody and the excess of tripropylamine, which generates photons . The entire reaction is carried on the near surface area between the spherical magnetic beads and an anode electrode . The detectable bacterial spores are at a linear range from 5 x 10(3) to 5 x 10(5) colony forming units (cfu) of Bacillus subtilis var . niger spores, 10(2) to 10(4) cfu of Bacillus anthrax spores and 10(2) to 10(6) cells of Escherichia coli O157:H7 in ECLIA . The unique MPFIA technique employs antibody-coated magnetic beads as solid phase in suspension for bacterial capture and concentration in a 96-well microplate format . Primary capturing antibodies, bacteria form a sandwich with alkaline phosphatase (AP)-labeled antibodies as reporter followed by a reaction with the AP substrate, AttoPhos to generate fluorescence for detection . Immunomagnetic separation permits direct isolating and concentrating bacterial cells from the crude samples, such as blood and environmental water . The results of MPFIA for detecting bacteria showed less sensitivity compared with that of ECLIA, however it provides a means for direct, high throughput screening bacteria from crude biological samples . Both ECLIA and MPFIA are rapid (less than one hour) and easy to use. Infect Immun, 1998 Oct, 66(10), 4696 - 9 Anthrax toxin as a molecular tool for stimulation of cytotoxic T lymphocytes: disulfide-linked epitopes, multiple injections, and role of CD4(+) cells; Ballard JD et al.; We have previously demonstrated that anthrax toxin-derived proteins, protective antigen (PA) and the amino-terminal portion of lethal factor (LFn), can be used in combination to deliver heterologous molecules to the cytosol of mammalian cells . In this study we examined the ability of an LFn-peptide disulfide-linked heterodimer to prime cytotoxic T lymphocytes (CTL) in the presence of PA . A mutant of LFn that contains a carboxy-terminal reactive cysteine was generated . This form of LFn could be oxidized with a synthetic cysteine containing peptide to form a heterodimer of the protein and peptide . Mice injected with the heterodimer plus PA mounted a peptide-specific CTL response, indicating that this molecule functioned similarly to the genetically fused forms used previously . We also report the results of an analysis of two aspects of this system important for the development of experimental vaccines . First, CD4 knockout mice were unable to generate a CTL response when treated with PA plus an LFn-epitope fusion protein, suggesting that CD4(+) helper responses are essential for stimulating specific CTL with the PA-LFn system . Second, we now show that primary injection with this system does not generate any detectable antibody response to the vaccine components and that prior immunization has no effect on priming a CTL response to an unrelated epitope upon subsequent injection. Emerg Infect Dis, 1998 Jul-Sep, 4(3), 488 - 92 Bioterrorism as a public health threat; Henderson DA; The threat of bioterrorism, long ignored and denied, has heightened over the past few years . Recent events in Iraq, Japan, and Russia cast an ominous shadow . Two candidate agents are of special concern--smallpox and anthrax . The magnitude of the problems and the gravity of the scenarios associated with release of these organisms have been vividly portrayed by two epidemics of smallpox in Europe during the 1970s and by an accidental release of aerosolized anthrax from a Russian bioweapons facility in 1979 . Efforts in the United States to deal with possible incidents involving bioweapons in the civilian sector have only recently begun and have made only limited progress . Only with substantial additional resources at the federal, state, and local levels can a credible and meaningful response be mounted . For longer-term solutions, the medical community must educate both the public and policy makers about bioterrorism and build a global consensus condemning its use. Vaccine, 1998 May-Jun, 16(9-10), 880 - 4 The effectiveness and safety of vaccines against human anthrax: a systematic review; Demicheli V et al.; We report on the results of a systematic review of existing controlled clinical trials undertaken to assess the effectiveness and safety of vaccines against human anthrax in relation to disease incidence and side-effects . Two articles retrieved by electronic and hand search fulfilling some of the inclusion criteria underwent a quality assessment by a group of reviewers . Data synthesized from the two trials showed that estimates of overall effectiveness and safety favour treatment (overall odds ratio 0.16; 95% confidence interval 0.07-0.34) . The route of inoculation appears to make little difference to the effectiveness of the vaccines; however, one study shows that the incidence and severity of side-effects are significantly higher with the killed vaccine than with the alum-based placebo (overall odds ratio 0.16; 95% confidence interval 2.38-27.17). Hist Philos Life Sci, 1997, 19(2), 181 - 209 The public science of Louis Pasteur: the experiment on anthrax vaccine in the popular press of the time; Bucchi M; The paper focuses on Pasteur's public experimentation of the anthrax vaccine (Pouilly-le-Fort, 1881) as portrayed in the English and French popular press of the time . It is argued that this 'popular' level of representation did not merely provide additional publicity for Pasteur's ideas . Rather, the nature and meaning of the experiment itself and of the related controversy on immunisation were substantially negotiated and shaped within the public arena . The multifold consequences of this framing at the public level are explored . In particular, attention is drawn to the relationships that in such process were established with other issues debated at the same time in the arena, namely homeopathy, vivisection and vaccination. J Appl Microbiol, 1998 Apr, 84(4), 667 - 76 Airborne movement of anthrax spores from carcass sites in the Etosha National Park, Namibia; Turnbull PC et al.; Tests for airborne movement of anthrax spores downwind from three heavily contaminated carcass sites were carried out under a range of wind conditions . Anthrax spores were detected in just three of 43 cyclone or gelatin filter air samples taken at distances of 6, 12 and 18 m from the sites . In addition, nine positives resulted during sampling sessions in which the site was mechanically disturbed, with a further five positives being found in sessions subsequent to those in which the site had been disturbed . The three positive samples not related to man-made disturbance were associated with the highest winds experienced during the study . Despite colony counts exceeding 100 on the culture plates in three instances, calculations showed that these represented very low worst case probable spore inhalation rates for animals or humans exposed to such levels . The low number of positives, the clear pattern of rapidly declining numbers of anthrax spores with distance downwind from the centres of the sites apparent on settle plates, and the persisting levels of contamination despite wind and rain, collectively suggest that the anthrax spores were associated with fairly heavy particles, although this was not seen by electron microscopy on soil samples from the sites . Overall, the findings are interpreted as indicating that it is very unlikely that Etosha animals contract anthrax by the inhalation route while simply in transit near or across a carcass site . The significance of the observations in relation to weather conditions in the Etosha, other studies on particulate aerosols in the region, and reports of long-distance airborne movement of microbes, is discussed. Autoimmunity, 1998, 27(3), 135 - 9 Cyclosporine induced autoimmunity in newborns prevented by early immunization; Classen JB; It has been shown in animal toxicity models that administration of Cyclosporine, CsA, to a pregnant mouse greatly increases the risk that the offspring will develop autoimmunity . Immunization starting at birth has been shown to prevent autoimmunity in other animal models of autoimmunity and early immunization is associated with the prevention of diabetes in humans . Experiments were performed to see if early immunization could also prevent CsA induced autoimmunity . Mice were injected with CsA during the first week of life and then immunized with killed human vaccines, including common pediatric vaccines, starting in the second week of life for a total of 3-4 doses . Administration of CsA during the first week of life resulted in the development of antigastric autoantibodies which were measured at week 8 of life . Only 12% of mice treated with CsA alone lacked anti-agastric antibodies compared to 61% in the group receiving the CsA and the diphtheria, tetanus, pertussis, and anthrax vaccines (p = 0.0005) . The results indicate early immunization can prevent CsA induced autoimmunity and provide further evidence that the effect of starting immunization in the first month should be compared to starting immunization after 2 months in humans. Biochemistry, 1998 Mar 17, 37(11), 3941 - 8 Identification of residues lining the anthrax protective antigen channel; Benson EL et al.; In its activated 63 kDa form, the protective antigen (PA) component of anthrax toxin forms a heptameric prepore, which converts to a pore (channel) in endosomal membranes at low pH and mediates translocation of the toxin's enzymic moieties to the cytosol . It has been proposed that the prepore-to-pore conversion involves a conformational rearrangement of a disordered amphipathic loop (D2L2; residues 302-325), in which loops from the 7 protomers combine to form a transmembrane 14-stranded beta barrel . To test this model, we generated Cys substitutions in 24 consecutive residues of the D2L2 loop, formed channels in artificial bilayers with each mutant, and examined changes in channel conductance after adding the thiol-reactive, bilayer-impermeant reagent methanethiosulfonate ethyltrimethylammonium (MTS-ET) to the trans compartment . The rationale for these experiments is that reaction of MTS-ET with a Cys residue adds a positively charged group and therefore would likely reduce channel conductance if the residue were in the ion-conducting pathway . We found alternating reduction and absence of reduction of conductance in consecutive residues over two stretches (residues 302-311 and 316-325) . This pattern is consistent with alternating polar and apolar residues of the two stretches projecting into the pore lumen and into the bilayer, respectively . Residues connecting these two stretches (residues 312-315) were responsive to MTS-ET, consistent with their being in a turn region . Single channels formed by selected mutants (H304C and N306C) showed multiple conductance step changes in response to MTS-ET, consistent with an oligomeric pore . We also found that the binding site for the channel-blocking tetraalkylammonium ions is located cis relative to the inserted D2L2 loops . These findings constitute strong evidence in favor of the model of conversion of the prepore to a 14-stranded beta barrel pore and solidify the foundation for studies to understand the mechanism of translocation by anthrax toxin. Arch Intern Med, 1998 Mar 9, 158(5), 429 - 34 Anthrax as a potential biological warfare agent; Pile JC et al.; Anthrax is a zoonotic illness recognized since antiquity . Today, human anthrax has been all but eradicated from the industrialized world, with the vast majority of practitioners in the United States unlikely to have seen a case . Unfortunately, the disease remains endemic in many areas of the world, and anthrax poses a threat as a mass casualty-producing weapon if used in a biological warfare capacity. Rev Sci Tech, 1996 Sep, 15(3), 853 - 62 {Vaccines against anthrax in animals, from Louis Pasteur to our day}; Shlyakhov E et al.; The authors outline the history of vaccination against anthrax in animals, from the end of the 19th century to the present time . The three main steps in the production of specific vaccines are described in detail: production of vaccines from live, encapsulated bacteria, followed by vaccines from live, unencapsulated bacteria and, finally, subunit vaccines . Advantages and disadvantages of these three types of vaccine, some of which are still in use today, are described and discussed. Clin Infect Dis, 1998 Jan, 26(1), 97 - 102 Cutaneous manifestations of anthrax in rural Haiti; Smego RA Jr et al.; In industrialized countries, the zoonotic disease anthrax has been virtually eradicated because of effective public health measures including animal vaccination and quality control of animal products . In developing parts of the world, however, anthrax remains an occupational hazard of herdsmen and workers who have direct contact with infected animals or who process animal hides, hair, bone and bone products, and wool . For clinicians unfamiliar with this interesting infectious disease, the major dermatologic characteristics and clinical evolution of five cases of cutaneous anthrax are reviewed in this study in both descriptive and photographic forms, to define the clinical spectrum of cutaneous disease. Infect Immun, 1998 Feb, 66(2), 615 - 9 Anthrax toxin-mediated delivery in vivo and in vitro of a cytotoxic T-lymphocyte epitope from ovalbumin; Ballard JD et al.; We reported earlier that a nontoxic form of anthrax toxin was capable of delivering a cytotoxic T-lymphocyte (CTL) epitope in vivo, such that a specific CTL response was primed against the epitope . The epitope, of bacterial origin, was fused to an N-terminal fragment (LFn) from the lethal-factor component of the toxin, and the fusion protein was injected, together with the protective antigen (PA) component, into BALB/c mice . Here we report that PA plus LFn is capable of delivering a different epitope--OVA(257-264) from ovalbumin . Delivery was accomplished in a different mouse haplotype, H-2Kb and occurred in vitro as well as in vivo . An OVA(257-264)-specific CTL clone, GA-4, recognized EL-4 cells treated in vitro with PA plus as little as 30 fmol of the LFn-OVA(257-264) fusion protein . PA mutants attenuated in toxin self-assembly or translocation were inactive, implying that the role of PA in epitope delivery is the same as that in toxin action . Also, we showed that OVA(257-264)-specific CTL could be induced to proliferate by incubation with splenocytes treated with PA plus LFn-OVA(257-264) . These findings imply that PA-LFn may serve as a general delivery vehicle for CTL epitopes in vivo and as a safe, efficient tool for the ex vivo expansion of patient-derived CTL for use in adoptive immunotherapy. Neuropediatrics, 1997 Dec, 28(6), 333 - 4 Ventricular shunt infection and meningitis due to Bacillus cereus; Berner R et al.; Non-anthrax Bacillus species are usually considered to be contaminants if found in clinical specimens . Only a few patients with systemic infections due to Bacillus cereus are reported . We present the case of a 18-month old boy with a primitive neuroectodermal tumor (PNET) in the brainstem and obstructive hydrocephalus that required an outlying and subsequently a ventriculoperitoneal drain . Following contamination at the site of entry of the external drain, shunt infection and meningitis with Bacillus cereus developed . Antibiotic treatment with vancomycin failed to eliminate the bacterium from the cerebrospinal fluid, so the shunt system had to be removed . Explantation of the shunt and addition of fosfomycin to the antibiotic regimen resulted in a complete cure of the infection. Mol Cell Biochem, 1997 Dec, 177(1-2), 7 - 14 Site directed mutagenesis of histidine residues in anthrax toxin lethal factor binding domain reduces toxicity; Arora N; Anthrax lethal toxin is a mixture of protective antigen (PA, 735 AA) and lethal factor (LF, 776 AA) . Earlier studies have shown that 254 residues of lethal factor are sufficient for PA binding to cause internalization (Arora N and Leppla SH, J Biol Chem 268: 3334-3342, 1993) . The present study was undertaken to determine residues which are important for binding of LF to PA . LF modification with diethyl pyrocarbonate (DEPC, modifies histidine residue primarily) results in the loss of binding and toxicity in mammalian cells . There are nine histidine residues in the binding domain . To locate the important residue(s), site-directed mutagenesis of these histidines were performed by recombinant methods . Replacement of His42 with Gly42 destablizes the protein and hence it could not be purified . His35 when mutagenized to Gly35 (mLF-DTA) diminishes the toxicity by 20 fold . Time dependent studies show that binding of mLF-DTA was reduced at shorter incubations and longer incubations taper off this difference . Gel shift assay suggested 8-10% less binding of mLF-DTA as compared to LF-DTA . In conclusion His35 is important for binding and His42 is critical and confers proper conformation for LF binding to PA. Przegl Epidemiol, 1997, 51(3), 317 - 9 {A case of cutaneous anthrax in the Lomza district}; Bacharz M et al.; A case of anthrax is reported in 46 year old man . Cases of anthrax in animals and human beings are rare in Poland and therefore the diagnosis of the disease can be difficult. Rev Sci Tech, 1997 Apr, 16(1), 111 - 6 Animal health risks associated with ostrich products; Huchzermeyer FW; Five diseases recorded in ostriches are regarded as posing a potential animal health threat to meat-importing countries . Newcastle disease causes an atypically low mortality in ostriches: infected birds display typical nervous symptoms but no pathognomonic lesions which could be detected during post-mortem inspection . The vaccination of feedlot birds and a thorough ante-mortem examination are regarded as necessary precautions to ensure virus carriers are not among those animals destined for slaughter and subsequent export . Avian influenza produces clinical depression and lesions can be detected at post-mortem examination . Borna disease appears to affect mainly younger birds, and the virus is probably not present in the meat of affected birds . Finally, there is little evidence to suggest that ostriches could play a role in the epidemiology of transmissible spongiform encephalopathies . Cases of anthrax are extremely rare . The importation of deboned ostrich meat reduces the risk of infected scraps being fed to susceptible animals. Rev Sci Tech, 1997 Apr, 16(1), 104 - 10 Animal health risks associated with the transportation and utilisation of wildlife products; Bengis RG; The animal health risks associated with the movement of wildlife products are infinitely less than those associated with the movement of live animals . Very few pathogens are sufficiently robust to survive the significant changes in temperature, pH, moisture content and osmolality which occur post mortem, or which are associated with preservation processes such as pickling, smoking or drying . Certain pathogens, however, (e.g . foot and mouth disease, classical swine fever {hog cholera} and African swine fever viruses and the anthrax bacillus) are hardy and resistant to these environmental changes and therefore constitute a finite animal health risk if raw, undercooked or under-preserved products from infected wild animals are imported . Other less robust pathogens, such as rinderpest virus, may remain infectious in animal products if these are obtained from acutely infected animals and frozen immediately . Macroparasitic diseases such as trichinellosis and echinococcosis-hydatidosis, if present in the unprocessed tissues of infected wildlife, are potentially infectious to carnivorous or omnivorous companion animals . The importation of untreated wet hides may result in the introduction of alien ectoparasites and/or the infectious diseases for which they are vectors . The author discusses the more significant pathogens found in free-ranging wildlife which should be taken into consideration when importing wildlife products from endemically or epidemically infected countries. Proc Natl Acad Sci U S A, 1997 Oct 28, 94(22), 12059 - 64 Targeting HIV proteins to the major histocompatibility complex class I processing pathway with a novel gp120-anthrax toxin fusion protein; Goletz TJ et al.; A challenge for subunit vaccines whose goal is to elicit CD8(+) cytotoxic T lymphocytes (CTLs) is to deliver the antigen to the cytosol of the living cell, where it can be processed for presentation by major histocompatibility complex (MHC) class I molecules . Several bacterial toxins have evolved to efficiently deliver catalytic protein moieties to the cytosol of eukaryotic cells . Anthrax lethal toxin consists of two distinct proteins that combine to form the active toxin . Protective antigen (PA) binds to cells and is instrumental in delivering lethal factor (LF) to the cell cytosol . To test whether the lethal factor protein could be exploited for delivery of exogenous proteins to the MHC class I processing pathway, we constructed a genetic fusion between the amino-terminal 254 aa of LF and the gp120 portion of the HIV-1 envelope protein . Cells treated with this fusion protein (LF254-gp120) in the presence of PA effectively processed gp120 and presented an epitope recognized by HIV-1 gp120 V3-specific CTL . In contrast, when cells were treated with the LF254-gp120 fusion protein and a mutant PA protein defective for translocation, the cells were not able to present the epitope and were not lysed by the specific CTL . The entry into the cytosol and dependence on the classical cytosolic MHC class I pathway were confirmed by showing that antigen presentation by PA + LF254-gp120 was blocked by the proteasome inhibitor lactacystin . These data demonstrate the ability of the LF amino-terminal fragment to deliver antigens to the MHC class I pathway and provide the basis for the development of novel T cell vaccines. Hum Immunol, 1997 May, 54(2), 129 - 36 Delivery of antigens to the MHC class I pathway using bacterial toxins; Goletz TJ et al.; Cytotoxic T lymphocytes (CTL) recognize antigens derived from endogenously expressed proteins presented on the cell surface in the context of major histocompatibility complex (MHC) class I molecules . Because CTL are effective in antiviral and antitumor responses, the delivery of antigens to the class I pathway has been the focus of numerous efforts . Generating CTL by immunization with exogenous proteins is often ineffective because these antigens typically enter the MHC class II pathway . This review focuses on the usefulness of bacterial toxins for delivering antigens to the MHC class I pathway . Several toxins naturally translocate into the cytosol, where they mediate their cytopathic effects, and the mechanisms by which this occurs has been elucidated . Molecular characterization of these toxins identified the functional domains and enabled the generation of modified proteins that were no longer toxic but retained the ability to translocate into the cytosol . Thus, these modified toxins could be examined for their ability to carry peptides or whole proteins into the cytosolic processing pathway . Of the toxins studied-diphtheria, pertussis, Pseudomonas, and anthrax-the anthrax toxin appears the most promising in its ability to deliver large protein antigens and its efficiency of translocation. Infect Immun, 1997 Aug, 65(8), 3370 - 5 A role for PACE4 in the proteolytic activation of anthrax toxin protective antigen; Gordon VM et al.; Several bacterial protein toxins require activation by eukaryotic proteases . Previous studies have shown that anthrax toxin protective antigen (PA), Pseudomonas exotoxin A (PE), and diphtheria toxin (DT) are cleaved by furin C-terminal to the sequences RKKR, RQPR, and RVRR, respectively . Because furin-deficient cells retain some sensitivity to PA and DT, it is evident that other cellular proteases can activate these toxins . Whereas furin has been shown to require arginine residues at positions -1 and -4 for substrate recognition, another protease with an activity which could substitute for furin in toxin activation, the furin-related protease PACE4, requires basic residues in the -1, -2, and -4 positions of the substrate sequence . To examine the relative roles of furin and PACE4 in toxin activation, we used furin-deficient CHO cells (FD11 cells) transfected with either the furin (FD11/furin cells) or PACE4 (FD11/PACE4 cells) gene . Mutant PA proteins containing the cleavage sequence RAAR or KR were cytotoxic toward cells expressing only PACE4 . In vitro cleavage data demonstrated that PACE4 can recognize RAAR and, to a much lesser extent, KR and RR . When extracts from PACE4-transfected cells were used as a source of proteases, PACE4 had minimal activity, indicating that it had been partially inactivated or did not remain associated with the cell membranes . Cleavage of iodinated PA containing the sequence RKKR or RAAR was detected on the surface of all cell types tested, but cleavage of a dibasic sequence was detected only intracellularly and only in cells that expressed furin or PACE4 . The data provide evidence that PACE4 is present at the exterior of cells, that it plays a role in the proteolytic activation of anthrax toxin PA, and that PACE4 can activate substrates at the sequence RAAR or KR. Mol Membr Biol, 1997 Apr-Jun, 14(2), 45 - 64 Membrane insertion: The strategies of toxins (review); Lesieur C et al.; Protein toxins are soluble molecules secreted by pathogenic bacteria which act at the plasma membrane or in the cytoplasm of target cells . They must therefore interact with a membrane at some point, either to modify its permeability properties or to reach the cytoplasm . As a consequence, toxins have the built-in capacity to adopt two generally incompatible states: water-soluble and transmembrane . Irrespective of their origin or function, the membrane interacting domain of most protein toxins seems to have adopted one out of two structural strategies to be able to undergo this metamorphosis . In the first group of toxins the membrane interacting domain has the structural characteristics of most known membrane proteins, i.e . it contains hydrophobic and amphipathic alpha-helices long enough to span a membrane . To render this 'membrane protein' water-soluble during the initial part of its life the hydrophobic helices are sheltered from the solvent by a barrel of amphipathic helices . In the second group of toxins the opposite strategy is adopted . The toxin is an intrinsically soluble protein and is composed mainly of beta-structure . These toxins manage to become membrane proteins by oligomerizing in order to combine amphipathic beta-sheet to generate sufficient hydrophobicity for membrane insertion to occur . Toxins from this latter group are thought to perforate the lipid bilayer as a beta-barrel such as has been described for bacterial porins, and has recently been shown for staphylococcal alpha-toxin . The two groups of toxins will be described in detail through the presentation of examples . Particular attention will be given to the beta-structure toxins, since four new structures have been solved over the past year: the staphyloccocal alpha-toxin channel, the anthrax protective antigen protoxin, the anthrax protective antigen-soluble heptamer and the CytB protoxin . Structural similarities with mammalian proteins implicated in the immune response and apoptosis will be discussed . Peptide toxins will not be covered in this review. Vaccine, 1997 Apr-May, 15(6-7), 631 - 6 Anthrax post-vaccinal cell-mediated immunity in humans: kinetics pattern; Shlyakhov E et al.; Seven groups (2596 subjects) were vaccinated with a human live anthrax vaccine (HLAV) by three different routes (scarification, subcutaneous and aerosol) . The vaccinees were tested for anthrax cell-mediated immunity using the "Anthraxin" skin test at 7, 15, 30, 90, 180 and 365 days following vaccination . The kinetic pattern obtained from all groups, shows a significant, five-phased curve: phase I (2-6 days post-vaccination) shows a slow increase in positive Anthraxin skin reactions . Phase II (7-15 days post-vaccination) shows an exponential rise to a maximum at day 15 . Phase III (16-30 days post-vaccination) shows a decrease to day 30 . Phase IV (31-90 days post-vaccination) leads to a relative restoration of the positive skin reactions . During phase V (91-365 days post-vaccination) there is a continuous decrease in positive Anthraxin skin reactions . The loss of the skin test reaction on day 30 is a characteristic feature of post vaccination anthrax cell-mediated immunity . It may be due to a blockade of macrophages by lethal anthrax toxin released by the multiplying vaccine strain . Epidemiological observations of HLAV protective rates correlate with the phases of the skin reaction kinetics. Commun Dis Intell, 1997 Feb 20, 21(4), 47 - 8 A case of human anthrax in Victoria; Lester R et al.; A human case of anthrax was identified through surveillance of knackery workers who had been exposed to infected cattle . The outbreak in cattle has affected 38 herds in the Stanhope/Tatura area of central northern Victoria . The human case, a 39 year old male, was treated in hospital and is recovering . Surveillance of other knackery workers has now been completed, and no other cases were found . Public health measures are in place to prevent further human cases. Biochemistry, 1996 Nov 26, 35(47), 14939 - 46 Secondary structure of anthrax lethal toxin proteins and their interaction with large unilamellar vesicles: a fourier-transform infrared spectroscopy approach; Wang XM et al.; Attenuated total reflection Fourier transform infrared spectroscopy has been used to study the secondary structure of anthrax lethal toxin proteins: protective antigen (PA) and lethal factor (LF), as a function of pH in the absence and in the presence of phospholipid vesicles . We first characterized the binding of LF and PA to the lipid membrane and demonstrated the strong pH dependence of the association of PA and LF to the lipid bilayer as well as the effect of pH neutralization on this binding . Binding of LF to the lipid membrane can be, at least partially, reversed when the pH is brought to neutral whereas in the same conditions PA binding is irreversible . Characterization of the conformational changes undergone by PA and LF upon pH lowering, lipid binding, and, in the case of LF, reversal of binding was carried out (i) by determining the secondary structure of the proteins and (ii) by evaluating their ability to undergo an hydrogen/deuterium exchange. Cent Afr J Med, 1996 Nov, 42(11), 312 - 5 Factors associated with human anthrax outbreak in the Chikupo and Ngandu villages of Murewa district in Mashonaland East Province, Zimbabwe; Mwenye KS et al.; OBJECTIVES: To determine factors associated with human anthrax . DESIGN: Unmatched restrospective case control . SETTING: General community of Chikupo and Ngandu villages of the Nyamutumbu area of Murewa district in Mashonaland East Province of Zimbabwe . SUBJECTS: 19 persons were identified using the clinical anthrax definition developed by the Centre for Diseases Control as cases, and 57 persons who reported not having taken antibiotics for anthrax were identified as controls . Both cases and controls had never been out of the village during the time of the epidemic . MAIN OUTCOME MEASURES: Percent of cases and controls handling, eating and drinking products of an alleged infected animal . RESULTS: Out of 19 cases of anthrax, five died, giving a case fatality rate of 26% (95% CI 5 to 47%) . The following factors were significantly associated with the disease: skinning and cutting meat of an animal alleged to have shown symptoms of anthrax (OR 29, 95% CI 3 to 707, p < 0.001), eating contaminated meat (OR 20, 95% CI 2 to 470, p < 0.001), belonging to a religious sect which does not restrict its followers from eating meat from a carcass (OR 6, 95% CI 1 to 21, p = 0.003), handling contaminated meat in the process of selling it (OR 5, 95% CI 1 to 31, p = 0.033) and attending a gathering (OR 4, 95% CI 1 to 21, p = 0.028) . CONCLUSIONS: Anthrax still remains endemic in Zimbabwe despite the public health and veterinary efforts to control the disease . Having knowledge of risk factors may help one to minimize chances of contracting anthrax. Proc Natl Acad Sci U S A, 1996 Oct 29, 93(22), 12531 - 4 Anthrax toxin-mediated delivery of a cytotoxic T-cell epitope in vivo; Ballard JD et al.; The protective antigen (PA) component of anthrax toxin mediates entry of the toxin's lethal factor (LF) and edema factor into the cytosolic compartment of mammalian cells . The amino-terminal domain of LF (LFn; 255 amino acids) binds LF to PA, and when fused to heterologous proteins, the LFn domain delivers such proteins to the cytoplasm in the presence of PA . In the current study, we fused a 9-amino acid cytotoxic T-lymphocyte (CTL) epitope (LLO91-99) from an intracellular pathogen, Listeria monocytogenes, to LFn and measured the ability of the resulting LFn-LLO91-99 fusion protein to stimulate a CTL response against the epitope in BALB/c mice . As little as 300 fmol of fusion could stimulate a response . The stimulation was PA-dependent and occurred with the peptide fused to either the amino terminus or the carboxyl terminus of LFn . Upon challenge with L . monocytogenes, mice previously injected with LFn-LLO91-99 and PA showed a reduction of colony-forming units in spleen and liver, relative to nonimmunized control mice . These results indicate that anthrax toxin may be useful as a CTL-peptide delivery system for research and medical applications. Curr Microbiol, 1996 Oct, 33(4), 224 - 7 Cytotoxic effects of anthrax lethal toxin on macrophage-like cell line J774A.1; Lin CG et al.; The cytotoxic effects of anthrax lethal toxin purified from an avirulent strain were examined on mouse macrophage-like J774A.1 cells . Cell death induced by high concentration of purified lethal toxin had the characteristics of necrosis . At lower concentrations, the toxin caused no morphological change and most of the cells were viable . Interestingly, apoptotic cells were observed when the cells were preincubated with a serine/threonine phosphatase inhibitor, calyculin A, and then exposed to a toxin concentration of 0.1 microg/ml . This is the first report that lethal toxin of the anthrax bacillus can induce both necrosis and apoptosis and that protein phosphatases are implicated in the regulation of bacterial toxin-induced apoptosis. Proc Natl Acad Sci U S A, 1996 Aug 6, 93(16), 8437 - 42 Fused polycationic peptide mediates delivery of diphtheria toxin A chain to the cytosol in the presence of anthrax protective antigen; Blanke SR et al.; The lethal factor (LF) and edema factor (EF) of anthrax toxin bind by means of their amino-terminal domains to protective antigen (PA) on the surface of toxin-sensitive cells and are translocated to the cytosol, where they act on intracellular targets . Genetically fusing the amino-terminal domain of LF (LFN; residues 1-255) to certain heterologous proteins has been shown to potentiate these proteins for PA-dependent delivery to the cytosol . We report here that short tracts of lysine, arginine, or histidine residues can also potentiate a protein for such PA-dependent delivery . Fusion of these polycationic tracts to the amino terminus of the enzymic A chain of diphtheria toxin (DTA; residues 1-193) enabled it to be translocated to the cytosol by PA and inhibit protein synthesis . The efficiency of translocation was dependent on tract length: (LFN > Lys8 > Lys6 > Lys3) . Lys6 was approximately 100-fold more active than Arg6 or His6, whereas Glu6 and (SerSerGly)2 were inactive . Arg6DTA was partially degraded in cell culture, which may explain its low activity relative to that of Lys6DTA . The polycationic tracts may bind to anionic sites at the cell surface (possibly on PA), allowing the fusion proteins to be coendocytosed with PA and delivered to the endosome, where translocation to the cytosol occurs . Excess free LFN blocked the action of LFNDTA, but not of Lys6DTA . This implies that binding to the LF/EF site is not an obligatory step in translocation and suggests that the polycationic tag binds to a different site . Besides elucidating the process of translocation in anthrax toxin, these findings may aid in developing systems to deliver heterologous proteins and peptides to the cytoplasm of mammalian cells. Mol Gen Mikrobiol Virusol, 1996 Jul-Sep, (3), 20 - 2 {Detection of the functionally active domains in the molecule of the lethal factor of the anthrax exotoxin}; Noskov AN et al.; Three functional domains were revealed in the molecule of the lethal factor of B . anthracis . They are located in the linear structure of the molecula as follows: the associative domain occupies the area from Lys39 to Met242, the stabilizing domain from Leu517 to Lys614, and the effector domain still further to the COOH-terminal Lys mino acid. Mol Gen Mikrobiol Virusol, 1996 Jul-Sep, (3), 16 - 20 {Detection of the functionally active domains in the molecule of protective antigen of the anthrax exotoxin}; Noskov AN et al.; Using the limited proteolysis method, we established that the protective antigen (PA) molecule consists of four functionally active domains . The shielding domain occupies an area in the linear structure of the molecule PA with NH4-terminal up to Lys166 and plays an important role in the proteolytic activation of PA . The associative domain situated in the Arg167-Met266 region is responsible for interactions with either lethal or edematous factors in self-assembly of the toxic complexes of the lethal or edematous toxin . The stabilizing domain occupies the Gly351 to Met434 area . On the one hand, this area promotes the formation of conformationally stable toxic complexes with the lethal factor, on the other, directly participates in the formation of the hydrophobic canal, through which the molecule of the lethal or edematous factor and, evidently, a fragment of PA molecule as well (from Arg167 to Gly314), including the associative gene, gets inside the target cell . The receptor domain representing a COOH-terminal region, starting from Leu663 amino acid, interacts with the specific receptors on macrophages and thus delivers the toxic complex to the target cell. Eur J Clin Microbiol Infect Dis, 1996 Mar, 15(3), 242 - 5 Evaluation of the anthraxin skin test for diagnosis of acute and past human anthrax; Shlyakhov E et al.; A skin test for the diagnosis of human anthrax was evaluated as an alternative to bacteriological confirmation of human anthrax, which is possible in 10-40% of cases within the first three weeks of the disease only . The anthraxin skin test, which detects anthrax cell-mediated immunity, was positive in 81.8% of cases in the first three days of the disease, and in 97-99% of cases in the next two to three weeks . The positivity rate was 98.5% in the first 1.5 months of convalescence, 92.8% in the next 3 years, 82.8% in the following 4 to 15 years, and 72.7% 16 to 31 years after recovery . Thus, the anthraxin skin test appears to be a valuable method for early diagnosis of acute anthrax as well as the only method available for retrospective diagnosis of human anthrax. Bull World Health Organ, 1996, 74(5), 465 - 70 Anthrax: memorandum from a WHO meeting. {Delayed hypersensitivity in man after booster anthrax vaccination} Shlyakhov E, Rubinstein E. Unite des Maladies Infectieuses, Chaim Sheba Medical Center, Tel-Hashomer, IsraelThe purpose of this study was to ascertain the need for boosters after administration of STI anthrax vaccine . Postvaccination dynamics was assessed by observing the intradermic reaction of anthraxine . The study included 138 subjects vaccinated 15 months earlier by subcutaneous injection or 128 subjects vaccinated 24 months earlier by aerosol inhalation . Subjects were tested using the anthraxine test in separate groups on D2, D7, D15, D90, D180, and D365 after administration of the booster via the same route as the primary vaccination . Immediately before administration of the booster residual positive skin reactions were observed in 14.3% of subjects in the subcutaneous vaccination group and 8.3% of subjects in the aerosol vaccination group . After the booster, the proportion of subjects with positive skin reactions increased rapidly . The proportion of positive skin reactions after the booster was not statistically different from the proportion after the primary vaccination . This study provides scientific evidence supporting the need for boosters after anthrax vaccination and demonstrated an absence of sensitization by anthraxine during tests to evaluate response kinetics in subjects after primary vaccination . Several questions remain to be answered concerning the optical booster dose, the most effective timing, and the value of acellular vaccines. Ann Hum Biol, 1996 Jan-Feb, 23(1), 1 - 21 The plague in Penrith, Cumbria, 1597/8: its causes, biology and consequences; Scott S et al.; Using a family reconstitution study the biology of the plague in Penrith, Cumbria in 1597/8 is described in detail; it was an explosive epidemic that spread rapidly within families and 606 individuals died of the plague, some 40% of the population . The age-specific mortality corresponded with the calculated age structure of the population and infection appeared to be random . The sex ratio of victims was 1.37 females to 1 male . The plague spread from the northeast via Richmond and then exploded in the Eden valley, appearing almost simultaneously in Penrith, Kendal and Carlisle . The details of the epidemics and the location and the climate of these widely separated small market towns show that bubonic plague was not the causative agent, and the possibility that anthrax was responsible for the drastic mortality is briefly considered . The population rapidly built up after the plague, largely by immigration and not by increased fertility, and steady-state conditions were re-established within 5 years and continued for 150 years . This severe mortality crisis of the plague had a profound effect on the population at Penrith, triggering long wavelength oscillations in both baptisms and burials in this population living under marginal conditions and maintained in steady-state by density-dependent factors. Berl Munch Tierarztl Wochenschr, 1996 Jan, 109(1), 6 - 7 {Technology of the production of antigens against soil-borne diseases in Madagascar}; Fatou-Rakotobe et al.; There are two important soilborne diseases in Madagascar: Anthrax and Blackleg . The history of vaccine production is described, which is especially marked by the success since the introduction of biofermenter technology. Can J Vet Res, 1995 Oct, 59(4), 256 - 64 Investigation, control and epizootiology of anthrax in a geographically isolated, free-roaming bison population in northern Canada; Gates CC et al.; In July 1993 anthrax caused significant mortality in an isolated, free-ranging population of bison (Bos bison athabascae) west of Great Slave Lake in the Northwest Territories . There was no previous record of anthrax in this area . An emergency response was undertaken to reduce the scale of environmental contamination and dissemination of anthrax spores and hence to reduce the likelihood of future outbreaks . One-hundred-and-seventy-two bison, 3 moose (Alces alces), and 3 black bear (Ursus americanus) carcasses were found . Visual detection of carcasses was enhanced with the use of an airborne, remote infrared sensing camera mounted externally on a helicopter . Fifty-five percent of the carcasses were located in forested or shrub-covered sites where detection would not have been likely without the thermal imaging equipment . Carcasses were disposed of by incineration and the sites were decontaminated with formaldehyde . Application of formaldehyde to carcasses prevented scavenging . The outbreak occurred after a prolonged period of drying between April and mid-July 1993 which followed several successive years of flooding of bison habitat . The "spore concentration hypothesis" provides the most conservative explanation for the occurrence of anthrax under the observed conditions. J Biol Chem, 1995 Aug 4, 270(31), 18626 - 30 Effect of anthrax toxin's lethal factor on ion channels formed by the protective antigen; Zhao J et al.; Protective antigen (PA), a component of anthrax toxin, mediates translocation of the toxin's lethal and edema factors (LF and EF, respectively) to the cytoplasm, via a pathway involving their release from an acidic intracellular compartment . PA63, a 63-kDa proteolytic fragment of PA, can be induced to form ionconductive channels in the plasma membrane of mammalian cells by acidification of the medium . These channels are believed to be comprised of dodecyl sulfate-resistant oligomers (heptameric rings) of PA63 seen by electron microscopy of the purified protein . Here we report that the PA63-mediated efflux of 86Rb+ from preloaded CHO-K1 cells under acidic conditions is strongly inhibited (> or = 70%) by LF or LFN, a PA-binding fragment of LF . Control proteins caused no inhibition . Evidence is presented that the inhibition involves partial blockage of ion conductance by the PA63 channel . Also, oligomer formation is slowed somewhat by LF at pH values near the pH threshold of channel formation (pH approximately 5.3), suggesting that channel formation may also be retarded under these conditions . The relevance of these results to the location of the LF-binding site on PA63 and the mechanism of LF and EF translocation is discussed. J Cell Biol, 1995 Jun, 129(6), 1533 - 41 Pseudomonas exotoxin-mediated selection yields cells with altered expression of low-density lipoprotein receptor-related protein; FitzGerald DJ et al.; The alpha 2-macroglobulin (alpha 2M) receptor/low-density lipoprotein receptor-related protein (LRP) is important for the clearance of proteases, protease-inhibitor complexes, and various ligands associated with lipid metabolism . While the regulation of receptor function is poorly understood, the addition of high concentrations of the 39-kD receptor-associated protein (RAP) to cells inhibits the binding and/or uptake of many of these ligands . Previously, we (Kounnas, M.Z., R.E . Morris, M.R . Thompson, D.J . FitzGerald, D.K . Strickland, and C.B . Saelinger . 1992 . J . Biol . Chem . 267:12420-12423) {corrected} showed that Pseudomonas exotoxin (PE) could bind immobilized LRP . Also, the addition of RAP blocked toxin-mediated cell killing . These findings suggested that PE might use LRP to gain entry into toxin-sensitive cells . Here we report on a strategy to select PE-resistant lines of Chinese hamster ovary cells that express altered amounts of LRP . An important part of this strategy is to screen PE-resistant clones for those that retain sensitivity to both diphtheria toxin and to a fusion protein composed of lethal factor (from anthrax toxin) fused to the adenosine diphosphate-ribosylating domain of PE . Two lines, with obvious changes in their expression of LRP, were characterized in detail . The 14-2-1 line had significant amounts of LRP, but in contrast to wild-type cells, little or no receptor was displayed on the cell surface . Instead, receptor protein was found primarily within cells, much of it apparently in an unprocessed state . The 14-2-1 line showed no uptake of chymotrypsin-alpha 2M and was 10-fold resistant to PE compared with wild-type cells . A second line, 13-5-1, had no detectable LRP mRNA or protein, did not internalize alpha 2M-chymotrypsin, and exhibited a 100-fold resistance to PE . Resistance to PE appeared to be due to receptor-specific defects, since these mutant lines showed no resistance to a PE chimeric toxin that was internalized via the transferrin receptor . The results of this investigation confirm that LRP mediates the internalization of PE. Bull Acad Natl Med, 1995 May, 179(5), 1009 - 21 {Introduction to the commemorative session of the centennial of the death of Louis Pasteur}; Le Minor L; When elected a member of Academie de medecine in 1873, Pasteur was well known for his achievements in chemistry, fermentation and silk worm diseases . He denied spontaneity of diseases, their cause are living germs . Passionate disputations took place in Academy on subjects of septicaemia, childbirth fever, or fowl cholera . In 1881, his success was obtained by vaccination of sheep against epidemic anthrax: his method of virus action softening was available for many human infections diseases, in spite of many disputations . The same method applied to rabies gave him universal glory . When he died in 1895, his views on infections diseases were accepted by Academie de medecine and all over the world. Mol Microbiol, 1995 Feb, 15(4), 661 - 6 Protective antigen-binding domain of anthrax lethal factor mediates translocation of a heterologous protein fused to its amino- or carboxy-terminus; Milne JC et al.; The edema factor (EF) and lethal factor (LF) components of anthrax toxin require anthrax protective antigen (PA) for binding and entry into mammalian cells . After internalization by receptor-mediated endocytosis, PA facilitates the translocation of EF and LF across the membrane of an acidic intracellular compartment . To characterize the translocation process, we generated chimeric proteins composed of the PA recognition domain of LF (LFN; residues 1-255) fused to either the amino-terminus or the carboxy-terminus of the catalytic chain of diphtheria toxin (DTA) . The purified fusion proteins retained ADP-ribosyltransferase activity and reacted with antisera against LF and diphtheria toxin . Both fusion proteins strongly inhibited protein synthesis in CHO-K1 cells in the presence of PA, but not in its absence, and they showed similar levels of activity . This activity could be inhibited by adding LF or the LFN fragment (which blocked the interaction of the fusion proteins with PA), by adding inhibitors of endosome acidification known to block entry of EF and LF into cells, or by introducing mutations that attenuated the ADP-ribosylation activity of the DTA moiety . The results demonstrate that LFN fused to either the amino-terminus or the carboxy-terminus of a heterologous protein retains its ability to complement PA in mediating translocation of the protein to the cytoplasm . Besides its importance in understanding translocation, this finding provides the basis for constructing a translocation vector that mediates entry of a variety of heterologous proteins, which may require a free amino- or carboxy-terminus for biological activity, into the cytoplasm of mammalian cells. Medinfo, 1995, 8 Pt 2, 1199 - 203 ALERT: a clinical case simulator program for serious, communicable, and rare diseases; Brai A et al.; ALERT is an intelligent Tutoring System (ITS) based on clinical case simulation . Its purpose is to assist in the training of general practitioners regarding the diagnosis and the control of serious, communicable, and rare diseases, such as anthrax, plague, and smallpox . ALERT provides both monitoring techniques and treatment protocols and is structured into two independent sections: one devoted to the simulation of the clinical case and the other to detailed description of disease. Infect Immun, 1995 Jan, 63(1), 82 - 7 Proteolytic activation of bacterial toxins by eukaryotic cells is performed by furin and by additional cellular proteases; Gordon VM et al.; Before intoxication can occur, anthrax toxin protective antigen (PA), Pseudomonas exotoxin A (PE), and diphtheria toxin (DT) must be activated by proteolytic cleavage at specific amino acid sequences . Previously, it was shown that PA and DT can be activated by furin . In Chinese hamster ovary (CHO) cells, wild-type (RKKR) and cleavage site mutants of PA, each administered with a modified form of anthrax toxin lethal factor (the N terminus of lethal factor fused to PE domain III), had the following potencies: RKKR (wild type) (concentration causing 50% cell death {EC50} = 12 ng/ml) > or = RAAR (EC50 = 18 ng/ml) > FTKR (EC50 = 24 ng/ml) > STRR (EC50 = 49 ng/ml) . In vitro cleavage of PA and cleavage site mutants of PA by furin demonstrated that native PA (RKKR) and PA with the cleavage sequence RAAR are substrates for furin . To characterize eukaryotic proteases that play a role in activating bacterial toxins, furin-deficient CHO cells were selected after chemical mutagenesis . Furin-deficient cells were resistant to PE, whose cleavage site, RQPR, constitutes a furin recognition site and to all PA cleavage site mutants, but were sensitive to DT (EC50 = 2.9 ng/ml) and PA (EC50 = 23 ng/ml), whose respective cleavage sites, RKKR and RVRR, contain additional basic residues . Furin-deficient cells that were transfected with the furin gene regained sensitivity to PE and PA cleavage site mutants . These studies provide evidence that furin can activate the three toxins and that one or more additional proteases contribute to the activation of DT and PA. Science, 1994 Nov 18, 266(5188), 1202 - 8 The Sverdlovsk anthrax outbreak of 1979; Meselson M et al.; In April and May 1979, an unusual anthrax epidemic occurred in Sverdlovsk, Union of Soviet Socialist Republics . Soviet officials attributed it to consumption of contaminated meat . U.S . agencies attributed it to inhalation of spores accidentally released at a military microbiology facility in the city . Epidemiological data show that most victims worked or lived in a narrow zone extending from the military facility to the southern city limit . Farther south, livestock died of anthrax along the zone's extended axis . The zone paralleled the northerly wind that prevailed shortly before the outbreak . It is concluded that the escape of an aerosol of anthrax pathogen at the military facility caused the outbreak. Br J Dermatol, 1994 Nov, 131(5), 598 - 607 Human cowpox 1969-93: a review based on 54 cases; Baxby D et al.; This survey of the clinical and epidemiological features of human cowpox, a rare but relatively severe zoonotic infection, is based on 54 cases, many unpublished, which we have studied since 1969 . Patients present with painful, haemorrhagic pustules or black eschars, usually on the hand or face, accompanied by oedema, erythema, lymphadenopathy, and systemic involvement . Severe, occasionally fatal, cases occur in eczematous and immunosuppressed individuals, although cowpox has not yet been reported in anyone infected with the human immunodeficiency virus . Variations in the clinical features are described, and the differential clinical diagnosis of cowpox, parapox, herpes virus, and anthrax infections is discussed . The role of the laboratory in diagnosis is described, and the value of electron microscopy in providing rapid confirmation is emphasized . Care in taking a detailed history will assist in the initial clinical diagnosis, and a history of contact with domestic cats, particularly during July-October, is important . The possible influence of smallpox vaccination on the incidence and severity is discussed and discounted. Infect Immun, 1994 Nov, 62(11), 4955 - 61 Fusions of anthrax toxin lethal factor with shiga toxin and diphtheria toxin enzymatic domains are toxic to mammalian cells; Arora N et al.; To investigate the ability of anthrax toxin lethal factor (LF) to translocate foreign proteins into the cytosol of eukaryotic cells and to characterize the structural requirements of this process, fusion proteins containing a portion of LF and the catalytic domains of either diphtheria toxin or Shiga toxin were constructed . Previous work showed that residues 1 to 254 of anthrax toxin lethal factor (LF1-254) are sufficient for binding to the protective antigen component of the toxin and that portions of Pseudomonas exotoxin A fused to LF1-254 are efficiently translocated to the cytosol of eukaryotic cells (N . Arora and S . H . Leppla, J . Biol . Chem . 268:3334-3341, 1993) . In this study, it was found that fusion proteins containing the ADP-ribosylation domain of diphtheria toxin fused at either the amino end or the carboxyl end of LF1-254 are highly toxic to Chinese hamster ovary (CHO) cells, indicating that translocation does not strictly require that the amino terminus of LF be free . A fusion protein containing the ribosome-inactivating A1 subunit of Shiga toxin fused to the carboxyl terminus of LF1-254 was also highly toxic for CHO cells . All fusion proteins were toxic only when administered with the anthrax toxin protective antigen component . The data show that the combination of protective antigen and LF fusion proteins can efficiently import polypeptides from diverse bacterial sources to the cytosol of eukaryotic cells and that LF fusion proteins may have the passenger polypeptides fused at either the amino terminus or the carboxyl terminus of LF1-254 . These LF fusion proteins could potentially be used as components of a therapeutic agent when the destruction of certain types of cells is desired (e.g., in treating cancer). J Biol Chem, 1994 Oct 21, 269(42), 26165 - 71 Cytotoxic effects of a chimeric protein consisting of tetanus toxin light chain and anthrax toxin lethal factor in non-neuronal cells; Arora N et al.; The light chain of tetanus toxin is a zinc endoprotease that inhibits neurotransmitter release by selective proteolysis of the synaptic vesicle-associated protein synaptobrevin/vesicle-associated membrane protein . Cellubrevin is a homologue of synaptobrevin that is found in most cell types and is also a substrate for tetanus toxin . The lack of receptors for tetanus toxin on most cell types has made studies of tetanus toxin action in non-neuronal cells difficult . To characterize tetanus toxin effects in non-neuronal cells, a fusion protein consisting of the 254 amino-terminal amino acids of lethal factor (LF) of anthrax toxin and tetanus toxin light chain (LC) was prepared . This protein (LF-LC) inhibited evoked glycine release from primary spinal cord neurons at concentrations between 1.0 and 100 ng/ml . LF-LC was cytotoxic to RAW 264.7, ANA-1 cells (mouse macrophage cell lines), and Chinese hamster ovary cells in a dose-dependent manner . These effects required the presence of protective antigen, the receptor binding component of anthrax toxin . In contrast, LF-LC was not cytotoxic to RBL-2H3, Vero, or mouse hybridoma cell lines . Mutagenesis of conserved amino acids (His237 and Glu234) in the zinc-binding motif of LC resulted in fusion proteins having no biological activity . LF-LC did not inhibit regulated secretion of serotonin in RBL-2H3 cells or constitutive secretion in any non-neuronal cell lines as measured in several different assays . We suggest that the cytotoxic effects of LF-LC result from inhibition of a specific intracellular membrane fusion event mediated by cellubrevin. Mol Microbiol, 1994 Sep, 13(6), 1093 - 100 Anthrax toxin lethal factor contains a zinc metalloprotease consensus sequence which is required for lethal toxin activity; Klimpel KR et al.; Comparison of the anthrax toxin lethal factor (LF) amino acid sequence with sequences in the Swiss protein database revealed short regions of similarity with the consensus zinc-binding site, HEXXH, that is characteristic of metalloproteases . Several protease inhibitors, including bestatin and captopril, prevented intoxication of macrophages by lethal toxin . LF was fully inactivated by site-directed mutagenesis that substituted Ala for either of the residues (H-686 and H-690) implicated in zinc binding . Similarly, LF was inactivated by substitution of Cys for E-687, which is thought to be an essential part of the catalytic site . In contrast, replacement of E-720 and E-721 with Ala had no effect on LF activity . LF bound 65Zn both in solution and on protein blots . The 65Zn binding was reduced for several of the LF mutants . These data suggest that anthrax toxin LF is a zinc metallopeptidase, the catalytic function of which is responsible for the lethal activity observed in cultured cells and in animals. J Biol Chem, 1994 Aug 12, 269(32), 20607 - 12 Anthrax protective antigen forms oligomers during intoxication of mammalian cells; Milne JC et al.; The protective antigen component (PA) of anthrax toxin binds to receptors on target cells and conveys the toxin's edema factor (EF) and lethal factor (LF) components into the cytoplasm . PA (83 kDa) is processed by a cellular protease, yielding a 63-kDa fragment (PA63), which binds EF and/or LF . When exposed to acidic pH, PA63 inserts into membranes and forms ion-conductive channels . By electron microscopy, a significant fraction of purified PA63 was found to be in the form of a multi-subunit ring-shaped oligomer (outer diameter, 10.4 nm) . The rings are heptameric, as judged by inspection and by rotational power spectra . Purified PA63 showed a high M(r) band, apparently corresponding to the oligomer, on SDS-polyacrylamide gels, and oligomer of similar size was formed in cells in a time-dependent manner after addition of full-length PA . Inhibitors of internalization and endosome acidification blocked conversion of cell-associated PA to a high molecular weight species, and medium at pH 5.0 induced oligomer formation in the presence or absence of the inhibitors . These results correlate PA63 oligomerization with conditions required for translocation of EF and LF across lipid bilayers, implying that the PA63 oligomer may function in translocation. Infect Immun, 1994 Jul, 62(7), 2958 - 62 Protein synthesis is required for expression of anthrax lethal toxin cytotoxicity; Bhatnagar R et al.; Anthrax lethal toxin, which is composed of two proteins, i.e., protective antigen and lethal factor, is cytolytic to mouse peritoneal macrophages and the macrophage-like cell line J774A.1 . After exposure of cells to lethal toxin, inhibition of protein synthesis occurred only slightly before the onset of cytolysis . Thus, cell death did not appear to be due to inhibition of protein synthesis . However, prior treatment of J774A.1 cells with cycloheximide or puromycin, which inhibited protein synthesis, protected them completely against lethal toxin-induced cytolysis, which suggested that continuous protein synthesis is required for the expression of lethal toxin activity . Inhibition of protein synthesis had no appreciable effect on the binding of protective antigen to the cell surface receptor or on proteolytic cleavage of surface-bound protective antigen . Furthermore, inhibition of protein synthesis did not alter the uptake of toxin, which suggested that protein synthesis is required at a later stage of the intoxication process . The protection provided by inhibition of protein synthesis was effective, even up to 1 h after exposure to anthrax lethal toxin . The increased uptake of calcium observed in cells exposed to lethal toxin did not occur when they were protected by blocking protein synthesis . Identifying the protein(s) synthesized during the intoxication process may help to understand the mechanism of cell death produced by anthrax lethal toxin. Vaccine, 1994 Jun, 12(8), 727 - 30 Human live anthrax vaccine in the former USSR; Shlyakhov EN et al.; The history of the development and use of the Soviet live spore human anthrax vaccine is described . Results of mass field trials on this vaccine following administration by scarification, by subcutaneous injection route or by aerosol exposure are presented . For the immunological assessment of these vaccinations a skin test with an original product 'Anthraxin' was used. Rev Sci Tech, 1994 Jun, 13(2), 537 - 43 Anthrax in Switzerland during the early 19th century; Sackmann W; The progress of a devastating case of enzootic anthrax is investigated by means of documents found in the archives of a private farm . These reports initiated a detailed historical study of the anthrax situation in Switzerland at the time, notably in the north-west of the country. Rev Sci Tech, 1994 Jun, 13(2), 397 - 416 Epidemiology, surveillance and control of the principal infectious animal diseases in Africa; Bizimana N; The author presents detailed information on traditional methods, the majority of which remain in use, for the recognition, prevention and treatment of the principal infectious diseases prevalent on the African continent . The information provided relates to the observations and practices of peoples in three main regions, namely West, East and Southern Africa . Data are presented for ten diseases of major importance, with the widest range of practices being recorded for the control of foot and mouth disease, rinderpest and anthrax. Am J Public Health, 1994 May, 84(5), 737 - 41 Health status differentials in the People's Republic of China; Lawson JS et al.; OBJECTIVES . This study sought to demonstrate that health status varies markedly in different parts of China . METHODS . The main source of data was statistics compiled by the Chinese Ministry for Public Health for 1978 to 1990 regarding causes of death . However, because mortality statistics in China are based on localities that have the capacity to provide data, they are not entirely representative . The international classification of disease categories was also used, together with anatomically based disease descriptions . Rates were calculated using the 1982 and 1990 population censuses . RESULTS . Death rates differ markedly between urban and rural areas . Deaths due to infectious diseases, respiratory diseases, pregnancy and childbirth, and injuries and poisoning are much higher in rural areas; those due to pertussis, dysentery, typhoid, hepatitis, rabies, and anthrax are much more common in the apparently poorer provinces . Schistosomiasis remains a major problem in some provinces . Goiter and cretinism are still major diseases in many parts of China, especially those areas with iodine deficiency . CONCLUSIONS . Cause-of-death patterns in Chinese cities are similar to those of industrially developed countries such as Australia, Japan, and the United States . Such patterns in the poorer rural areas are much more typical of those of developing countries. J Infect, 1994 May, 28(3), 311 - 4 Temporal artery inflammation as a complication of anthrax; Doganay M et al.; A 41-year-old male patient was treated with penicillin for cutaneous anthrax affecting the region of the right eye . He was also given dexamethasone for 3 days to combat extensive oedema which was causing respiratory difficulty because of tracheal compression . After the oedema had resolved and the typical necrotic black eschar of anthrax had evolved, he developed acute inflammation of the right temporal artery . We believe this is the first report of this type of complication of anthrax. Berl Munch Tierarztl Wochenschr, 1994 May, 107(5), 145 - 9 {Preliminary trials of oral immunization of wild animals against anthrax}; Rengel J et al.; As a pilot trial for the vaccination of game in African game parks against anthrax, trials with guinea-pigs were undertaken to vaccinate the animals orally against anthrax . The vaccine has been prepared with the Goettingen Bioreactor Technology obtaining sporulation in suspension . Guinea-pigs vaccinated orally and subcutaneously with the vaccine resisted a challenge of 1000 spores with a pathogen field strain isolated from elephants in Zambia . With a challenge dosis of 2500 spores orally and subcutaneously immunized animals died . A technique has been developed to identify anthrax organisms excreted with the faeces by means of gas chromatography. Trans R Soc Trop Med Hyg, 1994 Mar-Apr, 88(2), 206 - 7 An outbreak of anthrax meningoencephalitis; George S et al.; We report a common-source outbreak of anthrax meningoencephalitis in Chittoor district in Andhra Pradesh, southern India, in October 1990 . The source of infection was the carcass of a sheep . Of 5 persons who skinned and cut up its meat for human consumption, 4 developed anthrax meningoencephalitis and one a malignant pustule . Another person who wrapped the meat in a cloth and carried it home on his head developed a malignant pustule on his forehead and also meningoencephalitis . All subjects with anthrax meningoencephalitis died, but the one with only a malignant pustule recovered . A large number of people who cooked or ate the cooked meat of the dead sheep remained well . The medical, public health and veterinary authorities were alerted and sheep, goats and cattle in the locality were immunized with anthrax vaccine . Although rules against consumption of meat of dead animals exist, their violation shows a lack of public awareness . Health education should be undertaken to correct this situation. Onderstepoort J Vet Res, 1994 Mar, 61(1), 71 - 83 Ecology and epidemiology of anthrax in the Etosha National Park, Namibia; Lindeque PM et al.; Analysis of mortality records has revealed distinct patterns in the incidence of anthrax in elephant and plains ungulates . The seasonal peak among the former is in November at the end of the dry season, while among the latter it occurs in March towards the end of the rainy season . Among elephants, there has been a notable spread of the disease to the west of the Park . Age and sex analyses indicate that, except for zebra, proportionally greater numbers of adult males die of anthrax among the species predominantly affected; however, zebra carcases are difficult to sex . In a study to identify possible environmental sources of infection, B . anthracis was detected in 3.3% of 92 water and 3.0% of 230 soil samples collected at different times of the year from 23 sites not associated with known cases of anthrax . Slight seasonal differences were noted with 5.7% positives occurring in the cold-dry period (May to August), 3.5% in the hot-dry season (September to December) and 1.4% in the hot-wet season (January to April) . Higher rates (26.0% of 73 samples) were found in water from waterholes in the western part of the Park at the time of an outbreak in elephants . The possible importance of scavenger faeces was confirmed with > 50% of vulture, jackal and hyaena faeces collected from the vicinity of confirmed anthrax carcases yielding B . anthracis, sometimes in substantial numbers, while no spores were found in faeces not associated with known anthrax carcases . Despite terminal B . anthracis levels of usually > 10(7) cfu/milliliters in the blood of animals dying of anthrax, spore levels in soil contaminated by such blood at sites of anthrax carcases ranged from undetectable to a few tens of thousands . The rapid loss of viability in soil and water of anthrax bacilli was monitored experimentally and the importance of soil type demonstrated . Survival and extent of sporulation of the bacilli in water were shown to be dependent on the rate at which the blood was diluted out . Other relevant parameters examined were background flora, pH and sunlight. Toxicology, 1994 Feb 28, 87(1-3), 29 - 41 The channel formed in planar lipid bilayers by the protective antigen component of anthrax toxin; Finkelstein A; Anthrax toxin consists of three proteins: edema factor (EF, 89 kDa), lethal factor (LF, 90 kDa), and protective antigen (PA, 83 kDa) . The former two gain access to the cytosol, where they exert their respective toxic effects on a cell, only in binary combination with PA . The proposed pathways of EF and LF transport consists of (i) PA attaching to a membrane receptor; (ii) its proteolytic cleavage into two fragments, of which the larger, 63 kDa piece (PA63) remains attached to the receptor; (iii) either EF or LF binding to PA63; (iv) the complex undergoing endocytosis, and EF or LF being translocated into the cytosol from an acidic vesicle compartment . In planar phospholipid bilayers, PA63 (but not whole PA) forms cation-selection channels; the channel-forming activity of PA63 dramatically increases when the pH of the solution to which it was added is lowered . Tetraalkylammonium ions block the PA63 channel by binding to a site within the channel lumen . Analysis of this blocking phenomenon reveals that these ions can pass through the channel from one side of the membrane to the other and that the diameter of the channel is about 12 A . The N-terminal 30 kDa end of EF, which contains the region of EF that binds to PA63, interacts with the PA63 channel in a voltage-dependent manner . The nature of the voltage-gating suggests that this binding fragment of EF can enter and block the channel and even pass through it, but further evidence will be required to establish this. Vet Parasitol, 1994 Feb, 51(3-4), 333 - 6 Observations on cattle ticks in Huila Province (Angola); Gomes AF et al.; In Huila Province of Angola, 3864 ticks were collected during a parasitological survey carried out in the rainy season from October 1990 to April 1991 . The samples were collected from cattle gathered for the annual vaccination campaign against contagious bovine pleuropneumonia, anthrax and blackleg in 18 veterinary stations of six municipalities . After tick classification, the following proportions of ticks were obtained: Rhipicephalus evertsi mimeticus (27.1%), Amblyomma pomposum (26.4%), Boophilus decoloratus (19.0%), Rhipicephalus zambesiensis (9.4%), Rhipicephalus duttoni (8.3%), Hyalomma marginatum rufipes (3.8%), Hyalomma truncatum (3.0%), Rhipicephalus punctatus (2.5%) and Ixodes cavipalpus, Rhipicephalus lunulatus, Rhipicephalus turanicus, Rhipicephalus evertsi evertsi, Rhipicephalus simus, each less than 1% . These ticks are well known in southern Africa as vectors of diseases caused by protozoa and rickettsiae (babesiosis, theileriosis, anaplasmosis and cowdriosis) . Control programmes against ticks and tick-borne diseases should be based on critical studies regarding costs/risks/benefits in relation to socio-economic and ecological frameworks. Bull World Health Organ, 1994, 72(1), 13 - 22 Anthrax control and research, with special reference to national programme development in Africa: memorandum from a WHO meeting. {Post anthrax vaccine delayed hypersensitivity . II--delayed hypersensitivity in humans vaccinated against anthrax} Shlyakhov E, Rubinstein E. l'Unite des Maladies Infectieuses, Centre medical Chaim Sheba, Universite de Tel Aviv, IsraelTo detect cell immunity characterized by delayed postvaccination hypersensitivity to anthrax in man and assess its dynamics, vaccination using unencapsulated live anthrax vaccine was performed in 668 healthy volunteers . Vaccination was performed either by scarification (n = 172), subcutaneous injection (n = 202), or low-dose (n = 202) or high-dose (n = 83) inhalation . The anthraxin intradermal tests were performed in each patient at various times during the year following vaccination (D7, D15, D90, D180, D365) . This study confirm that, regardless of the mode of administration, the vaccine induces cell-mediated immunity in man, as determined by positive anthraxine skin test . The incidence of positive tests decreases with time regardless of the mode of vaccination . After one year, the test remained positive in 34.8% of subjects vaccinated by subcutaneous injection, 37.5% vaccinated by low-dose inhalation, 34.2% vaccinated by high-dose inhalation, and 22.4% vaccinated by scarification . These findings are in agreement with those obtained in clinical epidemiological studies documenting the effectiveness of encapsulated live anthrax vaccine in man. Ter Arkh, 1994, 66(11), 35 - 7 {The differential diagnosis of anthrax in man}; Nisnevich EB et al.; The paper reports group cases of the disease running as anthrax . The disease was not identified etiologically . The authors hold that it is important to make differential diagnosis of anthrax with necrobacillosis, pasteurellosis and contact pustular viral dermatitis . Laboratory and clinical diagnostic techniques are specified. Rev Sci Tech, 1993 Dec, 12(4), 1093 - 107 Risk analysis and the importation of animals and animal products; MacDiarmid SC; Importation of animals or animal products cannot take place without some element of risk . Risk analysis is a blend of art and science and is a tool intended to provide decision-makers with an objective, repeatable and defensible assessment of the risks posed by a particular import proposal . Risk analysis comprises risk identification, risk assessment, risk management and risk communication . Examples are presented of risk analysis involving anthrax in green hides, slow virus diseases and sheep embryos, and Office International des Epizooties List A diseases and embryos . The author proposes that, by sharing methodologies, quarantine services should be able to harmonize approaches to the problem of risk analysis. Br J Dermatol, 1993 Nov, 129(5), 625 - 7 Cowpox can mimic anthrax; Lewis-Jones MS et al.; We report a patient suffering from cowpox infection, in whom the clinical features mimicked those of anthrax . The infection may have been acquired as a result of contact with a rodent. Vet Microbiol, 1993 Nov, 37(3-4), 343 - 51 Potency testing of bacterial vaccines for human use; Habig WH; The potency tests for bacterial vaccines are quite diverse . For some products (pertussis, cholera, anthrax, typhoid and BCG vaccines) these are specified as Additional Standards in the Code of Federal Regulations . For other products (tetanus and diphtheria toxoids, plague vaccine) the testing is done according to so-called Minimum Requirements, which have less regulatory authority than Additional Standards . Still other products (e.g., polysaccharide conjugate vaccines, acellular pertussis vaccine, live oral typhoid) are tested according to individualized criteria that are contained in their specific Product License Applications . For some products there is inadequate knowledge of the pathogenic mechanisms and/or protective factors to design valid in vitro potency tests . In these cases, animal testing with subsequent serologic evaluation or challenge testing is often necessary . Examples would include vaccines such as cholera and plague vaccines . The FDA supports the elimination of animal testing when suitable alternatives are available . Thus, many of the potency tests, especially for newer products, rely on in vitro characterization . For example, the immunogenicity of conventional polysaccharide vaccines is largely proportional to their molecular weight . Potency testing therefore relies heavily on physical characterization in terms of composition, molecular weight, and quantity. Mol Microbiol, 1993 Nov, 10(3), 647 - 53 pH-dependent permeabilization of the plasma membrane of mammalian cells by anthrax protective antigen; Milne JC et al.; Protective antigen (PA) of anthrax toxin forms ion-conductive channels in planar lipid bilayers and liposomes under acidic pH conditions . We show here that PA has a similar permeabilizing action on the plasma membranes of CHO-K1 and three other mammalian cell lines (J774A.1, RAW264.7 and Vero) . Changes in membrane permeability were evaluated by measuring the efflux of the K+ analogue, 86Rb+, from prelabelled cells, and the influx of 22Na+ . The permeabilizing activity of PA was limited to a proteolytically activated form (PAN) and was dependent on acidic pH for membrane insertion (optimal at pH 5.0), but not for sustained ion flux . The flux was reduced in the presence of several known channel blockers: tetrabutyl-, tetrapentyl-, and tetrahexylammonium bromides . PAN facilitated the membrane translocation of anthrax edema factor under the same conditions that induced changes in membrane permeability to ions . These results indicate that PAN permeabilizes cellular membranes under conditions that are believed to prevail in the endosomal compartment of toxin-sensitive cells; and they provide a basis for more detailed studies of the relationship between channel formation and translocation of toxin effector moieties in vivo. Rev Sci Tech, 1993 Mar, 12(1), 137 - 46 Surveillance and control of anthrax and rabies in wild herbivores and carnivores in Namibia; Berry HH; Anthrax has been studied intensively in Etosha National Park, Namibia since 1966; in addition, since 1975, mortality due to rabies and all other causes has been recorded, totalling 6,190 deaths . Standard diagnostic procedures demonstrated that at least 811 deaths (13%) were due to anthrax and 115 deaths (2%) were caused by rabies . Of the total number of deaths due to anthrax, 97% occurred in zebra (Equus burchelli), elephant (Loxodonta africana), wildebeest (Connochaetes taurinus) and springbok (Antidorcas marsupialis) while 96% of rabies deaths occurred in kudu (Tragelaphus strepsiceros), jackal (Canis mesomelas), bat-eared fox (Otocyon megalotis) and lion (Panthera leo) . Anthrax deaths were highest in the rainy season for zebra, wildebeest and springbok, while elephant mortality peaked during dry seasons . No statistical relationship existed between seasonal rainfall and overall incidence of either anthrax or rabies . Control of anthrax is limited to prophylactic inoculation when rare or endangered species are threatened . Incineration of anthrax carcasses and chemical disinfection of drinking water are not feasible at Etosha . Rabies control consists of the destruction of rabid animals and incineration of their carcasses when possible. Zh Mikrobiol Epidemiol Immunobiol, 1993 Mar-Apr, (2), 89 - 92 {The nonspecific resistance indices of different species of laboratory animals immunized with STI anthrax vaccine}; Kogotova OI et al.; A complex comparative study of the characteristics of nonspecific resistance in different species of laboratory animals immunized with vaccine STI against anthrax has revealed the existence of marked interspecific differences between noninbred white mice, guinea pigs, rabbits and noninbred white rats in such characteristics as phagocytic activity, oxygen-dependent function of polymorphonuclear blood leukocytes, serum beta-lysin and lysozyme. J Biol Chem, 1993 Feb 15, 268(5), 3334 - 41 Residues 1-254 of anthrax toxin lethal factor are sufficient to cause cellular uptake of fused polypeptides; Arora N et al.; Anthrax lethal toxin is a complex of protective antigen (PA, 735 amino acids) and lethal factor (LF, 776 amino acids) that lyses certain eukaryotic cells . LF interacts with PA to gain access to the cytosol to assert its toxicity . The internalization of LF requires that PA bind to a specific membrane receptor and be cleaved by a cell-surface protease (probably furin), so as to expose a site on PA to which LF binds with high affinity . To localize LF functional domains, amino, carboxyl, and internal deletions of LF were made . Toxicity was eliminated by deletion of 40 and 47 residues from the amino and carboxyl termini, respectively . Similarly, deleting the first of the four imperfect repeats of 19 amino acids located at residues 308-383 made LF non-toxic, showing that this region is also essential for activity . To identify the minimum region of LF which is required for binding to PA, varying amino-terminal portions of LF were fused to the ADP-ribosylation domain of Pseudomonas exotoxin A . Fusion proteins containing residues 1-254 of LF were toxic when administered with PA, while those having only residues 1-198 of LF were inactive, showing that the PA-binding domain of LF lies within residues 1-254. Med Trop (Mars), 1993 Jan-Mar, 53(1), 55 - 9 {Retrospective diagnosis of anthrax by intradermal reaction}; Shlyakhov E et al.; The authors report a detailed study of utilization of a skin test with anthraxin to perform a retrospective diagnosis of anthrax in humans who suffered from anthrax 45 days to 31 years after recovery . For a total of 884 persons studied, 762 showed a positive skin test (86.2%) . This index was 92.8% for persons tested 45 days to 3 years after convalescence, 82.8% 4 to 15 years after convalescence and 72.7% 16-31 years after recovery . The site of the primary carbuncle on the skin of the fingers, hands, face and neck gave an index of positivity (88.2%) statistically greater (p = 99%) than by localization of the carbuncle on arms, forearms, trunk and legs (77.3%). HNO, 1993 Jan, 41(1), 37 - 40 {Pharyngeal tuberculosis as a differential diagnosis to carcinoma}; Meuthen I et al.; The differential diagnosis of pharyngeal tumors includes malignomas as well as chronic inflammatory processes . Squamous cell carcinoma is the most prevalent malignoma of the pharynx, representing about 90% of all malignomas of the head and neck . Malignant lymphomas, lymphoepithelial tumors (Schmincke's tumor) and anaplastic carcinomas are less prevalent . Amelanotic melanoma, rhabdomyosarcoma and extramedullary plasmocytoma are rare malignomas of the pharynx . Infectious diseases may also be a cause of pharyngeal tumors which have been reported to be associated with mycobacterial infections, syphilis, leproma, malleus and anthrax . Sarcoidosis and Wegener's granulomatosis are chronic inflammatory diseases of unknown etiology . We report a case of a 65-year-old female with an 11-year history of a slowly progressing tumor of the nasopharynx who had been admitted to hospital with suspicion of a malignoma. J Wildl Dis, 1993 Jan, 29(1), 130 - 5 An enzyme-linked immunosorbent assay for detecting anthrax antibody in white-tailed deer (Odocoileus virginianus): evaluation of anthrax vaccination and sera from free-ranging deer; Peterson MJ et al.; An enzyme-linked immunosorbent assay for anthrax antibody in white-tailed deer (Odocoileus virginianus) was developed and used to evaluate a vaccination study and compare sera from hunter-killed deer in anthrax endemic and non-endemic areas . Deer subcutaneously vaccinated with anthrax avirulent spore vaccine developed specific antibody to protective antigen (PA) which was significantly higher than the non-vaccinated controls at 30, 60, 90, and 240 days post-vaccination . There was no difference between the levels of antibody to PA between deer in anthrax endemic and non-endemic areas. Infect Immun, 1993 Jan, 61(1), 245 - 52 Characterization of macrophage sensitivity and resistance to anthrax lethal toxin; Friedlander AM et al.; Anthrax lethal toxin, which consists of two proteins, protective antigen and lethal factor, is cytolytic for macrophages . Macrophages from different mouse strains were found to vary in their sensitivities to toxin . C3H mouse macrophages lysed by lethal factor concentrations of 0.001 micrograms/ml were 100,000 times more sensitive than those from resistant A/J mice . We analyzed various stages of the intoxication process to determine the basis for this resistance . Direct binding studies with radioiodinated protective antigen revealed that the affinity (Kd, approximately 0.5 nM) and number of receptors per cell (25,000 to 33,000) were the same in sensitive and resistant cells . Proteolytic activation of protective antigen by a cell surface protease and subsequent binding of lethal factor were also the same in both sensitive and resistant macrophages . Resistant A/J macrophages were not cross-resistant to other toxins and a virus which, like lethal toxin, require vesicular acidification for activity, implying that resistance is not due to a defect in vesicular acidification . When introduced into the cytosol by osmotic lysis of pinosomes, lethal factor in the absence of protective antigen was cytolytic for the sensitive macrophages while resistant cells were unaffected . Thus, lethal factor by itself possesses the toxic activity of lethal toxin . These results suggest that macrophage resistance is due to a defect at a stage occurring after toxin internalization . A/J macrophages may lack the putative lethal factor target in the cytosol or be defective in the further processing or activation of lethal factor in the cytosol or in endocytic vesicles. Cent Afr J Med, 1993 Jan, 39(1), 10 - 20 The current epidemiology and control of trypanosomiasis and other zoonoses in Tanzania; Kilonzo BS et al.; The epidemiology and