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Human neutrophils produce small amounts of O2- when stimulated with the chemotactic peptide F-Met-Leu-Phe; preincubating neutrophils with low concentrations of lipopolysaccharide (LPS) markedly increases this response, an effect referred to as priming. Neutrophil suspensions without mononuclear cells and platelets were insusceptible to priming by 10 ng of LPS; susceptibility was restored by reintroducing platelets, approximately five platelets per neutrophil. Incubation of platelets with 10 ng of LPS/mL released a soluble factor that produced graded priming responses of at least fivefold in neutrophils. The priming factor had the properties of a labile protein and did not resemble previously described mediators derived from platelets. Anthrax toxin, which inhibits priming of neutrophils by LPS, inhibited priming by the platelet factor but not release of the factor from platelets. Thus, the platelet factor mediates a portion of the overall priming effect of LPS and thereby modulates the level of O2- generation by neutrophils.

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We studied the pretreatment of human polymorphonuclear neutrophils (PMN) with purified preparations of the anthrax toxin components--protective antigen (PA), edema factor (EF), and lethal factor (LF)--and their effects on release of superoxide anion (O-2) after stimulation with the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). PMN isolated in the absence of lipopolysaccharide (LPS) (less than 0.1 ng/ml) released only small amounts of O-2 after FMLP stimulation; pretreatment with anthrax toxin had little effect. The release of O-2 was increased fivefold by prior treatment with 3 ng/ml LPS for 1 h at 37 degrees C, an effect referred to as priming. PMN were primed to an equivalent extent by treatment with 100 ng/ml N-acetyl-muramyl-L-alanyl-D-isoglutamine (muramyl dipeptide [MDP]). Pretreatment of PMN with anthrax toxin components PA plus EF or PA plus LF inhibited priming by LPS or MDP, as shown by the reduction in the release of O-2 up to 90% relative to controls not treated with toxin; single toxin components were inactive. The inhibition was markedly reduced when priming with LPS or MDP was carried out before exposure to toxin. O-2 release after stimulation by phorbol myristate acetate was not increased by priming, and pretreatment with toxin did not inhibit O-2 release after this stimulus. Evidently, anthrax toxin inhibits the priming that is normally induced in PMN by bacterial products and is necessary for the full expression of antimicrobial effects.

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Effects of the three-component toxin of Bacillus anthracis on chemotaxis of human polymorphonuclear leukocytes (PMN) were investigated in an effort to determine the basis of the reported antiphagocytic effect of the toxin. The three toxin components, edema factor (EF), protective antigen (PA), and lethal factor (LF), were tested alone and in various combinations for their effect on PMN chemotaxis under agarose to formyl peptides and zymosan-activated serum. No component was active alone; combinations of EF + PA, LF + PA, and EF + LF + PA markedly stimulated chemotaxis (directed migration), but had little or no effect on unstimulated random migration. The toxin components were not themselves chemoattractants. EF in combination with PA had previously been identified as an adenylate cyclase in Chinese hamster ovary (CHO) cells. We found that EF + PA produced detectable cyclic adenosine 3'-5'monophosphate (cAMP) in PMN, but the level of cAMP was less than 1% of that produced in CHO cells by EF + PA, and in PMN by other bacterial adenylate cyclases. LF + PA (which stimulated chemotaxis to an equivalent extent) had no effect on cAMP levels. Thus, the enhancement of chemotaxis by anthrax toxin (at least by LF + PA) does not seem to be related to adenylate cyclase activity.

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In an effort to prevent cytomegalovirus infection among seronegative patients having marrow transplants, a globulin with high antibody levels against cytomegalovirus was given before and for 11 weeks after transplantation in a randomized trial. Among 36 patients who received no prophylactic granulocyte transfusions, globulin recipients had significantly fewer infections than controls (2 of 17 versus 8 of 19, p = 0.05 by Fisher's exact test and p = 0.03 by Mantel-Cox test). Conversely, infection rates were high and unchanged by globulin use among patients who received granulocytes from seropositive donors (7 of 8 recipients versus 6 of 7 controls). The lack of effect of the globulin among patients receiving transfusions of granulocytes from seropositive donors may suggest that the dose of antibody was insufficient or that antibody is ineffective against virus transmitted in granulocytes. We conclude that cytomegalovirus infection can be prevented by immunoprophylaxis in seronegative patients having marrow transplants who are not given granulocyte transfusions.

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Varicella-zoster immune globulin (VZIG), an immunoglobulin prepared from normal donor plasma selected for high titer of antibody to varicella-zoster virus (VZV), and zoster immune globulin (ZIG), prepared from the plasma of donors convalescing from herpes zoster, were compared in a double-blind, randomized clinical trial to determine their relative efficacy in protecting immunosuppressed children from severe varicella. VZV infection occurred in 49 (60.4%) of 81 recipients of VZIG and in 57 (68.6%) of 83 recipients of ZIG. These rates and the clinical severity of varicella were not significantly different; however, the subclinical infection rate was significantly higher in ZIG recipients (31.3% vs. 16.0%). This difference was accounted for by a subgroup of patients receiving immunosuppressive therapy for nonneoplastic diseases. Doubling the dose of VZIG administered reduced the rate of subclinical infection. These data indicate that VZIG can be used to protect immunosuppressed children from severe chicken pox.

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Single-dose immunization against tetanus was studied in 511 previously non-immunized residents of rural villages in Upper Volta. Males and females were equally represented and a wide age range was covered. A single dose of adsorbed tetanus toxoid containing 17.5 Lf units of toxoid and 3.86 mg of aluminium phosphate per 0.5 ml dose was used. Blood samples were taken 7 days, 2 months, and 12 months after immunization, and serum antitoxin titres were determined by neutralization titrations in mice. Adverse reactions were negligible. Only 2 participants gave evidence of prior immunization by developing detectable antitoxin titres after 7 days; they were eliminated from the study. After 12 months, 59% of the participants had antitoxin titres of >/=0.01 IU/ml, a titre usually considered protective. The mean titre and the proportion of those protected decreased substantially with increasing age; overall, females gave somewhat greater serological responses than males. Mean titre increased by 25% between 2 months and 1 year after immunization; the increase was greater in females than in males. In children under 6 years of age, 100% of females and 82% of males had protective titres after 1 year.

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Outdated blood from blood banks in Massachusetts was screened for complement-fixing antibody to varicella-zoster virus (VZV). Approximately 15% of the plasma units had a titer greater than or equal to 16, and one-half of these had a titer of greater than or equal to 32. Plasma units with titers of CF antibody to VZV of greater than or equal to 16 were pooled. This pool had a CF antibody titer of 32 and a titer of fluorescent antibody of 64. Varicella-zoster immune globulin was prepared from the selected plasma pool by alcohol fractionation and analyzed for VZV-specific antibody. Varicella-zoster immune globulin had a fluorescent antibody titer of 2,048 and a neutralizing antibody titer of 1,024. This antibody concentration is equivalent to that in preparations of zoster immune globulin made from plasma of donors recovering from recent VZV infection.

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Accumulation of protective antigen was essentially independent of culture volume, provided the organisms were kept in suspension by stirring. Agitation by orbited shaking was unsatisfactory.

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A method was developed for identification of Bacillus anthracis based on elaboration of protective antigen by individual colonies and its detection by double-diffusion precipitation in agar plates.

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Retention of protective antigen on columns of diethylaminoethyl cellulose was inhibited by the 0.01 m phosphate salts in the standard growth medium. Reduction in the concentration of phosphate to 0.001 m allowed satisfactory retention of antigen on diethylaminoethyl cellulose but decreased significantly the elaboration of antigen during growth of the cultures. Growth remained normal at phosphate concentrations as low as 0.0001 m. Inhibition of antigen elaboration in media containing reduced concentrations of phosphate was overcome by addition of 0.006% (w/v) charcoal. It is suggested that antigen elaboration, like encapsulation of virulent strains, is stimulated in the presence of an adsorbent. However, a nonencapsulated strain derived from an F mutant exhibited the same requirement for an adsorbent as nonencapsulated strains derived from the parent strain.

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The ammonium sulfate coprecipitation technique of Farr was applied in a study of the purified enterotoxins of Staphylococcus aureus. Ammonium sulfate coprecipitation of iodine-131-labeled enterotoxins A, B, and C, with the use of a 1.6 m concentration of (NH(4))(2)SO(4), revealed differences in the antigen-binding capacity of normal and immune rabbit sera for the enterotoxins. The coprecipitation technique provided a quantitative test for detecting antibody to enterotoxin that was more sensitive than agar-gel diffusion methods. Antigen-binding tests suggested the presence of similar antigenic determinant groups in all three toxins. Measurable antigenbinding capacities for enterotoxins A, B, and C were detected in sera of normal human subjects and became elevated in several subjects accidently exposed to enterotoxin.

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