Cellular and cytokine responses in the circulation and tissue reactions in the lung of rhesus monkeys (Macaca mulatta) pretreated with cyclosporin A and challenged with staphylococcal enterotoxin B.

Cyclosporin A (CsA), an inhibitor of T cell cytokine production, protects mice against staphylococcal enterotoxin B (SEB) intoxication. To determine whether CsA treatment would work in a species closer to humans. 4 rhesus monkeys were given 50 mg/kg CsA followed by an intratracheal challenge with approximately 6 LD50 of SEB. The CsA was not protective: one of the monkeys died and the other three had to be euthanised when they became moribund. All monkeys made IL-2, TNF, and IFN-gamma in response to SEB. In addition, there was about a 10-fold increase in ACTH levels 2 hr after SEB challenge. CsA significantly suppressed in vitro proliferation of lymphocytes from treated monkeys. Both CsA-treated monkeys and monkeys that had been challenged in a previous experiment with a lethal dose of SEB but had received no cyclosporin had pathologic changes in several organs. The most prominent changes were marked edema and leukocytic infiltration of the bronchial and bronchiolar mucosa. The CsA treatment appeared to reduce the intensity of lung inflammation, but this effect was not sufficient to protect the monkeys. The results suggest that CsA alone may not be an effective therapeutic agent for humans suffering from SEB intoxication or gram-positive septic shock.

Detection of interleukin-6 and interleukin-2 in serum of rhesus monkeys exposed to a nonlethal dose of staphylococcal enterotoxin B.

The immune response to a nonlethal dose of aerosolized staphylococcal enterotoxin B (SEB) was studied in nonhuman primates to define the potential human host response to a nonlethal exposure of SEB on the battlefield. Serum levels of the cytokines interleukin 2 (IL-2) and interleukin 6 (IL-6) increased significantly (p < 0.01) in six juvenile rhesus monkeys 4 hours after receiving a nonlethal, inhaled dose of SEB. The mean (+/-SD) peak serum levels of IL-2 and IL-6 were 63 +/- 39 units/ml and 514 +/- 234 pg/ml, respectively, post-SEB treatment. Tumor necrosis factor, known to be associated with SEB-mediated lethal toxic shock, was undetectable in all samples. gamma-Interferon concentrations were also elevated, but not significantly [p < 0.089]. Hence, elevated levels of IL-2 and IL-6 might be used as a serological marker for a nonlethal, incapacitating exposure to SEB.

Medical knowledge overload: a disturbing trend for physicians.

The article examines responses of more than 500 physicians questioned about the time available for reading professional journals over the past 5 years as well as their utilization of information retrieval systems. It also examines physicians' perceptions of professional medical sales personnel as medical information sources.

Immediate responses of leukocytes, cytokines and glucocorticoid hormones in the blood circulation of monkeys following challenge with aerosolized staphylococcal enterotoxin B.

The immediate responses to aerosolized staphylococcal enterotoxin B (SEB) in respiratory toxic shock were studied in the circulation of rhesus monkeys with low antibody levels following immunization with SEB toxoid-containing microspheres. Both the surviving and dying monkeys had toxic shock syndrome 4-48 h after SEB challenge and all showed three distinctive patterns of immediate responses. The first pattern, characterized by the responses of all T cells, HLA-DRlo cells, monocytes, IL-2R+ cells, IFN-gamma, and augmented lymphocyte mitotic responses to lipopolysaccharide (LPS) and SEB in culture, was a rapid increase at 20 min followed by a quick decrease at 90 min to approximately the original levels. The second pattern, which included responses of HLA-DRhi cells, NK cells, adrenocorticotropic hormone (ACTH) and cortisol, was characterized by a moderate decrease at 20 min and a further decrease at 90 min. The third pattern, the inverse of the second pattern, including responses of polymorphonuclear leukocytes (PMN), concanavalin A (Con A) mitogenesis, IL-6 and IL-2, was a moderate increase at 20 min and a further increase at 90 min. Between the surviving and dying monkeys, the responses of T cells, HLA-DRhi cells, PMN and cortisol did not differ significantly, suggesting that they are the basic causes that initiated toxic shock. However, significant differences were seen in the responses of HLA-DRlo cells, monocytes, IL-2R+ cells and lymphocyte mitogenesis in culture at 20 min, and of Con A mitogenesis, NK cells, IL-2, IL-6 and ACTH at 90 min. These different responses are apparently the exacerbating causes of death of the monkeys. All together, the immediate responses seem to be caused by the combined effects of SEB superantigenicity, activation of NK cells and non-lymphoid cells, and depression of the neuroimmune defense system.

Immunogenicity and efficacy against lethal aerosol staphylococcal enterotoxin B challenge in monkeys by intramuscular and respiratory delivery of proteosome-toxoid vaccines.

Staphylococcal enterotoxin B (SEB), a primary cause of food poisoning, is also a superantigen that can cause toxic shock after traumatic or surgical staphylococcal wound [correction of would] infections or viral influenza-associated staphylococcal superinfections or when aerosolized for use as a potential biologic warfare threat agent. Intranasal or intramuscular (i.m.) immunization with formalinized SEB toxoid formulated with meningococcal outer membrane protein proteosomes has previously been shown to be immunogenic and protective against lethal respiratory or parenteral SEB challenge in murine models of SEB intoxication. Here, it is demonstrated that immunization of nonhuman primates with the proteosome-SEB toxoid vaccine is safe, immunogenic, and protective against lethal aerosol challenge with 15 50% lethal doses of SEB. Monkeys (10 per group) were primed i.m. and given booster injections by either the i.m. or intratracheal route without adverse side effects. Anamnestic anti-SEB serum immunoglobulin G (IgG) responses were elicited in all monkeys, but strong IgA responses in sera and bronchial secretions were elicited both pre- and post-SEB challenge only in monkeys given booster injections intratracheally. The proteosome-SEB toxoid vaccine was efficacious by both routes in protecting 100% of monkeys against severe symptomatology and death from aerosolized-SEB intoxication. These data confirm the safety, immunogenicity, and efficacy in monkeys of parenteral and respiratory vaccination with the proteosome-SEB toxoid, thereby supporting clinical trials of this vaccine in humans. The safety and enhancement of both bronchial and systemic IgA and IgG responses by the proteosome vaccine delivered by a respiratory route are also encouraging for the development of mucosally delivered proteosome vaccines to protect against SEB and other toxic or infectious respiratory pathogens.

Potentiation of inhaled staphylococcal enterotoxin B-induced toxicity by lipopolysaccharide in mice.

Nonhuman primates are the established model for evaluating toxic responses to staphylococcal enterotoxins (SEs), as they react similarly to humans. Rodents are generally considered unresponsive to SEs. Binding affinities and T-cell reactivity suggest that SE binds more efficiently to primate major histocompatability complex class II receptors than to mouse receptors. We investigated the potentiation of staphylococcal enterotoxin B (SEB) inhalation toxicity by lipopolysaccharide (LPS) in BALB/c mice. Lethality occurred only when SEB was potentiated by LPS. Neither SEB nor LPS produced lethal effects alone. Temporal responses of interleukin 1 alpha, tumor necrosis factor alpha, interleukin 2, and interferon-gamma evoked by inhaled SEB were enhanced by LPS. By 24 hr after intoxication, serum cytokines decreased to baseline levels, and consistent pulmonary perivascular leukocytic infiltrates were evident histologically. Histologic lesions induced by inhalation exposure to SEB by mice, with or without potentiation by LPS, were similar to those in the rhesus monkey. Predominant pulmonary lesions included severe, diffuse interstitial and alveolar pulmonary edema, leukocytic infiltrates, mild perivascular edema, and alveolar fibrin deposition. Although the mechanism of aerosolized SEB-induced toxicity has not been completely resolved, similarities in histologic lesions, cytokine responses, and acute dose-response suggest the LPS-potentiated mouse model may be a credible alternative to the nonhuman primate model.

Intranasal and intramuscular proteosome-staphylococcal enterotoxin B (SEB) toxoid vaccines: immunogenicity and efficacy against lethal SEB intoxication in mice.

Intranasal or intramuscular (i.m.) immunization of mice and i.m. immunization of rabbits with formalinized staphylococcal enterotoxin B (SEB) toxoid in saline elicited higher anti-SEB serum immunoglobulin G (IgG) titers when the toxoid was formulated with proteosomes. In addition, intranasal immunization of mice with this proteosome-toxoid vaccine elicited high levels of anti-SEB IgA in lung and intestinal secretions, whereas the toxoid without proteosomes did not. Two i.m. immunizations with proteosome-toxoid plus alum also induced higher murine serum responses than alum-adjuvanted toxoid without proteosomes. Furthermore, proteosome-toxoid delivered intranasally in saline or i.m. with either saline or alum afforded significant protection against lethal SEB challenge in two D-galactosamine-sensitized murine models of SEB intoxication, i.e., the previously described i.m. challenge model and a new respiratory challenge model of mucosal SEB exposure. Efficacy correlated with the induction of high serum levels of anti-SEB IgG. In contrast, intranasal or i.m. immunization with toxoid in saline without proteosomes was not significantly protective in either challenge model. Proteosome-toxoid plus alum given i.m. also elicited more significant protection against respiratory challenge than the alum-adjuvanted toxoid alone. The capacity of proteosomes to enhance both i.m. and intranasal immunogenicity and efficacy of SEB toxoid indicates that testing such proteosome-SEB toxoid vaccines in the nonhuman primate aerosol challenge model of SEB intoxication prior to immunogenicity trials in humans is warranted. These data expand the applicability of the proteosome mucosal vaccine delivery system to protein toxoids and suggest that respiratory delivery of proteosome vaccines may be practical for enhancement of both mucosal and systemic immunity against toxic or infectious diseases.

Protective effects of niacinamide in staphylococcal enterotoxin-B-induced toxicity.

Staphylococcal enterotoxins (SE) interact with major histocompatibility complex (MHC) class II cell-surface receptors, eliciting signal transduction in antigen-presenting cells (APC). Subsequent toxin-class II complex interaction with specific T-cell receptors induces T-cell activation. We investigated the effect of niacinamide and interleukin (IL)-10 on SEB-induced responses. In a macrophage cell line, niacinamide (ED50--2mM) and IL-10 (ED50--7U/ml) inhibited interferon (IFN)-gamma-induced MHC class II expression in a dose-dependent manner. Also, niacinamide was a potent inhibitor of T-cell proliferation induced by SEB (ED50-- 1 mM) while IL-10 has minimal effects. In mice, the temporal responses of IL-1alpha, tumor necrosis factor (TNF)-alpha, IL-2, and IFN-gamma evoked by SEB were synergistically potentiated by lipopolysaccharide (LPS). Lethality occurred only when SEB was potentiated by LPS. Niacinamide or IL-10 improved survival of mice after lethal SEB challenge. Niacinamide reduced cytokine serum levels, although the pattern differed from that of IL-10. Niacinamide primarily reduced IL-2 and IFN-gamma, while IL-10 predominantly reduced IL-1alpha and TNF-alpha. The immunomodulatory effects of niacinamide observed on SEB-induced activation of APC and T-cells in vitro and in the LPS potentiated murine model for SEB-induced toxicity suggest it may have therapeutic value.

Divergence of human and nonhuman primate lymphocyte responses to bacterial superantigens.

We compared T cell responses of human, rhesus monkey (Macaca mulatta), and chimpanzee (Pan troglodytes) to four bacterial superantigens. When lymphocytes were cultured in media supplemented with species-specific sera, chimpanzee T cells were stimulated by lower doses of staphylococcal enterotoxin (SE) A and toxic shock syndrome toxin 1 (TSST1) than were human T cells, while chimpanzee responses to SEB and SEC1 were nearly equivalent to the human response. Interestingly, rhesus lymphocytes responded to 10,000 times lower amounts of SEA, SEB, and SEC1 and to 100 times lower concentrations of TSST1 than human cells. The greater sensitivity of rhesus T cells to these toxins was not a result of differences in class II binding affinities and was only partly attributable to the presence of anti-SE and TSST1 antibodies in human serum. These results suggest that rhesus T lymphocytes are more sensitive toward these bacterial superantigens than human T cells.

Humoral immunity to aerosolized staphylococcal enterotoxin B (SEB), a superantigen, in monkeys vaccinated with SEB toxoid-containing microspheres.

Staphylococcal enterotoxin B (SEB) toxoid-containing microspheres were tested for efficacy in rhesus monkeys as a vaccine candidate for respiratory SEB toxicosis and toxic shock. Forty monkeys were randomly separated into 10 groups of four monkeys each: 9 groups were vaccinated with the microspheres via combinations of mucosal and nonmucosal routes, and 1 group served as nonvaccinated controls. Both vaccinated and nonvaccinated monkeys were then challenged with a high lethal dose of SEB aerosol. Monkeys primed with an intramuscular dose of the microspheres followed by an intratracheal booster all survived the SEB challenge. Overall, monkeys with an intratracheal booster generally had the highest antibody levels, which is consistent with their high survival rate and lower rate of illness. Protective immunity was correlated with antibody levels in both the circulation and the respiratory tract. The protection was not due to the depletion or anergy of SEB-reactive T cells, since SEB-induced proliferation in cultures of circulating lymphocytes was not significantly reduced after the microsphere vaccination. It is evident that the nonsurvivors did not die of systemic anaphylaxis or hypersensitivity because the monkeys did not die immediately after SEB challenge and there were no significant differences in histamine levels between the vaccinated and control monkeys before and after SEB challenge. The antibodies seemed to neutralize the SEB that got into the airway and the circulation.

Aerosolized staphylococcal enterotoxin B-induced pulmonary lesions in rhesus monkeys (Macaca mulatta).

The pathology of aerosolized staphylococcal enterotoxin B (SEB) was studied in the nonhuman primate. Six juvenile rhesus monkeys that received multiple lethal inhaled doses of SEB developed diarrhea and vomiting within 24 hr followed by depression, dyspnea, and shock. Three of 6 animals died by 52 hr. The most striking gross lesion in all 6 monkeys was diffuse severe pulmonary edema. Histologically, edema fluid was present within the peribronchiolar, peribronchial, and perivascular interstitium, alveolar septa, and alveoli. The adventitia of pulmonary vessels was infiltrated by lymphocytes, macrophages, and fewer neutrophils. Numerous large lymphocytes with occasional mitotic figures were within pulmonary vessels, often occluding alveolar capillaries. These cells were strongly immunoreactive with monoclonal antibodies against CD3, establishing them as T cells. Ultrastructurally, endothelial cell junctions were intact, and endothelial cells and type I pneumocytes contained numerous pinocytotic vesicles. Alveolar septal interstitial spaces were expanded by edema. The mechanism of these SEB-induced pulmonary lesions was not determined. We hypothesize that cytokine production by activated T cells may have caused vascular permeability changes leading to widespread pulmonary edema and shock.

Identification of staphylococcal enterotoxin B sequences important for induction of lymphocyte proliferation by using synthetic peptide fragments of the toxin.

A series of 13 synthetic peptides, approximately 30 amino acids each, which spanned the entire sequence of staphylococcal enterotoxin B (SEB) were tested to evaluate their effects on T-cell proliferation in a culture system containing elutriated human peripheral blood lymphocytes incubated with a specific ratio of mononuclear cells. Four peptide regions were found to inhibit SEB-induced proliferation; they included sequences 1 to 30 (previously thought to be involved in major histocompatibility complex class II binding), 61 to 92 (sequences which relate to the T-cell receptor site), 93 to 112 (a linear sequence corresponding to the cysteine loop), and 130 to 160 (containing a highly conserved sequence, KKKVTAQEL). Antisera raised to this last peptide were capable of neutralizing SEB-induced proliferation. Antisera raised against the peptides which overlapped this sequence also were somewhat inhibitory. Neutralizing antisera were not produced from any other peptide sequence tested. To determine if any of these effects were nonspecific with regard to SEB-induced proliferation, the peptides were tested for inhibition of phorbol dibutyryl ester-induced proliferation, and only the sequence 93 to 112 (corresponding to the cysteinyl loop region) was consistently inhibitory (40%). Of the regions which displayed inhibition of SEB-induced proliferation, the peptide 130 to 160 inhibited binding of 125I-SEB to lymphocytes. These data suggest that the residues containing and surrounding the sequence KKKVTAQEL may be critical in the SEB-induced proliferation and may be useful for developing neutralizing antisera to SEB.

Immunity and responses of circulating leukocytes and lymphocytes in monkeys to aerosolized staphylococcal enterotoxin B.

Rhesus monkeys immunized intramuscularly or orally with staphylococcal enterotoxin B (SEB) toxoid or SEB toxoid incorporated in microspheres made of poly(DL-lactide-co-glycolide) were challenged with a lethal dose of aerosolized SEB to study their immunity and cellular responses in the circulation. It was found that circulating antibodies play a critical role in preventing SEB from triggering toxicosis. Monkeys with high levels of antibodies survived, while those with low levels underwent 2 to 3 days of toxicosis and died. Intramuscular immunization induced high levels and oral immunization induced low levels of antibodies. The circulating antibodies in surviving monkeys decreased dramatically within 20 min and started to rebound at 90 min after SEB challenge. At 90 min, the dying monkeys showed in the circulation a dramatic increase of polymorphonuclear leukocytes and decreases of NK cells and monocytes (CD16 and CD56 markers) as well as of lymphocytes with HLA-DR, CD2, CD8, and IL2R alpha (CD25) markers. The number of polymorphonuclear leukocytes showed an inverse correlation with the numbers of monocytes and various lymphocyte subpopulations which, except for IL-2R, CD16, and CD56(+) cells, showed a direct correlation with one another. The changes in the populations of leukocytes, monocytes, NK cells, and lymphocytes seem to be an indication of initial toxicosis; however, the roles of these cells in toxicosis and death remain to be defined.

Different high-frequency ventilator strategies: effect on the propagation of tracheobronchial histopathologic changes.

To assess the role of high-frequency ventilator strategy in the propagation of airway injury, we compared the tracheobronchial histologic alterations in 20 newborn piglets ventilated for 8 hours with high-frequency flow interruption (HFFI). Ten animals were assigned to HFFI with a strategy of continuous pulsations at a frequency of 10 Hz and a mean airway pressure of 16 cm H2O. Ten piglets were treated at identical settings except for 10 one-second baseline pauses per minute to a positive end-expiratory pressure of 5 cm H2O. A semiquantitative scoring system was used to grade light microscopic tissue alterations in the trachea, carina, and mainstem bronchi. Ultrastructural changes were evaluated with scanning electron microscopy. The HFFI-continuous-treated piglets had significantly more damage in all areas than the HFFI-baseline pause group (P less than .001). The upper tracheas of animals in both groups were altered to a greater extent than the lower tracheas (P less than .007). In addition, numerous "skip" areas of injury were noted throughout the tracheas. High-frequency ventilator strategy is a determinant of the severity of airway histologic changes. Factors that adversely affect tissue oxygenation or cause direct mechanical trauma may also influence the degree of injury. Optimal operating characteristics and limitations of different high-frequency devices must be assessed before their use in human neonates.

Genetic aspects of interpopulational differences in pheromone blend of cabbage looper moth,Trichoplusia ni.

The genetic basis of interpopulational differences in the pheromone blend emitted by the cabbage looper moth,Trichoplusia ni (Hübner), was examined by crossing individuals from a field-derived population (P1) with individuals from a long-maintained laboratory colony (P2). These colonies differed in the emission rate and relative proportions of four of the five known minor pheromone components, but not in the emission rate of the major component, (Z)-7-dodecenyl acetate (Z7-12∶Ac). These differences in pheromone blend were quantitatively small but biologically significant, because in the field, males responded preferentially to traps baited with a pheromone blend that is similar to that emitted by P1 females relative to a blend similar to that emitted by P2 females. In initial crosses, variation in the quantity and quality of pheromone blends among families of P1, P2, and F1 hybrid females was examined. In F1 females the relative proportions (quantity relative to the major component) of (Z)-5-dodecenyl acetate (Z5-12∶Ac) and (Z)-7-tetradecenyl acetate (Z7-14∶Ac) were intermediate to parental lines. In a second more extensive set of crosses, analyses included P1, P2, F1, F2, and selected backcrosses. The relative proportion of Z5-12∶Ac, Z7-14∶Ac, and Z9-14∶Ac emitted by F1 females were intermediate to parental lines. The frequency distributions of relative proportions of these components emitted by females were not consistent with those expected under a single autosomal or sex-linked gene hypothesis, suggesting that more than one gene is involved in the quantitative differences in the pheromone blend.

Interpopulational variation in emitted pheromone blend of cabbage looper moth,Trichoplusia ni.

Female cabbage looper moths,Trichoplusia ni, from laboratory colonies initiated from three locations across the United States emitted similar quantities and blend ratios of the six known pheromone components. In contrast, females from a long-established laboratory colony emitted a greater proportion of four of the five minor components relative to the major component, (Z)-7-dodecenyl acetate; only the relative proportion of 11-dodecenyl acetate was similar in all of the populations sampled. Females from this population emitted (Z)-7-dodecenyl acetate at a rate similar to that from females from field-collected colonies. Within each population there were highly significant correlations among the quantities of pheromone components of similar molecular weights. Correlations between components of different molecular weights were not as great, but often were significant. Similarities of blend ratios among field populations may indicate that the chemical signal in this species is conservative. The difference of the blend ratios in our laboratory population from the other populations may indicate a decrease in the intensity of selection pressure that usually would maintain these values.

A mutation in pheromonal communication system of cabbage looper moth,Trichoplusia ni.

A mutation that results in a dramatic change in the relative proportions of the pheromone components produced by female cabbage looper moths has been found. The most notable changes involve reduction in the emission rate of the major component [(Z)-7-dodecenyl acetate], near absence of a component [(Z)-5-dodecenyl acetate] that is normally present at about 20% of the major component, and a remarkable ca. 20-fold increase in a trace component [(Z)-9-tetradecenyl acetate]. In spite of the multiplicity of changes in the pheromone blend, a genetic analysis indicates that the condition is controlled by a single autosomal gene. These mutants are ineffective in attracting conspecifics, but do attract another distantly related noctuid moth. These results suggest that the evolutionary process that leads to distinct chemical signals in sibling species may include single gene mutations that lead to major changes in the species-specific blend.

Determinants of tracheobronchial histologic alterations during conventional mechanical ventilation.

It was hypothesized that diverse mechanisms may influence upper airway injury during mechanical ventilation. To assess the roles of several factors in the propagation of such injury, the tracheobronchial histologic changes in 53 newborn piglets were compared following conventional positive pressure ventilation. Eight animals were assigned to each of four positive pressure ventilation groups at "low" settings (an FiO2 of 0.25, a frequency of 10 breaths per minute, a peak inspiratory pressure of 20 cm H2O, a positive end-expiratory pressure of 4 cm H2O, a flow rate of 10 L/min, and an inspiratory time to expiratory time ratio of 1:2): (1) positive pressure ventilation with no hypotension or hypoxemia; (2) positive pressure ventilation with hypotension; (3) positive pressure ventilation with hypoxemia; and (4) positive pressure ventilation with both hypotension and hypoxemia. In addition, eight piglets were assigned to each of two positive pressure ventilation groups at "high" settings (greater frequency [40 breaths per minute], higher peak inspiratory pressure [40 cm H2O], and greater flow rate [17 L/min]): (1) positive pressure ventilation with no hypotension or hypoxemia; and (2) positive pressure ventilation with both hypotension and hypoxemia. The changes were mild and similar among the first three positive pressure groups at low settings. However, the injury scores of the combined hypotension and hypoxemia group (group 4) were greater than those of the former three positive pressure ventilation groups (P less than .004). The piglets receiving positive pressure ventilation at high settings with no hypotension or hypoxemia (group 5) had no more injury than those in the first three groups receiving positive pressure ventilation.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid purification of staphylococcal enterotoxin B by high-pressure liquid chromatography.

The Staphylococcus aureus enterotoxins represent a group of proteins that cause emesis and diarrhea in humans and other primates. We have developed a rapid two-step high-pressure liquid chromatography (HPLC) procedure for purification of staphylococcal enterotoxin B (SEB). Sterile filtrates (2.5 liters) of strain 10-275 were adsorbed directly onto a reversed-phase column (50 mm by 30 cm Delta Pak; 300 A [30 nm], 15 microns, C18). SEB was obtained by using a unique sequential gradient system. First, an aqueous ammonium acetate to acetonitrile gradient followed by an aqueous trifluoroacetic acid (TFA) wash was used to remove contaminants. A subsequent TFA to acetonitrile-TFA gradient eluted the bound SEB. Further purification was obtained by rechromatography on a cation-exchange column. From 35 to 45% of the SEB in starting filtrates was recovered. Analysis by immunoblotting of samples separated on sodium dodecyl sulfate-polyacrylamide gels indicated that HPLC-purified SEB exhibited immunological and biochemical properties similar to those of the SEB standard. Induction of an emetic response in rhesus monkeys showed that the HPLC-purified toxin also retained biological activity.