Association of Bacillus anthracis capsule with lethal toxin during experimental infection.

Bacillus anthracis lethal toxin (LT) was characterized in plasma from infected African Green monkeys, rabbits, and guinea pigs. In all cases, during the terminal phase of infection only the protease-activated 63-kDa form of protective antigen (PA(63)) and the residual 20-kDa fragment (PA(20)) were detected in the plasma. No uncut PA with a molecular mass of 83 kDa was detected in plasma from toxemic animals during the terminal stage of infection. PA(63) was largely associated with lethal factor (LF), forming LT. Characterization of LT by Western blotting, capture enzyme-linked immunosorbent assay, and size exclusion chromatography revealed that the antiphagocytic poly-gamma-d-glutamic acid (gamma-DPGA) capsule released from B. anthracis bacilli was associated with LT in animal blood in variable amounts. While the nature of this in vivo association is not understood, we were able to determine that a portion of these LT/gamma-DPGA complexes retained LF protease activity. Our findings suggest that the in vivo LT complexes differ from in vitro-produced LT and that including gamma-DPGA when examining the effects of LT on specific immune cells in vitro may reveal novel and important roles for gamma-DPGA in anthrax pathogenesis.

Pathology of inhalational anthrax infection in the african green monkey.

There is a critical need for an alternative nonhuman primate model for inhalational anthrax infection because of the increasingly limited supply and cost of the current model. This report describes the pathology in 12 African green monkeys (AGMs) that succumbed to inhalational anthrax after exposure to a low dose (presented dose 200-2 x 10(4)colony-forming units [cfu]) or a high dose (presented dose 2 x 10(4)-1 x 10(7) cfu) of Bacillus anthracis (Ames strain) spores. Frequent gross lesions noted in the AGM were hemorrhage and edema in the lung, mediastinum, and mediastinal lymph nodes; pleural and pericardial effusions; meningitis; and gastrointestinal congestion and hemorrhage. Histopathologic findings included necrohemorrhagic lymphadenitis of mediastinal, axillary, inguinal, and mesenteric lymph nodes; mediastinal edema; necrotizing splenitis; meningitis; and congestion, hemorrhage, and edema of the lung, mesentery, mesenteric lymph nodes, gastrointestinal tract, and gonads. Pathologic changes in AGMs were remarkably similar to what has been reported in rhesus macaques and humans that succumbed to inhalational anthrax; thus, AGMs could serve as useful models for inhalation anthrax studies.

Differential stimulation of IgE production, STAT activation and cytokine and CD86 expression by 2,4-dinitrochlorobenzene and trimellitic anhydride.

It has been reported that dermal exposure to trimellitic anhydride (TMA, 50%), a respiratory allergen, induced greater production of serum IgE and expression of Th2 cytokines than 2,4-dinitrochlorobenzene (DNCB, 1%), a potent contact sensitizer, in female BALB/C mice. To determine if there is any strain difference, four strains (B6C3F1, C57BL/6, BDF1 and BALB/C) of female mice were employed in this study to compare the differential effects of these chemicals on the hypersensitivity responses. Serum IgE levels were increased in TMA-treated B6C3F1, C57BL/6 and BDF1 mice when compared with the DNCB treatment and vehicle controls; in contrast, no difference was observed between TMA- and DNCB-treated BALB/C mice, although both chemicals induced greater IgE production than vehicle controls. In vitro expression of interleukin 4 (IL-4) and IL-13 mRNA by overnight concanavalin A (ConA)-stimulated draining lymph node cells was enhanced following in vivo treatment with TMA but not with DNCB in the B6C3F1, C57BL/6 and BDF1 mice. In contrast, TMA and DNCB induced similar levels of IL-4 and IL-13 mRNA in the BALB/C mice. The IL-4 protein levels in the supernatants of overnight ConA-treated draining lymph node cells were also increased in TMA-treated B6C3F1 and C57BL/6 mice when compared with the DNCB treatment and vehicle controls. Further mechanistic evaluation in the B6C3F1 mice indicated that the activation of STAT6 but not STAT4 by ConA plus IL-2-treated draining lymph node cells was increased in TMA- but not DNCB-treated mice when compared with the vehicle controls. Furthermore, surface expression of B7.2 (CD86) by B cells was increased in both TMA- and DNCB-treated B6C3F1 mice when compared with the vehicles; however, greater B7.2 expression was observed in TMA-treated compared with DNCB-treated. Overall, these results demonstrate that a similar pattern of IgE and cytokine production was observed in these strains of mice except for BALB/C. Furthermore, differential activation of STAT6 and expression of CD86 following exposure to TMA and DNCB may contribute to the differential production of IgE and cytokines.

Sodium metasilicate hypersensitivity in BALB/c mice.

BACKGROUND: Sodium metasilicate (SMS) is a key ingredient for a number of industrial and consumer products. Although little is known about potential for this chemical to cause allergic reactions, a similar silicate compound, sodium silicate, was reported to elicit IgE-mediated contact urticaria. OBJECTIVE: The aim of this study was to evaluate the potential for sodium metasilicate to elicit an allergic response in female BALB/c mice after dermal exposure. METHODS: The primary irritancy assay (IA), local lymph node assay (LLNA), and a mouse ear swelling test (MEST) were used to evaluate the hypersensitivity response elicited by SMS exposure. An evaluation of lymph node subpopulations, cytokine mRNA expression, and serum IgE levels was also conducted. RESULTS: SMS caused significant dermal irritation at concentrations >or=6% and an allergic response after mice were sensitized with 4% SMS then challenged with 6% SMS in the MEST. Lymph node cell proliferation was not observed in the LLNA after treatment with SMS (2% to 6% SMS). Increases in lymph node cellularity, the percentage of B cells, and the expression of certain cytokine mRNAs were observed in mice treated with SMS. Changes in the concentration of serum IgE after SMS treatment, however, were not observed. CONCLUSIONS: SMS appears to elicit a chemical hypersensitivity response in mice, as indicated by the MEST, but not by the LLNA. Increases in auricular lymph node cellularity, the percentage of B cells, and certain cytokine mRNAs support classifying SMS as a weak chemical allergen.

Dermal exposure to cinnamaldehyde alters lymphocyte subpopulations, number of interferon-gamma-producing cells, and expression of B7 costimulatory molecules and cytokine messenger RNAs in auricular lymph nodes of B6C3F1 mice.

BACKGROUND: Although the Murine Local Lymph Node Assay (LLNA) is efficient in identifying chemicals with sensitizing potential, there is increasing need for alternative end points. Cinnamaldehyde (CIN) was chosen for evaluation based on its moderate potency and extensive use in fragrance materials. OBJECTIVES: The purpose of the present studies is to incorporate some alternative end points, such as phenotypic analysis and cytokine production, into a modified LLNA/irritancy assay (IA) to evaluate the sensitization of female B6C3F1 mice to CIN. METHODS: Several nontraditional end points, including the analysis of lymphocyte subpopulations, B7 costimulatory molecule and cytokine messenger RNA (mRNA) expression, and intracellular interferon-gamma (IFN-gamma) levels, were incorporated into a modified murine local lymph node (LLNA)/irritancy assay (IA) to evaluate the sensitization of female B6C3F1 mice to cinnamaldehyde (CIN). RESULTS: The alternate end points used in these studies support the classification of CIN as a moderately potent sensitizer. Dermal treatment with CIN resulted in an increase in the percentage of B cells in the auricular lymph nodes (ALNs) and expression of the costimulatory molecule, B7-2, on B cells. Lymph node cells also showed increased transforming growth factor-beta1, migration-inhibition factor, and mild increases in IFN-gamma and interleukin-2 cytokine mRNA expression. Although the increase in IFN-gamma mRNA expression did not translate into increased intracellular IFN-gamma levels, the absolute number of T cells producing IFN-gamma in the ALNs increased. Conversely, the MEST did not classify CIN as a contact allergen. CONCLUSION: The nontraditional end points used in the LLNA/IA were not as sensitive as the traditional radioisotope method used to assess cell proliferation. However, they may help identify compounds inappropriately classified as sensitizers or nonsensitizers by the LLNA and MEST.

Simplified method for producing permanent ruminal fistulae.

A quick and simple method for establishing permanent rumimal fistulae is described. A frozen cannula is forced into the rumen in a manner similar to the use of a trocar. Minimal equipment is required, and the entire operation can be performed in 15 to 20 min in sheep or 30 min in cattle. The technique has been used successfully to fistulate seven sheep and two cows.