Kinetics of cellular and cytokine responses in a chimeric mouse model for the study of staphylococcal enterotoxin B pathogenesis.

The mechanisms by which superantigens, such as staphylococcal enterotoxin B (SEB), contribute to microbial pathogenicity have been poorly defined. The study of such pathogenic processes has been hampered by the lack of an adequate animal model. We utilized a previously described murine chimeric model to determine the cytokines and cell populations that might be involved in SEB toxicity. In the absence of bone marrow transplantation (BMT), all total body irradiated (TBI) mice died, while all transplanted mice survived up to 6 months. Compared with non-TBI and non-BMT mice, chimeric mice had an increased percentage of CD11b (Mac-1)-positive splenocytes (17 vs. 59%, P < 0.05) and decreased CD45R-positive (B) cells (33 vs. 6%, P < 0.05) at 6 weeks after BMT. The relative numbers of splenocyte CD4 and CD8 cells were similar in chimeric and normal mice. Susceptibility of chimeric animals to 10 or 100 microg SEB was time-dependent: no mice challenged at 2 weeks post-BMT died, but 15% of mice challenged at 4 weeks and 50% of those challenged at 6-8 weeks died. Compared with TBI and non-BMT C3H/HeJ mice, SEB-challenged chimeric mice at 6-8 weeks had (1) increased splenocyte mRNA expression for: IFN-gamma (3.5 x optimally at 1 h), TNF-alpha (6.5 x at 2 h), IL-6 (4.8 x at 4 h), IL-1beta (8.4 x at 4 h), IL-2 (4.7 x at 4 h), and IL-10 (3 x at 16 h), and (2) increased and earlier peak serum levels of IFN-gamma, IL-6, IL-1beta and IL-2, but no increase in serum TNF-alpha or IL-4. These data support the hypothesis that the decreased percentage of B cells and increased macrophages in chimeric mice lead to enhanced T cell-macrophage interactions after SEB administration and a lethal burst of T cell and macrophage cytokine release. This model will provide insight into cell populations and mechanisms that mediate superantigen-induced toxicity.

Role of plasminogen activator inhibitor-1 in promoting fibrin deposition in rabbits infused with ancrod or thrombin.

The role of defective fibrinolysis caused by elevated activity of plasminogen activator inhibitor-1 (PAI-1) in promoting fibrin deposition in vivo has not been well established. The present study compared the efficacy of thrombin or ancrod, a venom-derived enzyme that clots fibrinogen, to induce fibrin formation in rabbits with elevated PAI-1 levels. One set of male New Zealand rabbits received intravenous endotoxin to increase endogenous PAI-1 activity followed by a 1-hour infusion of ancrod or thrombin; another set of normal rabbits received intravenous human recombinant PAI-1 (rPAI-1) during an infusion of ancrod or thrombin. Thirty minutes after the end of the infusion, renal fibrin deposition was assessed by histopathology. Animals receiving endotoxin, rPAI-1, ancrod, or thrombin alone did not develop renal thrombi. All endotoxin-treated rabbits developed fibrin deposition when infused with ancrod (n = 4) or thrombin (n = 6). Fibrin deposition occurred in 7 of 7 rabbits receiving both rPAI-1 and ancrod and in only 1 of 6 receiving rPAI-1 and thrombin (P < .01). In vitro, thrombin but not ancrod was inactivated by normal rabbit plasma and by purified antithrombin III or thrombomodulin. The data indicate that elevated levels of PAI-1 promote fibrin deposition in rabbits infused with ancrod but not with thrombin. In endotoxin-treated rabbits, fibrin deposition that occurs with thrombin infusion may be caused by decreased inhibition of procoagulant activity and not increased PAI-1 activity.

A surgical technique for bilateral cochleotomy in the Long-Evans rat.

A bilateral cochleotomized surgical rat model, needed for a study involving microwave effects, was developed, standardized, and assessed for reproducibility. After a review of the literature concerning attempts and approaches with various species, a technique involving an approach through the external auditory canal was chosen and modified. Using a stereomicroscope, a cutaneous incision in the intertragic notch was made and extended medially along the ventral aspect of the external auditory canal to the depth of the external auditory meatus. The tympanic membrane was ruptured and the malleus removed with splinter forceps, allowing visualization of the cochlea. The lateral wall of the cochlea was penetrated with a 0.024-in. wire gauge drill bit and endolymph was suctioned from the cochlea. A 5-mm piece of 3-O silk suture, inserted into the cochlear opening, maintained patency. Appraisal of the reliability and standardization of the procedure was performed utilizing startleometry. Histology assessed completeness of the procedure and any evidence of cochlear infection.

Enhancement of tissue plasminogen activator-induced fibrinolysis by activated protein C in endotoxin-treated rabbits.

Endotoxin-treated rabbits produce high levels of plasminogen activator inhibitor-1 (PAI-1), which inhibits fibrinolysis by neutralizing endogenous tissue-type plasminogen activator (t-PA). These animals will develop renal fibrin deposition when infused with ancrod, an enzyme that acts directly on fibrinogen. In normal rabbits with an intact fibrinolytic system, ancrod induces hypofibrinogenemia without fibrin deposition. Rabbit PAI-1 activity can be neutralized by recombinant human t-PA or by bovine activated protein C. The present study determined the efficacy of these two agents used alone or in combination in neutralizing increased PAI-1 activity and in preventing renal fibrin deposition in a rabbit model. Male New Zealand rabbits first received intravenous endotoxin to increase PAI-1 activity. Ancrod was infused intravenously during hour 4 to 5, and the kidneys were examined at hour 5.5. Renal fibrin deposition occurred in 100% (6 out of 6) of the endotoxin-treated rabbits that received ancrod; this was reduced to 14% (1 out of 7) for rabbits receiving t-PA (170 micrograms/kg) before and during the ancrod infusion. Fibrin deposition occurred in only 12% (1 out of 8) of the rabbits that received a 10-fold lower dose of t-PA (17 micrograms/kg) combined with activated protein C (1 mg/kg) before and during the ancrod. Activated protein C at this dose completely neutralized plasma PAI-1 activity. However, low-dose t-PA and activated protein C did not prevent fibrin deposition when used as single agents, with fibrin deposition occurring in 75% and 100% of rabbits, respectively. The data indicate that activated protein C can neutralize plasma PAI-1 activity in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)