Failure of endotoxin-free superoxide dismutase to reduce some paw edemas and adjuvant arthritis in rats.

OBJECTIVE AND DESIGN: To elucidate whether superoxide anion plays a major role in inflammation models in rats. Endotoxin-free CuZn superoxide (SOD) was used in the present study, because we had shown previously that even a trace of endotoxin contamination of SOD would carry anti-inflammatory effects. MATERIAL AND TREATMENT: Male rats were treated with saline (n=8-10), recombinant human SOD (n=8-10), and dexamethasone or indomethacin (n=8-10). METHODS: Foot volume was determined by mercury displacement. RESULTS: Two administrations of SOD (5 and 50 mg/kg), 2 h and just before irritant injection, had no effect on rat paw edema induced by phorbol, zymosan or carrageenan. Two administrations of dexamethasone (1 mg/kg) or indomethacin (3 mg/kg), 2 h and just before irritation, inhibited paw edema significantly (p<0.05). Administration of SOD (10 mg/kg) once daily for 3 weeks from the day of mycobacterium injection had no effect on injected paw swelling and non-injected paw swelling 7, 14, 21 days after irritation. Indomethacin (2 mg/kg) once daily for 3 weeks inhibited this arthritis significantly (p < 0.05). CONCLUSIONS: Superoxide anion may not play a major role in the genesis of inflammation in these rat inflammation models, and endotoxin-like contaminants may have contributed to some protective effects of SOD administration in previous studies.

Hyperthermic potentiation of bleomycin cytotoxicity in the presence of Bacillus thuringiensis subsp. israelenis delta-endotoxin.

We previously reported that a 25-kDa Bacillus thuringiensis subsp. israelensis (BTI) delta-endotoxin potentiated the antitumor activity of bleomycin (BLM) in both in vitro and in vivo systems. We report here a characteristic hyperthermic synergy toward the cytocidal activity of BLM in the presence of BTI endotoxin in cultured L1210 cells. Cytocidal gain, i.e., the ratio of the IC50 of BLM in the absence versus the presence of BTI delta-endotoxin, reached ca. 20 at 40 degrees C, suggesting that a more effective heat-BLM combination treatment may be achieved in the presence of BTI delta-endotoxin.

Endotoxin or cytokines attenuate ozone-induced DNA synthesis in rat nasal transitional epithelium.

Pretreatment of rats with endotoxin (E), a potent inducer of tumor necrosis factor alpha (TNF), and interleukin 1 beta (IL 1), or a combination of TNF and IL1, has been shown to increase levels of lung antioxidant enzymes and protect against pulmonary toxicity associated with hyperoxia. Inhalation of ozone (O3) induces cell injury, followed by increased DNA synthesis, cell proliferation, and secretory cell metaplasia in rat nasal transitional epithelium (NTE). This study was designed to test the effects of E, TNF, and IL1 pretreatment on acute O3-induced NTE cell injury as measured by changes in NTE cell DNA synthesis. Rats were exposed to either 0.8 ppm O3 or air for 6 hr in whole-body inhalation chambers. Immediately before exposure, rats in each group were injected intraperitoneally (ip) with either saline alone or saline containing E (1 microgram/g body wt), TNF (10 micrograms), IL1 (10 micrograms), or both TNF and IL1 (TNF/IL1; 10 micrograms each). Eighteen hours postexposure, rats were injected ip with bromodeoxyuridine (BrdU; 50 micrograms/g body wt) to label cells undergoing DNA synthesis and were euthanized 2 hr later. NTE was processed for light microscopy and immunochemically stained to identify cells that had incorporated BrdU into nuclear DNA. The number of BrdU-labeled NTE nuclei per millimeter of basal lamina was quantitated. There were no significant differences in the number of BrdU-labeled NTE nuclei in air-exposed rats that were injected with E, TNF, IL1, or TNF/IL1 compared with those in saline-injected, air-exposed controls. Rats that were injected with saline and exposed to O3 had approximately 10 times the number of BrdU-labeled NTE nuclei than saline-injected, air-exposed control rats. O3 exposure also induced a significant increase in labeled nuclei in rats that were pretreated with TNF alone. In contrast, pretreatment with E, IL1, or TNF/IL1 attenuated the O3-induced increase in NTE DNA synthesis. These results indicate that both E and the cytokines TNF and IL1 have physiologic effects that can attenuate O3-induced injury or modify the response to NTE cells to O3 exposure.

Endotoxin treatment protects rats against ozone-induced lung edema: with evidence for the role of manganese superoxide dismutase.

Ozone is a strong oxidizing agent that can cause lung damage and edema. There is evidence that it does so by causing peroxidation of membrane lipids. However, the elevation in lung activity of copper, zinc superoxide dismutase (Cu, ZnSOD), and manganese superoxide dismutase (MnSOD) during exposure to ozone suggests that increased production of superoxide could contribute to lung edema caused by ozone. This latter observation, and preliminary evidence that treatment of rats with endotoxin elevates lung activity of MnSOD without elevation of the activity of Cu, ZnSOD, catalase (CAT), or glutathione peroxidase (GP), led to the present study. We treated rats with endotoxin, exposed them to different concentrations of ozone, measured lung wet weight to dry weight ratio, thiobarbituric acid-reactive material (TBAR), and assayed lung tissue for Cu, ZnSOD, MnSOD, CAT, and GP activity. Our major findings are, (1) a strongly edemogenic concentration of ozone-lowered MnSOD activity; (2) endotoxin treatment of air-breathing rats did not decrease lipid peroxidation as indicated by the lung concentration of TBAR; (3) induction of increased MnSOD activity in lung by treatment with endotoxin was associated with virtually complete protection against an otherwise edemogenic concentration of ozone, with less lipid peroxidation, and with less loss of weight; and (4) this protection occurred without elevated Cu, ZnSOD, CAT, or GP activity.

Response of tumors to thermodynamic stimulation of the immune system.

Cytokines such as interferon, interleukin and tumor necrosis factor are natural body defense proteins which have been used individually in recent years to produce a few complete responses of some tumors in a few patients but their overall effect has been limited. The hypotheses is that a biologic stimulus such as endotoxin will stimulate the immune system in a more natural way and hence will be more likely to have an effect especially in the presence of a naturally produced fever than treatment from just one lymphokine. One of the side effects of current lymphokine studies has been fever usually spiking in nature but there has been no obvious effort to relate the extent of the fever to any tumor responses. Clinical experience has shown that increased temperature can enhance the results of radiotherapy and chemotherapy in some situations. Therefore it is logical to put this hypothesis to a test clinically. It is surprising that the combined effect of fever and lymphokine stimulation has not been reported aside from Coley's work, and it is only in retrospect that fever has been suggested to be essential to the tumor control noted. Most of the literature, animal and human, has focused on the destructive effect of temperature on tumor cells and not as part of an immune response to control tumor growth. Endotoxin is the pyrogen on which most current information is available. More effective and more available pyrogens may be developed later.

The endotoxin-pretreated, oxygen-adapted rat model in hyperbaric hyperoxia.

Rats pretreated with 500 micrograms X kg-1 endotoxin are resistant to the pulmonary toxic effects of normobaric hyperoxia (greater than 95% O2). After endotoxin-pretreatment and exposure to 1.0 ATA O2 for 72 h, such rats are found to have elevated total superoxide dismutase, glutathione peroxidase, and catalase activities in homogenates of whole lungs. Despite increases in these protective antioxidant enzymes which persist in 2.0 ATA O2 (4 h) and 4.0 ATA O2 (1.0 h), such rats do not have improved survival in hyperbaric hyperoxia. Likewise, endotoxin-pretreatment immediately prior to 2.0 or 4.0 ATA O2 exposure does not prolong survival compared to controls. It is likely that lung injury during the normobaric oxygen preexposure and the central nervous system toxicity of hyperbaric oxygen interact to limit survival.

Studies on host defenses enhanced by endotoxins: a brief review.

The findings reported here indicate that toxic reactions to endotoxin do not have to be part of the mechanisms leading to clinically improved status of patients or of experimental animals. It seems to be possible to mobilize the host defenses without the unwanted or dangerous side effects which were encountered during fever therapy. For the demonstration of activated host defenses, several experimental models are available. Non-specific resistance to bacterial infections, to lethal irradiation or to experimental challenge with viable tumors are clear manifestations of enhanced capacity of the endotoxin-treated host to compensate for the induced damage, or to cope with an otherwise overwhelming number of pathogenic cells. Immunopotentiation is obviously one of the contributing factors in some of the above parameters. How these results are applicable to human therapy remains to be seen. Such use of endotoxin derivatives, or better, the mediators of beneficial endotoxic effects, must wait until we learn more about the nature of the alternating positive and negative effects of endotoxin application. The importance of the proper timing of such a therapeutic modality cannot be overemphasized.