(ii) E. coli-induced peritonitis (model ii).
The rate of lethality in control group C1
was 100%. All intraperitoneal antibiotic treatments given immediately after challenge were better than no treatment (P
< 0.05). Specifically, survival rates were 73.4, 60.0, 46.7, and 86.7% in the groups treated with buforin II, indolicidin, KFFKFFKFF, and imipenem, respectively (Table 2
). Bacteriological evaluation showed 100% positive blood and intra-abdominal fluid cultures for control group C1
: the average bacterial count in the peritoneal fluid from dead or surviving animals at 72 h was 8.9 × 108
± 2.2 × 108
CFU/ml. Imipenem had the highest antimicrobial activities and therapeutic efficacies. Similar to the lethality endpoint, there were significant differences in the results for the quantitative bacterial cultures when the data obtained for the imipenem-treated groups were compared with those obtained for the peptide-treated groups (P
< 0.05). Endotoxin and TNF-α concentrations increased constantly in control group C1
, with mean peak levels achieved at 6 h postinjection. All peptide-treated groups showed significant reductions in plasma endotoxin and TNF-α levels compared to those in the control and the imipenem-treated groups. In contrast, no significant differences in plasma endotoxin and TNF-α concentrations were observed between the imipenem-treated group and control group C1
. The results observed at 6 h postinjection are summarized in Table 2
(iii) Cecal ligation and puncture (model iii).
The rate of lethality in control group C2
was 100%. All intravenous antibiotic treatments given immediately after challenge were better than no treatment (P
< 0.05). Specifically, rates of survival were 66.7, 46.7, 33.3, and 80.0% in the groups treated with buforin II, indolicidin, KFFKFFKFF, and imipenem, respectively (Table 3
). Bacteriological evaluation showed 100% positive blood and intra-abdominal fluid cultures for control group C2
. Specifically, the peritoneal fluid of the control group demonstrated high bacterial numbers, averaging 2.9 × 109
± 0.7 × 109
CFU/ml. Gram-negative and gram-positive bacteria were simultaneously isolated from more than 90% of the animals. The pathogens isolated from both blood and the abdominal fluid were mainly members of the family Enterobacteriaceae
, including Escherichia coli
spp., and gram-positive cocci (primarily enterococci). The most frequently isolated anaerobic isolates were Bacteroides
spp. As in model ii, imipenem had the highest antimicrobial activities and therapeutic efficacies. Among the peptides tested, buforin II was confirmed to be the most active. As shown with the other experimental models, constant increases in plasma endotoxin and TNF-α concentrations were found in the animals in model iii, with mean peak levels achieved at 6 h postinjection. Overall, the peptides produced significant reductions in plasma endotoxin and TNF-α levels compared to the levels in the controls, while imipenem produced significant increases. The results are summarized in Table 3
Although several papers have been devoted to the study of animal models of septic shock, there continues to be a lack of a reliable model system. For this reason, extrapolation of results obtained with animal models to human pathologies should be regarded with caution. Peritonitis models have been proposed as the “gold standard”; nevertheless, studies with a single dose of endotoxin and studies with a single inoculum of one gram-negative species have been the models of sepsis most often used for the screening of antiendotoxin and antimicrobial drugs. However, these models are not representative of the situation during clinical infections: humans are usually exposed not to only one species of gram-negative bacteria but to gram-positive organisms as well and are exposed in different ways. For these reasons, the animal model of cecal ligation and puncture, which resembles the clinical situation of bowel perforation and mixed bacterial infection of intestinal origin, seems to be the more realistic model of sepsis with a clear resemblance to the clinical situation. Analysis of the data from our three experiments showed that the different models did not have different essential effects on the parameters evaluated. Actually, the only clear difference in the models concerned the plasma TNF-α concentration. In particular, in agreement with previous results, model iii, in which intra-abdominal sepsis was induced via cecal ligation and a single puncture, TNF-α appeared slightly later and its levels were lower, which was different from the results obtained with the other two models. Nevertheless, it was evident that the efficacies of the compounds were not affected by the animal models used and that these were retained regardless of the system used.
In the present study the effects of buforin II, indolicidin, and KFFKFFKFF were evaluated by the use of different endpoints. Since severe infections caused by gram-negative organisms involve the release of the endotoxic component responsible for the increase in cytokine levels in plasma and this phenomenon is thought to be a pathogenic element of sepsis (2
), we were interested in investigating compounds that could neutralize endotoxins. Current treatments for septic shock caused by gram-negative bacteria rely on antibiotics to control the infection and intensive-care support to correct dysfunctions of the cardiovascular, endocrine, and other organ systems. Nevertheless, it is known that many antibiotics used clinically can be harmful when administered for the treatment of severe infections cause by gram-negative bacteria, in that they can stimulate the release of endotoxin and thus increase the rates of occurrence of symptoms and life-threatening complications (4
). Recent reports have described a large collection of antimicrobial peptides that comprise a widespread effector arm of the immune system (14
). Actually, they have been identified in organisms as diverse as bacteria, protozoa, plants, insects, and vertebrates. Use of the antimicrobial polycationic peptides might offer the opportunity to inhibit not only bacterial growth but also the biological activity of the endotoxin (7
). Actually, these cationic molecules act on gram-negative bacteria by initially binding to their surface polyanionic LPS, followed by self-promoted uptake across the outer membrane. By virtue of this affinity for LPS, the cationic peptides operate as antiendotoxin agents and inhibit the production of cytokines such as TNF-α by macrophages stimulated with LPS (8
The results of this study have demonstrated that single intraperitoneal and intravenous doses of buforin II, indolicidin, and KFFKFFKFF produced significant reductions in plasma endotoxin and TNF-α levels compared to the levels in the control and the carbapenem-treated groups. Specifically, buforin II showed much stronger antiendotoxin activity than indolicidin and KFFKFFKFF in all models evaluated in the study. These differences in the antiendotoxin potentials between the peptides, although statistically not significant, can be ascribed in part to the differences in their mechanisms of action. As mentioned above, the killing mechanism detected for most polycationic peptides consists of attacks on the outer and inner membranes, ultimately resulting in lysis of the organisms (12
). Otherwise, buforin II kills bacteria without cell lysis, suggesting the possibility that its target is not the cell membranes (19
). On the other hand, the two models (models ii and iii) that also evaluated the antimicrobial roles of the compounds showed that the peptides lack the antibiotic potency of imipenem. In fact, our data demonstrated that, despite the high plasma endotoxin and TNF-α levels, the strong antimicrobial activity of this clinically used antibiotic resulted in the lowest rate of lethality by inhibition of bacterial growth at the highest levels. The relevance of the pivotal role of antimicrobial activity was pointed out by the third experimental model, in which various gram-negative and gram-positive bacterial species were involved in the pathogenesis of the sepsis. In fact, lethality data demonstrated that the strong activity and the broad antimicrobial spectrum of imipenem prevailed against the peptides' antiendotoxin activities exclusively directed against the LPS component of the membranes of the gram-negative bacteria. Nevertheless, on the basis of our results, the use of cationic peptides might have certain potential advantages. First, the use of single doses of peptides resulted in significant inhibition of bacterial growth compared with that in the control groups. In addition, they produced significant reductions in plasma endotoxin levels compared to those achieved in the imipenem-treated groups, confirming their double antimicrobial and antiendotoxin activities, while the administration of imipenem significantly increased the plasma endotoxin and TNF-α levels compared with those in the control groups and the peptide-treated animals. Finally, there is a substantial interest in identifying novel strategies to overcome not only infections but also the underlying conditions. For this reason, agents that can modulate the effects of endotoxin and the inflammatory response, in addition to having direct antimicrobial activity, would be advantageous.