help button home button
AJRCCM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on October 16, 2003, doi:10.1164/rccm.200307-971OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
200307-971OCv1
169/2/187    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Giacometti, A.
Right arrow Articles by Scalise, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Giacometti, A.
Right arrow Articles by Scalise, G.
American Journal of Respiratory and Critical Care Medicine Vol 169. pp. 187-194, (2004)
© 2004 American Thoracic Society

Cathelicidin Peptide Sheep Myeloid Antimicrobial Peptide-29 Prevents Endotoxin-induced Mortality in Rat Models of Septic Shock

Andrea Giacometti, Oscar Cirioni, Roberto Ghiselli, Federico Mocchegiani, Giuseppina D'Amato, Raffaella Circo, Fiorenza Orlando, Barbara Skerlavaj, Carmela Silvestri, Vittorio Saba, Margherita Zanetti and Giorgio Scalise

Institute of Infectious Diseases and Public Health; Department of General Surgery, I.N.R.C.A. I.R.R.C.S., Università Politecnica delle Marche; Biotechnology Centre, Research Department, I.N.R.C.A. I.R.R.C.S., Ancona; Department of Biomedical Sciences and Technology, University of Udine, Udine; and National Laboratory CIB, Area Science Park, Padriciano, Trieste, Italy

Correspondence and requests for reprints should be addressed to Andrea Giacometti, M.D., Clinica delle Malattie Infettive, c/o Ospedale Regionale, Via Conca, 60020 Torrette AN, Italy. E-mail: anconacmi{at}interfree.it


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study was designed to investigate the antiendotoxin activity and therapeutic efficacy of sheep myeloid antimicrobial peptide (SMAP)-29, a cathelicidin-derived peptide. The in vitro ability of SMAP-29 to bind LPS from Escherichia coli 0111:B4 was determined using a sensitive limulus chromogenic assay. Two rat models of septic shock were performed: (1) rats were injected intraperitoneally with 1 mg E. coli 0111:B4 LPS and (2) intraabdominal sepsis was induced via cecal ligation and single puncture. All animals were randomized to receive parenterally isotonic sodium chloride solution, 1 mg/kg SMAP-29, 1 mg/kg polymyxin B or 20 mg/kg imipenem. The main outcome measures were: abdominal exudate and plasma bacterial growth, plasma endotoxin and tumor necrosis factor-{alpha} concentrations, and lethality. The in vitro study showed that SMAP-29 completely inhibited the LPS procoagulant activity at approximately 10 µM peptide concentration. The in vivo experiments showed that all compounds reduced the lethality when compared with control animals. SMAP-29 achieved a substantial decrease in endotoxin and tumor necrosis factor-{alpha} plasma concentrations when compared with imipenem and saline treatment and exhibited a slightly lower antimicrobial activity than imipenem. No statistically significant differences were noted between SMAP-29 and polymyxin B. SMAP-29, because of its double antiendotoxin and antimicrobial activities, could be an interesting compound for septic shock treatment.

Key Words: LPS • cathelicidins • sheep myeloid antimicrobial peptide-29 • septic shock • cationic peptides

Lipopolysaccharides (LPSs) are major structural and functional components of gram-negative bacteria. Their prominent harmful role as initiators of septic shock is well recognized. In fact, LPSs, composed of an O-polysaccharide chain, a core sugar, and a lipophilic fatty acid (lipid A), exhibit a variety of toxic and proinflammatory activities that are related to the pathogenesis of gram-negative infection (1–6). Many of these pathophysiologic phenomena result from the ability of LPS to activate host effector cells through stimulation of receptors on their surface. These target cells secrete large quantities of inflammatory cytokines, such as tumor necrosis factor (TNF), interleukin-1, interleukin-6, and interleukin-8, platelet-activating factor, arachidonic acid metabolites, erythropoitin, and endothelin (1, 7–9). Current treatments for gram-negative septic shock are founded on prompt institution of antimicrobial agents to control the infection and intensive care support to correct the dysfunction of the main organ systems, but despite these advances the mortality from sepsis-related diseases has remained substantially unchanged (10, 11). Moreover, several studies have shown that exposure of gram-negative organisms to antibacterial agents can result in endotoxin release and suggest that this phenomenon could have deleterious effects (12, 13).

Among various therapeutic strategies aimed at improving outcomes of septic shock, administration of endotoxin-blocking agents such as monoclonal antibodies to endotoxin, anti–TNF-{alpha} antibody, interleukin-1 receptor antagonists, and antioxidants have been recently proposed (14–17). On the basis of its highly conserved molecular structure among gram-negative bacteria, lipid A may be an appropriate target for agents designed to bind to LPS. The anionic and amphiphilic nature of lipid A enables it to bind either to compounds that are positively charged or to molecules that possess an amphipathic character (18, 19). Antimicrobial peptides are positively charged and amphipathic molecules isolated from a wide variety of animals and plants in which they act as natural defense mechanisms. In recent years, these molecules have received increased attention as they possess a broad spectrum of activity against bacteria, fungi and protozoa, and antiendotoxin activity (20–25).

A variety of antimicrobial peptides are present in mammals (26). Most of them belong to the defensin or the cathelicidin peptide families. Cathelicidins are bipartite molecules with an N-terminal cathelin domain and a C-terminal domain of varied structure, that displays antimicrobial activity after being freed by proteolytic processing of the holoprotein (27). The {alpha}-helical cathelicidin-derived peptides display potent and broad-spectrum antimicrobial activity against gram-negative and gram-positive bacteria and fungi (28). These peptides kill bacteria by thinning and disrupting the bacterial membrane (29). Sheep myeloid antimicrobial peptide (SMAP)-29 is an {alpha}-helical cathelicidin-derived peptide deduced from sheep myeloid messenger RNA (30). This compound is a potent antibacterial and antifungal agent, active under both low- and high-ionic strength conditions (28, 31, 32), and induces significant morphologic alterations in the bacterial surface (31). Similar to other polycationic peptides, SMAP-29 binds LPS as the initial step in the killing mechanism of gram-negative organisms (28), and recent studies have demonstrated that it has two LPS-binding sites and a central hinge (33).

The present experimental study was designed to investigate the antiendotoxin activity and the in vivo efficacy of SMAP-29 in two rat models of septic shock.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Adult male Wistar rats weighting 250 to 300 g were used for all the experiments. All animals were housed in individual cages under constant temperature (22°C) and humidity with 12 hours light/dark cycle and had access to chow and water as much as desired throughout the study. The study was approved by the animal research ethics committee of the I.N.R.C.A. I.R.R.C.S., University of Ancona, Ancona, Italy.

Reagents
Endotoxin (Escherichia coli serotype 0111:B4; Sigma-Aldrich S.r.l., Milan, Italy) was prepared in sterile saline, aliquoted, and stored at -80°C for short periods. Peptide amide linker (PAL)-polyethylene glycol (PEG)-polystyrene (PS) resins, coupling reagents for peptide synthesis, and Fmoc-amino acids were purchased from Applied Biosystems (Foster City, CA). Peptide synthesis–grade N,N-dimethylformamide, N-methyl-2-pyrrolidone, dichloromethane, and high-performance liquid chromatography–grade acetonitrile were from Biosolve (Valkenswaard, The Netherlands). Trifluoroacetic acid, N-methyl-morpholine and trifluoroethanol were from Acros Chimica (Beerse, Belgium).

Agents
SMAP-29 (RGLRRLGRKIAHGVKKYGPTVLRIIRIA) was chemically synthesized as a C-terminally amidated peptide on PAL-PEG-PS resin (0.16 meq/g), using a Milligen 9050 synthesizer (Bio-Tek Instruments, Inc., Winooski, VT) and the Fmoc chemistry. Fmoc-protected amino acids were added in a sixfold molar excess with respect to resin substitution for most coupling steps, and coupling reactions (30 minutes) were performed with 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetraflouro-borate. As several couplings were predicted to be difficult, the synthesis was performed at 48°C by heating the jacketed column and the solvent solutions. An eightfold molar excess of Fmoc-protected amino acid and the highly efficient acylating reagent O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate were used for difficult couplings. After Fmoc deprotection and before addition of the next residue, the resin was saturated with a solution of dichloromethane/N,N-dimethylformamide/N-methyl-2-pyrrolidone (1:1:1) containing 1% Triton X-100 and 2 M ethylencarbonate. Amino acid side chains were protected as follows: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Arg), t-butoxycarbonyl (Lys), trityl (His), and t-butyl (Tyr, Thr). Cleavage from the resin and deprotection of synthesized peptides were performed with a solution of 90% trifluoroacetic acid, 3% water, 1% triisopropylsilane, and 2% each of phenol, 1,2-ethanedithiol, and thioanisole. After repeated precipitation with methylbutylether, the peptides were purified by reversed phase–high-performance liquid chromatography on a C18 Delta-Pak column (Waters, Bedford, MA), using an appropriate 0 to 60% acetonitrile gradient in 0.05% trifluoroacetic acid. The correct peptide was obtained in greater than 60% yield and with measured mass of 3,198.0 ± 0.3 versus a calculated mass of 3,197.99 D and was homogeneous after preparative purification, as confirmed by mass spectrometry and analytic reversed phase–high-performance liquid chromatography. The peptide concentration was determined measuring the absorbance of SMAP-29 and dissolved in 0.05% trifluoroacetic acid, at 280 nm, using a molar extinction coefficient of 1,280 (Tyr). SMAP-29 was dissolved in distilled water at 20 times the required maximal concentration. Successively, serial dilutions of the peptide were prepared in pyrogen-free water for limulus amebocyte assay assays, in 0.01% acetic acid containing 0.2% bovine serum albumin in polypropylene tubes for in vitro susceptibility tests, and in physiologic saline for in vivo experiments. Polymyxin B (Sigma-Aldrich) and imipenem (Merck, Sharp and Dohme, Milan, Italy) powders were diluted in accordance with manufacturers' recommendations. Solutions were made fresh on the day of assay.

LPS-binding Activity
A quantitative chromogenic limulus amebocyte assay was performed using the QCL-1000 kit (BioWhittaker, Walkersville, MD). Incubations were performed using flat-bottom, nonpyrogenic 96-well tissue culture plates. Stock solutions of SMAP-29 at 64 µg/ml or polymyxin B at 50 µg/ml were prepared in pyrogen-free water and serially diluted in the same solvent. Twenty-five microliter peptide solution and 25 µl of 1 U/ml of LPS from E. coli O111:B4 were mixed in each well, and plates were incubated for 30 minutes at 37°C to allow LPS binding. The mixture was next incubated for 10 minutes in the presence of 50 µl of amebocyte lysate reagent, and 100 µl of the chromogenic substrate (acetyl-Ile-Glu-Ala-Arg-p-nitroanilide) was then added. Incubation was continued at 37°C for 6 minutes, and release of p-nitroaniline was monitored during this time at 405 nm with an Ultra Microplate Spectrophotometer (Bio-Tek Instruments, Inc.). The change in optical density ({Delta}OD) between 0 and 6 minutes was calculated for the control sample, which contained the peptide with no LPS, and this value was subtracted from the {Delta}OD between 0 and 6 minutes for samples containing both the peptide and LPS. Percent peptide–LPS binding (inhibition) was calculated from the quotient (Q) of the {Delta}OD, with peptide divided by the {Delta}OD peptide-free control samples, using the formula (1-Q) x 100. Standard curves generated with increasing amounts of LPS were linear between 0.1 and 1.0 endotoxin units/assay. Statistical comparisons between SMAP-29 and polymyxin B were made by t test analysis of the peptide concentrations that exhibit half-maximal LPS-binding activity (median effective concentration). Significance was accepted when the p value was less than or equal to 0.05.

Experimental Design
Septic shock was induced by two different experimental methods: (1) by intraperitoneal administration of LPS and (2) by cecal ligation and puncture.

Model 1.
Four groups, each containing 15 animals, were anesthetized by intramuscular injection of ketamine (30 mg/kg of body weight) and injected intraperitoneally with 1.0 mg E. coli serotype 0111:B4 LPS in a total volume of 500 µl of sterile saline. Immediately after injection, the animals in the four groups received intraperitoneally isotonic sodium chloride solution (control group C0), 1 mg/kg of SMAP-29, 20 mg/kg of imipenem, and 1 mg/kg of polymyxyn B, respectively.

Model 2.
All animals (four groups, each containing 15 animals) were anesthetized as described previously. The abdomen of each animal was shaved and prepared with iodine. Through a midline laparotomy, the cecum was filled with feces by milking the stools back from the descending colon and then ligated just below the ileocaecal valve with a 3-0 silk ligature. The antimesenteric cecal surface was punctured twice with a 23-gauge needle below the ligature, the bowel was placed back into the peritoneal cavity, and the abdomen was closed in two layers. The operative procedure was done under aseptic conditions. For administration of antibiotics, a catheter was placed into the jugular vein and was sutured to the back of the rat. The drugs were given immediately after the surgical procedure. The rats received isotonic sodium chloride solution (control group C1), 1 mg/kg of SMAP-29, 20 mg/kg of imipenem, and 1 mg/kg of polymyxyn B, respectively.

The same experiments were performed with administration of the drugs 360 minutes after the surgical procedure to better investigate the clinical situation where there is an interval between the onset of sepsis and the initiation of therapy.

For each animal model, toxicity was evaluated on the basis of the presence of any drug-related adverse effects, i.e., local signs of inflammation, anorexia, weight loss, vomiting, diarrhea, fever, and behavioral alterations. In particular, to evaluate the physiologic effects of SMAP-29, temperature, pulse, blood pressure, respirations and oxygenation were monitored in a supplementary SMAP-29–treated group without infection or LPS.

Serum Antibiotic Concentration Measurements and Kinetics
Preventive experiments were performed to measure serum polymyxin B, SMAP-29, and imipenem levels in uninfected animals. Blood samples were obtained from the tail vein of 18 rats (6 rats for each agent) 1, 2, and 4 hours after a single intravenous dose of SMAP-29 (1 mg/kg), polymyxyn B (1 mg/kg), and imipenem (20 mg/kg). Drug levels were measured by bioassay: a spore suspension of Bacillus subtilis ATCC 6633 suspended in tryptic soy agar was used. The plates were read after incubation at 30°C for 18 hours.

Evaluation of Treatment
On the basis of the kind of experiment, at the end of the study the rate of blood culture positivity, the quantities of bacteria in the intraabdominal fluid, the rate of lethality, and plasma endotoxin and TNF-{alpha} levels were evaluated. The animals were monitored for the subsequent 72 hours.

The surviving animals (Model 2) were killed with chloroform, and blood samples for culture were obtained by aseptic percutaneous transthoracic cardiac puncture. In addition, to perform quantitative evaluations of the bacteria in the intraabdominal fluid, 10 ml of sterile saline was injected intraperitoneally, samples of the peritoneal lavage fluid were serially diluted, and a 0.1 ml volume of each dilution was spread onto blood agar plates. The limit of detection was less than or equal to 1 log10 cfu/ml. The plates were incubated both in air and under anaerobic conditions at 35°C for 48 hours. The bacterial isolates were identified by biochemical assay.

For blood cultures and determination of endotoxin and TNF-{alpha} in plasma, 0.2 ml blood samples were collected from a tail vein 0, 2, 6 and 12 hours after injection into a sterile syringe and transferred to tubes containing ethylenediaminetetraacetic acid tripotassium salt.

Biochemical Assays
Endotoxin concentrations were measured by the commercially available limulus amebocyte lysate test (E-TOXATE; Sigma-Aldrich). Plasma samples were serially twofold diluted with sterile endotoxin-free water and were heat treated for 5 minutes in a water bath at 75°C to destroy inhibitors that can interfere with the activation. The endotoxin content was determined as described by the manufacturer. Endotoxin standards (0, 0.015, 0.03, 0.06, 0.125, 0.25, and 0.5 endotoxin units/ml) were tested in each run, and the concentration of endotoxin in the text samples was calculated by comparison with the standard curve. TNF-{alpha} levels were measured with a commercially available solid-phase sandwich ELISA (Nuclear Laser Medicine, S.r.l., Settala, Italy) according to the protocol supplied by the manufacturer. The standards and samples were incubated with a TNF-{alpha} antibody coating 96-well microtitre plate. The wells were washed with buffer and then incubated with biotinylated anti-TNF-{alpha} antibody conjugated to streptavidin–peroxidase.

This was washed away and color was developed in the presence of chromogen (tetramethylbenzidine) substrate. The intensity of the color was measured in a MR 700 Microplate Reader (Dynatech Laboratories, Guernsey, UK) by reading the absorbance at 450 nm. The results for the samples were compared with the standard curve to determine the amount of TNF-{alpha} present. All samples were run in duplicate. The lower limit of sensitivity for TNF-{alpha} by this assay was 0.05 ng/ml. The intraassay and interassay coefficients of variation were 5.0 and 7.1%, respectively.

Statistical Analysis
Survival data were compared by the log-rank test. Qualitative results for blood cultures were analyzed by the {chi}2 test, Yates' correction, or Fisher's exact test, depending on the sample size. Quantitative evaluations of the bacteria in the intraabdominal fluid cultures were presented as means ± SDs of the mean and statistical comparisons between groups were made by analysis of variance. Post hoc comparisons were performed by Bonferroni's test. Plasma endotoxin and TNF-{alpha} mean values were compared between groups by analysis of variance. Each comparison group contained 15 rats. Significance was accepted when the p value was less than or equal to 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Endotoxin Binding
The ability of SMAP-29 to bind LPS from E. coli O111:B4 was determined using a sensitive limulus chromogenic in vitro assay. Polymyxin B, a lipopeptide antibiotic known to bind LPS with high affinity, was used as a positive control in these experiments. As shown in Figure 1 , SMAP-29 binds LPS in the low micrometer range of peptide concentrations and completely inhibits the LPS procoagulant activity at 8 to 10 µM concentration. When compared with polymyxin B on a molar basis, SMAP-29 showed an approximately 10-fold lower inhibition activity (median effective concentration values of 0.45 and 3.2 µM for polymyxin B and SMAP-29, respectively).



View larger version (20K):
[in this window]
[in a new window]
  		Figure 1. Binding of sheep myeloid antimicrobial peptide (SMAP)-29 (open diamonds) and of polymyxin B (POL-B; closed diamonds) to LPS from Escherichia coli O111:B4, as determined by the chromogenic limulus amebocyte assay. Results are means ± SDs from four independent experiments.

 
In Vivo Studies
Model (1): intraperitoneal administration of LPS.
Intraperitoneal SMAP-29 and polymyxin B treatments given immediately after administration of 1.0 mg E. coli serotype 0111:B4 LPS resulted in marked decrease (p < 0.05) of TNF-{alpha} and virtually undetectable levels of endotoxin in the plasma of peptide-treated groups, compared with those of control group (C0), whereas no substantial differences in the plasma levels of both LPS and TNF-{alpha} were observed between imipenem-treated and untreated groups (Table 1) .


View this table:
[in this window]
[in a new window]
  		TABLE 1. Endotoxin and tumor necrosis factor-{alpha} plasma levels in a rat model 6 hours after intraperitoneal administration of 1.0 mg escherichia coli serotype 0111:B4 lps

 
Model (2): cecal ligation and puncture.
The rate of lethality in control groups C1 (antibiotics administered immediately after the surgical procedure, Figure 2) and C2 (antibiotics administered 360 minutes after the surgical procedure, Figure 3) was 100%. All intravenous antibiotic treatments led to decreased mortality (p < 0.05). Specifically, a survival rate of 80.0% was observed for groups treated immediately after intervention with SMAP-29 or with imipenem, whereas a rate of 73.4% was observed in the polymyxin B–treated group (Table 2) . In the same groups, bacteriologic evaluation of C1 showed 100% positive blood cultures, and 9.1 x 108 ± 3.1 x 108 cfu/ml were counted in the intraabdominal fluid. Similar effects on lethality and bacterial counts were observed when the drugs were administered 360 minutes after intervention (Table 3) . Overall, gram-negative and gram-positive bacteria were simultaneously isolated from more than 95% of the rats. The microorganisms isolated from both blood and abdominal fluid were primarily Enterobacteriaceae, including E. coli, Enterobacter spp., Klebsiella spp., and gram-positive cocci. The most frequent anaerobic isolates were Bacteroides spp. SMAP-29 and imipenem showed the highest antimicrobial activities, although no significant differences were observed among the antibiotic-treated groups.




View larger version (28K):
[in this window]
[in a new window]
  		Figure 2. Effects on endotoxin plasma levels of 1 mg/kg SMAP-29, 1 mg/kg POL-B, and 20 mg/kg imipenem (IMP) administered intravenously at 0 and 360 minutes after the operative procedure. Control C1 (diamonds), SMAP-29 (squares), POL-B (triangles), and IMP (circles).

 



View larger version (28K):
[in this window]
[in a new window]
  		Figure 3. Effects on tumor necrosis factor (TNF)-{alpha} levels of 1 mg/kg SMAP-29, 1 mg/kg POL-B, and 20 mg/kg IMP administered intravenously at 0 and 360 minutes after the operative procedure. Control C1 (diamonds), SMAP-29 (squares), POL-B (triangles), and IMP (circles).

 

View this table:
[in this window]
[in a new window]
  		TABLE 2. Efficacy of administration at 0 minutes after the operative procedure of intravenous sheep myeloid antimicrobial PEPTIDE-29, polymyxin b, and imipenem in a rat model of cecal ligation and puncture-induced peritonitis

 

View this table:
[in this window]
[in a new window]
  		TABLE 3. Efficacy of administration at 360 minutes after the operative procedure of intravenous sheep myeloid antimicrobial PEPTIDE-29, polymyxin b, and imipenem in a rat model of cecal ligation and puncture-induced peritonitis

 
In contrast, the administration of drugs at different times after surgical procedures had different impacts on plasma endotoxin and TNF-{alpha} levels. In fact, significant increase in plasma endotoxin and TNF-{alpha} concentrations with mean peak levels achieved at 6 hours postinjection were observed in the control group and imipenem-treated animals when the antibiotics were injected immediately after intervention (Tables 4 and 5) , whereas a constant decrease was produced by administration of SMAP-29 and polymyxin B, 360 minutes after surgical procedure (Tables 6 and 7) . Overall, SMAP-29 and polymyxin B produced significant reductions in plasma endotoxin and TNF-{alpha} levels compared with control and imipenem-treated groups. No significant differences were observed between SMAP-29 and polymyxin B.


View this table:
[in this window]
[in a new window]
  		TABLE 4. Effect of administration at 0 minutes after the operative procedure of intravenous sheep myeloid antimicrobial PEPTIDE-29, polymyxin b, and imipenem on endotoxin plasma levels in a rat model of cecal ligation and puncture-induced peritonitis

 

View this table:
[in this window]
[in a new window]
  		TABLE 5. Effect of administration at 0 minute after the operative procedure of intravenous sheep myeloid antimicrobial PEPTIDE-29, polymyxin b, and imipenem on tumor necrosis factor-{alpha} plasma levels in a rat model of cecal ligation and puncture-induced peritonitis

 

View this table:
[in this window]
[in a new window]
  		TABLE 6. Effect of administration at 360 minutes after the operative procedure of intravenous sheep myeloid antimicrobial PEPTIDE–29, polymyxin b, and imipenem on endotoxin plasma levels in a rat model of cecal ligation and puncture-induced peritonitis

 

View this table:
[in this window]
[in a new window]
  		TABLE 7. Effect of administration at 360 minutes after the operative procedure of intravenous sheep myeloid antimicrobial PEPTIDE-29, polymyxin b, and imipenem on tumor necrosis factor-{alpha} plasma levels in a rat model of cecal ligation and puncture-induced peritonitis

 
Thirty minutes after a single intravenous injection, SMAP-29, polymyxin B, and imipenem reached the peak levels of 1.9 mg/L, 2.1 mg/L, and 62.2 mg/L, respectively.

Finally, none of the polymyxin B– and imipenem-treated animals had clinical evidence of drug-related adverse effects, and only one rat belonging to the SMAP-29–treated group (Model 1) showed anorexia, weight loss, vomiting, and diarrhea. Nevertheless, no changes in physiologic parameters were observed in the supplementary 1 mg/kg SMAP-29–treated group without infection.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Septic shock is a severe clinical syndrome characterized by cytokine release, increased expression of adhesion molecules, chemotactic recruitment of lymphoid cells, increased phagocytotic activity of macrophages, release of reactive oxygen species, and expression of acute-phase proteins (1, 3, 9). LPS is one of the major toxins responsible for initiating this pathophysiologic cascade. For this reason, a bolus of endotoxin has been widely used as a model of sepsis by gram-negative bacteria for initial screening of protective agents (34). Gram-positive or mixed gram-negative and gram-positive infections are also common in humans (35, 36). Therefore, in this study, we used both the animal model of intraperitoneal administration of LPS and the model of cecal ligation and puncture. The latter mimics the clinical condition of bowel perforation and mixed bacterial infection and can be considered a model of peritonitis similar to clinical situations (37, 38). Moreover, to better investigate the efficacy of the compounds in a more common clinical situation, where there is an interval between the onset of sepsis and the initiation of therapy, the same experiments were performed with administration of the drugs 360 minutes after the surgical procedure.

SMAP-29 is a 28-residue, C-terminally amidated cationic peptide belonging to the cathelicidin peptide family (30). This molecule is active in vitro at 0.25 to 4 µM concentrations against a wide panel of bacteria, also including vancomycin-resistant Enterococcus faecalis and methicillin-resistant Staphylococcus aureus isolates, and several fungal species (29, 31, 39), and is toxic to Cryptosporidium parvum sporozoites (40). Preliminary in vivo experiments have shown that SMAP-29, injected intraperitoneally at 0.2 to 1.6 mg/kg, completely protects mice from intraperitoneal doses of E. coli O18:K7:H1, P. aeruginosa, or methicillin-resistant S. aureus that cause over 90% mortality of control animals (29). SMAP-29 is toxic in vitro to human erythrocytes, although at concentrations that are significantly higher than those antimicrobial (67% hemolysis at 100 µM) (31) and shows a mean lethal dose of 5.7 mg/kg when injected intravenously in mice (29). Nevertheless, the peptide is well tolerated when inoculated in sheep lung in a model of acute pulmonary infection (41) and efficiently reduces the bacterial load and the histopathologic lesions at this infection site.

In the present study, the antimicrobial and endotoxin-neutralizing effects of SMAP-29 were compared with those of polymyxin B and imipenem both in vitro and in vivo.

Polymyxin B was used as a control agent because it is well known that polymyxins have direct antimicrobial activity and bind stoichiometrically (1:1) to the lipid A moiety of bacterial LPS. This binding results in the complete neutralization of endotoxin activity (42). Imipenem is one of the most used antimicrobial agents in the empiric antibiotic therapy in septic shock. This compound kills all pathogens that can cause intraabdominal infections including anaerobes, gram-positive cocci, and Enterobacteriaceae (43). It is well known, however, that many clinically used antibiotics such as imipenem can be harmful when administered for the treatment of severe gram-negative bacteria infections, in that they can stimulate the release of endotoxin and thus increase the rates of occurrence of symptoms and life-threatening complications (12, 13). For this reason, alternative compounds such as cationic peptides, that can kill bacteria and neutralize the effects of endotoxin such as cytokine production by LPS-stimulated macrophages, could be very useful in the treatment of gram-negative sepsis. Furthermore, the need for new antiinfective therapies is also incentivized by the recent increase in the rate of multiple-antibiotic resistance among bacteria coupled with increasing immunocompromised and elderly patient populations.

The compounds used in this study were effective versus all parameters considered, regardless of the animal model used, although, in Model 2, in which intraabdominal sepsis was induced via cecal ligation and single puncture, TNF-{alpha} appeared slightly later and its levels were lower than those obtained from Model 1.

Importantly, single intravenous doses of SMAP-29 produced a significant reduction in the TNF-{alpha} plasma levels, compared with both control and imipenem-treated groups. When compared with polymyxin B, SMAP-29 produced a similar decrease in the activity of plasma endotoxin, despite showing a 10-fold lower LPS-binding activity in the in vitro study. Model 2, that also evaluated the antimicrobial effects of the three compounds at different times, showed that SMAP-29 and imipenem exhibit comparable antibacterial activity and both are more effective than polymyxin B (see bacterial counts in Table 2). No significant differences however were observed in the rate of lethality among SMAP-29–, imipenem-, and polymyxin B–treated groups in this model.

Overall, these results highlight the high therapeutic potential of SMAP-29. Due to the potent antimicrobial activity and the ability to neutralize the biological effects of endotoxin, SMAP-29, alone or in combination with other compounds, may offer an important opportunity for a new approach in the therapeutic strategies of this lethal condition.


   FOOTNOTES
 
Supported by the Italian Ministry for University and Research (COFIN 2002, PRIN 2003), C.I.B. 2002, Regione FVG.

Conflict of Interest Statement: A.G. has no declared conflict of interest; O.C. has no declared conflict of interest; R.G. has no declared conflict of interest; F.M. has no declared conflict of interest; G.D. has no declared conflict of interest; R.C. has no declared conflict of interest; F.O. has no declared conflict of interest; B.S. has no declared conflict of interest; C.S. has no declared conflict of interest; V.S. has no declared conflict of interest; M.Z. has no declared conflict of interest; G.S. has no declared conflict of interest.

Received in original form July 17, 2003; accepted in final form October 15, 2003


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bone RC. Pathophysiology of sepsis. Ann Intern Med 1991;115:457–469.
  2. Iwagaki A, Porro M, Pollack M. Influence of synthetic antiendotoxin peptides on lipopolysaccharide (LPS) recognition and LPS-induced proinflammatory cytokine responses by cells expressing membrane-bound CD14. Infect Immun 2000;68:1655–1663.[Abstract/Free Full Text]
  3. Glauser MP, Zanetti G, Baumgartner JD, Cohen J. Septic shock: pathogenesis. Lancet 1991;338:732–736.[CrossRef][Medline]
  4. Hardaway RM. A review of septic shock. Am Surg 2000;66:22–29.[Medline]
  5. Annane D, Aegerter P, Jars-Guincestre MC, Guidet B for CUB-Réa Network. Current epidemiology of septic shock. Am J Respir Crit Care Med 2003;168:165–172.[Abstract/Free Full Text]
  6. Tobin MJ. Critical care medicine in AJRCCM 2002. Am J Respir Crit Care Med 2003;167:294–305.[Free Full Text]
  7. Oberholzer AU, Steckholzer M, Kurimoto O, Trentz Ertel W. Interleukin-18 plasma levels are increased in patients with sepsis compared to severely injured patients. Shock 2001;16:411–414.[Medline]
  8. Villa P, Sartor G, Angelini M, Sironi M, Conni M, Gnocchi P, Isetta AM, Grau G, Buurman W, van Tits LJH, et al. Pattern of cytokines and pharmacomodulation in sepsis induced by cecal ligation and puncture compared with that induced by endotoxin. Clin Diagn Lab Immunol 1995;2:549–553.[Abstract]
  9. Wenzel RP, Pinsky MR, Ulevitch RJ, Young L. Current understanding of sepsis. Clin Infect Dis 1996;22:407–413.[Medline]
  10. Brun-Buisson C, Doyan F, Carlet J, Dellamonica P, Gouin F, Lepoutre A, Mercier JC, Offenstadt G, Regnier B. Incidence, risk factors, and outcome of severe sepsis and septic shock in adults. JAMA 1995;274:968–974.[Abstract]
  11. Wheeler AP, Bernard GR. Treating patients with severe sepsis. N Engl J Med 1999;340:207–214.[Free Full Text]
  12. Prins JM, Kuijper EJ, Mevissen MLCM, Speelman P, van Deventer SJH. Release of tumor necrosis factor alpha and interleukin 6 during antibiotic killing of Escherichia coli in whole blood: influence of antibiotic class, antibiotic concentration, and presence of septic serum. Infect Immun 1995;63:2236–2242.[Abstract]
  13. Byl B, Clevenbergh P, Kentos A, Jacobs F, Marchant A, Vincent JL, Thys JP. Ceftazidime- and imipenem-induced endotoxin release during treatment of gram-negative infections. Eur J Clin Microbiol Infect Dis 2001;20:804–807.[CrossRef][Medline]
  14. Manocha S, Feinstein D, Kumar A, Kumar A. Novel therapies for sepsis: antiendotoxin therapies. Expert Opin Investig Drugs 2002;11:1795–1812.[CrossRef][Medline]
  15. Sharma VK, Dellinger RP. Recent developments in the treatment of sepsis. Expert Opin Investig Drugs 2003;12:139–152.[CrossRef][Medline]
  16. Kotanidou A, Xagorari A, Bagli E, Kitsanta P, Fotsis T, Papapetropoulos A, Roussos C. Luteolin reduces lipopolysaccharide-induced lethal toxicity and expression of proinflammatory molecules in mice. Am J Respir Crit Care Med 2002;165:818–823.[Abstract/Free Full Text]
  17. Dinarello CA, Abraham E. Does blocking cytokines in sepsis work? Am J Respir Crit Care Med 2002;166:1156–1157.[Free Full Text]
  18. Yethon JA, Whitfield C. Lipopolysaccharide as a target for the development of novel therapeutics in gram-negative bacteria. Curr Drug Targets Infect Disord 2001;1:91–106.[CrossRef][Medline]
  19. Lamping N, Dettmer R, Schröder NWJ, Pfeil D, Hallatscheck D, Burger R. LPS-binding protein protects mice from septic shock caused by LPS or Gram-negative bacteria. J Clin Invest 1998;101:2065–2071.[Medline]
  20. Hancock REW. Cationic peptides: effectors in innate immunity and novel antimicrobials. Lancet Infect Dis 2001;1:156–164.[CrossRef][Medline]
  21. Giacometti A, Cirioni O, Greganti G, Quarta M, Scalise G. In vitro activity of membrane-active peptides against Gram-positive and Gram-negative aerobic bacteria. Antimicrob Agents Chemother 1998;42:3320–3324.[Abstract/Free Full Text]
  22. Hancock REW. Peptides antibiotics. Lancet 1997;349:418–422.[CrossRef][Medline]
  23. Gough M, Hancock REW, Kelly NM. Antiendotoxin activity of cationic peptide antimicrobial agents. Infect Immun 1996;64:4922–4927.[Abstract]
  24. Giacometti A, Cirioni O, Ghiselli R, Viticchi C, Mocchegiani F, Riva A, Saba V, Scalise G. Effect of mono-dose intraperitoneal cecropins in experimental septic shock. Crit Care Med 2001;29:1666–1669.[CrossRef][Medline]
  25. Giacometti A, Cirioni O, Ghiselli R, Mocchegiani F, D'Amato G, Del Prete MS, Orlando F, Kamysz W, Lukasiak J, Saba V, et al. Administration of protegrin peptide IB-367 to prevent endotoxin-induced mortality in bile duct-ligated rats. Gut 2003;52:874–878.[Abstract/Free Full Text]
  26. Gallo RL, Murakami M, Ohtake T, Zaiou M. Biology and clinical relevance of naturally occurring antimicrobial peptides. J Allergy Clin Immunol 2002;110:823–831.[CrossRef][Medline]
  27. Gennaro R, Zanetti M. Structural features and biological activities of the cathelicidin-derived antimicrobial peptides. Biopolymers 2000;55:31–49.[CrossRef][Medline]
  28. Travis SM, Anderson NN, Forsyth WR, Espiritu C, Conway BD, Greenberg EP, McCray PB Jr, Lehrer RI, Welsh MJ, Tack BF. Bactericidal activity of mammalian cathelicidin-derived peptides. Infect Immun 2000;68:2748–2755.[Abstract/Free Full Text]
  29. Zanetti M, Gennaro R, Skerlavaj B, Tomasinsig L, Circo R. Cathelicidin peptides as candidates for a novel class of antimicrobials. Curr Pharm Des 2002;8:779–793.[CrossRef][Medline]
  30. Bagella L, Scocchi M, Zanetti M. cDNA sequences of the three sheep myeloid cathelicidins. FEBS Lett 1995;376:225–228.[CrossRef][Medline]
  31. Skerlavaj B, Benincasa M, Risso A, Zanetti M, Gennaro R. SMAP-29: a potent antibacterial and antifungal peptide from sheep leukocytes. FEBS Lett 1999;463:58–62.[CrossRef][Medline]
  32. Guthmiller JM, Vargas KG, Srikantha R, Schomberg LL, Weisroffel PL, McCray PB Jr, Tack BF. Susceptibilities of oral bacteria and yeast to mammalian cathelicidins. Antimicrob Agents Chemother 2001;45:3216–3219.[Abstract/Free Full Text]
  33. Tack BF, Sawai MV, Kearney WR, Robertson AD, Sherman MA, Wang W, Hong T, Boo LM, Wu H, Waring AJ, et al. SMAP-29 has two LPS-binding sites and a central hinge. Eur J Biochem 2002;269:1181–1189.[Medline]
  34. Parker SJ, Watkins PE. Experimental models of Gram-negative sepsis. Br J Surg 2000;88:22–30.
  35. Saklayen MG. CAPD peritonitis: incidence, pathogens, diagnosis, and management. Med Clin North Am 1990;74:997–1010.[Medline]
  36. Castillo M, Toledo-Pereyra LH, Gutierrez R, Prough D, Shapiro E. Peritonitis after cecal perforation. Am Surg 1991;57:313–316.[Medline]
  37. Remick DG, Newcomb DE, Bolgos GL, Call DR. Comparison of the mortality and inflammatory response of two models of sepsis: lipopolysaccharide vs. cecal ligation and puncture. Shock 2000;13:110–116.[Medline]
  38. Tracey KJ, Abraham E. From mouse to man: or what have we learned about cytokine-based anti-inflammatory therapies? Shock 1999;11:224–225.[Medline]
  39. Sambri V, Marangoni A, Giacani L, Gennaro R, Murgia R, Cevenini R, Cinco M. Comparative in vitro activity of five cathelicidin-derived synthetic peptides against Leptospira, Borrelia and Treponema pallidum. J Antimicrob Chemother 2002;50:895–902.[Abstract/Free Full Text]
  40. Giacometti A, Cirioni O, Del Prete MS, Skerlavaj B, Circo R, Zanetti M, Scalise G. In vitro effect on Cryptosporidium parvum of short-term exposure to cathelicidin peptides. J Antimicrob Chemother 2003;51:843–847.[Abstract/Free Full Text]
  41. Brogden KA, Kalfa VC, Ackermann MR, Palmquist DE, McCray PB Jr, Tack BF. The ovine cathelicidin SMAP29 kills ovine respiratory pathogens in vitro and in an ovine model of pulmonary infection. Antimicrob Agents Chemother 2001;45:331–334.[Abstract/Free Full Text]
  42. Drabick JJ, Bhattacharjee AK, Hoover DL, Siber GE, Morales VE, Young LD, Brown SL, Cross AS. Covalent polymyxin B conjugate with human immunoglobulin G as an antiendotoxin reagent. Antimicrob Agents Chemother 1998;42:583–588.[Abstract/Free Full Text]
  43. Jaccard C, Troillet N, Harbarth S, Zanetti G, Aymon D, Schneider R, Chiolero R, Ricou B, Huber O, Ambrosetti P, et al. Prospective randomized comparison of imipenem-cilastatin and piperacillin-tazobactam in nosocomial pneumonia or peritonitis. Antimicrob Agents Chemother 1998;42:2966–2972.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
200307-971OCv1
169/2/187    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Giacometti, A.
Right arrow Articles by Scalise, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Giacometti, A.
Right arrow Articles by Scalise, G.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Cell Mol. Biol.
Copyright © 2004 American Thoracic Society