| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
), and bacteriemia. Recipients of BZL, 25 mg/kg, had an increased survival rate at 24 hours after CLP, showing a better clinical situation and a significant reduction of TNF- levels and bacteriemia, with respect to the other groups. BZL failed to inhibit in vitro bacterial growth, suggesting that these effects may be partly caused by the immunomodulatory effects of BZL.
Benznidazole (BZL) has been used for the treatment of acute Chagas’ disease for more than three decades. In addition to its trypanocidal activity, our studies indicate that BZL also affects the synthesis of important biological response modifiers with potential implications in inflammatory processes. By using different experimental approaches, it was shown that BZL downregulated NO and cytokine synthesis by lipopolysaccharide (LPS) and/or interferon (IFN)-
–stimulated murine macrophages.1–3 Systemic treatment with BZL in Trypanosoma cruzi–infected rats inhibited the production of pro-inflammatory cytokines and NO-derived metabolites. Beneficial effects of BZL were also seen in murine experimental endotoxemia. C57BL/6 mice given BZL orally and challenged with LPS intraperitoneally had decreased mortality, reduced serum levels of pro-inflammatory cytokines, diminished number of interleukin (IL)-6–producing peritoneal macrophages, and lowered IL-12 and inducible NO synthase (iNOS) mRNA expression in liver samples.4 These findings provided a stimulating background for analyzing the usefulness of BZL during infections, accompanied by a strong and ultimately detrimental inflammatory reaction.
Ineffective clearance of pathogens or failure of regulatory mechanisms may result in systemic inflammation, further progressing to an aggravated situation such as septic shock.5 Septic shock seems to have a multifactorial basis, with tissue damage being not only caused by the presence of pathogens but also to mediators released in response to infection. Tumor necrosis factor (TNF) is a major inflammatory cytokine in this regard.6 Factors involved in anti-microbial mechanisms also include NO, which plays diverse biological roles (i.e., vascular tone, neurotransmission, and immune response). NO overproduction may be relevant for the development of some pathological events occurring during septic shock.
Given this background, we analyzed the effect of BZL in a closer situation to sepsis, the cecal ligation and puncture (CLP) model. The CLP murine model mirrors more closely the clinical course of abdominal sepsis in humans, because it is triggered by an endogenous septic focus progressing to a polymicrobial infection with systemic inflammatory response syndrome.7–10
Male C57BL/6 mice, 8–10 weeks of age, were provided by the School of Veterinarian Sciences, National University of La Plata. Experiments were conducted according to internationally accepted guidelines for animal handling. Mice were divided into eight experimental groups: 1) sham control plus vehicle (V); 2) sham control plus treatment with BZL 10 mg/kg body weight; 3) sham control plus BZL 25 mg/kg body weight; 4) sham control plus BZL 200 mg/kg body weight; 5) CLP + V; 6) CLP + BZL 10 mg/kg body weight; 7) CLP + BZL 25 mg/kg body weight; and 8) CLP + BZL 200 mg/kg body weight.
The CLP studies were performed according to Baker and others,11 with slight modifications. Mice were anesthetized with a mixture of ketamine (100 mg/kg) and xylacin (10 mg/ kg). Under aseptic conditions, the abdomen was opened to extract and ligate the cecum directly under the ileum–cecum valve. A double puncture using a 21-gauge needle was done to spread the cecal content into the peritoneal cavity. The ligated and perforated cecum was placed in the peritoneal cavity, and the surgical incision was closed with 4-0 silk sutures. All mice received 0.1 mL of normal saline subcutaneously for fluid resuscitation and were placed on a heating pad until they recovered from anesthesia. Sham controls underwent the same surgical procedures, but the cecum was neither ligated nor punctured.
BZL (Roche Laboratories, Buenos Aires, Argentina) was dissolved in carboxymethylcellulose (CMC; Sigma-Aldrich, Buenos Aires, Argentina). Mice were given BZL suspended in 1% CMC through the oral route with an intubation syringe for animal feeding in a 0.1-mL volume. Therapy with BZL was given 2 hours before CLP and every 12 hours after surgery. Control animals received vehicle alone. Preventive therapy was applied to allow drug absorption to further reach pharmacologically active concentrations because it was administered by the oral route. The amount of mice surviving at different time points after the surgical procedure is shown in Table 1
. Most animals were dead by the end of the experiment (96 hours after CLP) regardless of the group under analysis. However, comparisons at 24 hours after CLP showed an increased survival rate in mice given BZL 25 mg/ kg body weight (P < 0.05). These animals showed a better clinical situation, as judged by their response to external stimuli, ability to maintain upright position, and normal breathing. Because recipients of 10- and 25-mg/kg body weight doses were in better condition, further studies concentrated on these two mice groups.
|
and NO-derived metabolites. Values of NO-derived metabolites were within baseline levels (16–29 µmol/L) at 90 minutes and 4 hours after CLP, irrespective of the group under analysis. The same was true for sham control mice at 24 hours after CLP. In contrast, increased amounts of nitrate were observed when analyzing samples from the CLP group taken at 24 hours after CLP compared with values seen in sham operated controls. As shown in Figure 1, we found no significant differences in levels of this mediator between animals with CLP treated with BZL and those who were not.
|
serum levels at 24 hours after CLP showed that mice given BZL 25 mg/kg body weight had the lowest cytokine values, which were significantly different from the CLP + V group (P = 0.04; Figure 2). No measurable amounts of TNF- were found when analyzing samples taken 90 minutes and 4 hour after CLP or in sera from sham control mice.
|
, mice from the CLP + V and CLP + BZL 10 mg/kg groups displayed increased levels of bacteriemia. In contrast, CLP mice given BZL at 25 mg/kg body weight had significantly decreased bacteriemia with respect to the CLP + V group (P < 0.05). To test whether BZL exerted a direct bactericidal effect, the drug was further added to cultures. Cecal content obtained under sterile conditions was cultured on Petri plates containing different culture media: chocolate-agar, blood-agar, cysteine-lactose electrolyte deficient (CLDE), and Salmonella-Shigella, in an attempt to recover habitual bacteria from intestinal flora. Five-millimeter-diameter disks were embedded with dimethyl sulfoxide (DMSO; Sigma) or BZL dissolved in DMSO at concentrations known to mediate in vitro immunomodulatory effects (0.1 and 1 mmol/L) or above that (10 mmol/L). Petri plates were incubated overnight at 37°C under aerobic or anaerobic conditions. In no case was BZL able to inhibit bacterial growth (data not shown).
|
NO overproduction is known to be toxic for different organs and systems; this is related to septic shock pathophysiology. In line with this, NO-derived metabolites were found to be increased in CLP mice 24 hours after surgery. The fact that serum nitrate represents the total amount of NO produced by the host may account for the inability of BZL to decrease its levels, because this compound may only inhibit the iNOS-driven production.
TNF- is involved in the early inflammatory response, and studies in human sepsis showed that increased TNF- concentrations are correlated with a lethal outcome.12 In this study, TNF- was clearly noticeable in circulation 24 hours after surgery, with mice receiving BZL at 25 mg/kg body weight showing significantly decreased levels of this cytokine. Mice given the 25 mg/kg dose also showed a better clinical condition at the same time point evaluation; this was not the case for those treated with 10 mg/kg body weight, for which it may be a suboptimal dose. The fact that mice subjected to BZL 25 mg/kg body weight had a lower bacteriemia by 24 hours after CLP may imply a direct or indirect anti-infectious effect of BZL, resulting in a decreased bacterial load and overall reduced levels of TNF-. Nevertheless, BZL failed to inhibit bacterial growth, for which beneficial effects may be explained by an immunomodulatory effect favoring microbial clearance. Some preliminary studies in peritoneal adherent cells showed fewer yeast-containing cells on treatment with BZL (data not shown). Future studies are needed to ascertain the mechanisms by which this effect is achieved. The fact that bacteriemia levels at an early point were not predictive of the final outcome in this sepsis model suggests that factors other than the pathogen "per se" may be implied in disease outcome (i.e., inflammatory compounds released in response to infection).
Although BZL has been used for > 30 years for treatment of Chagas disease, the mechanisms accounting for its beneficial effects are not yet fully understood. Results from this initial report provide an additional clue for such protective action and show the need for a more in-depth analysis of the mechanisms involved.
Treatment with BZL can result in side effects, such as anorexia, vomiting, peripheral polyneuropathy, hypersensitivity dermatitis, and hematologic disorders, sometimes leading to discontinuation of treatment.13 Because an immunomodulatory-based approach may require a shorter administration period with lower doses of BZL, and hence less toxic, the possibility of testing its use as an adjunct therapy in situations characterized by a systemic inflammatory response should be considered.
Received March 6, 2008. Accepted for publication July 6, 2008.
Acknowledgments: The authors thank Dr. María Teresa Ronco for expert assistance and Dr. Stella Pezzotto for critical help with statistical analysis.
Financial support: This work was supported by grants from Agencia Nacional de Promoción Científica y Tecnológica (BID 1728/OC-AR PICT 17500), Argentina.
* Address correspondence to Romina Manarin, Instituto de Inmunologi, Facultad de Ciencias Médicas, Universidad Nacional de Rosario, Santa Fe 3100, 2000 Rosario, Argentina. E-mail: r.manarin{at}yahoo.com.ar 
Authors’ addresses: Romina Manarin, Emanuel Bottasso, Oscar Bottasso, and Silvia Revelli, Instituto de Inmunologia, Facultad de Ciencias Médicas, Universidad Nacional de Rosario, Santa Fe 3100, 2000 Rosario, Argentina. Romina Manarin and Esteban Serra, Instituto de Biología Molecular y Celular de Rosario (IBR)-CONICET, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, 2100 Rosario, Argentina.
T lymphocytes contributes to mortality and immunosuppression in sepsis. Am J Physiol Regul Integr Comp Physiol 291: R1338–R1343.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
