Vipera aspis venom reduces lethality and down-regulates tumor necrosis factor-a in a rat model of LPS-induced sepsis Inna Frolkis b,1 , Yifat Klein a,1 , Chaim Locker a , Nimrod Adi a , Esther Dahan a , Gideon Uretzsky b , Itzhak Shapira b , Patrick Sorkine a, * a Department of Anesthesiology and Intensive Care, Tel Aviv-Sourasky Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel b Department of Cardiothoracic Surgery, Tel Aviv-Sourasky Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel article info Article history: Received 7 August 2009 Received in revised form 27 October 2009 Accepted 22 November 2009 Keywords: Septic shock Vipera aspis venom (VAV) TNF-a abstract Objectives: Sepsis and septic shock are major causes of morbidity and mortality in critically-ill patients. Sepsis constitutes the systemic response to infection, that is predominantly mediated by the pro-inflam- matory cytokines TNF-a and IL-1b. Hence, cytokine modulation provides a promising target for the treat- ment of sepsis. In this work we evaluated the effect of a low-dose Vipera aspis venom (VAV) vaccine on survival and cytokine serum levels in a rat model of lipopolysaccharide (LPS)-induced septic shock. Meth- ods: Adult male Wistar rats were given either VAV vaccine or saline, and 2 weeks later half of each group received LPS challenge, and were monitored for mortality, cytokine levels, blood count and chemistry. Results: Survival rate was significantly higher in venom-treated, compared to non-vaccinated septic rats. Furthermore, VAV treatment significantly reduced LPS-associated TNF-a and LDH, without affecting IL-6 and IL-10 levels, and modified WBC and platelet counts. Conclusions: Our data suggest that sub-toxic doses of VAV have a protective effect against LPS-induced septic shock that may be mediated, at least par- tially, by the modulated TNF-a activity. This study thus offers a novel therapeutic approach for the atten- uation of bacteremia-induced septic shock through the modulation of a central pro-inflammatory cytokine by VAV vaccination in mammals. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Septic shock is one of the leading causes of death in critically-ill patients and the main cause of death in non-coronary intensive care units [1]. Sepsis occurs in approximately 2% of hospitalized patients and up to 75% of intensive care unit (ICU) patients, with a mortality rate of 20%–50%, despite advances in supportive care and the availability of potent antimicrobials [2–7]. Sepsis constitutes the systemic response to infection. This re- sponse encompasses both pro-inflammatory and anti-inflamma- tory phases that are marked by the sequential generation of pro- and anti-inflammatory cytokines. Among the most important pro-inflammatory cytokines are TNF-a and IL-1b [8–10]. LPS challenge given to experimental animals is commonly used as a preclinical model for gram-negative bacteria-induced septic shock, where it induces various pro-inflammatory cytokines including tumor necrosis factor-a (TNF-a), interleukin-1 (IL-1) and IL-6 [11–13]. An early study demonstrated that passive infusion of antibodies directed against a conserved region of LPS molecule was highly protective against gram-negative bacteremia and shock in human subjects [14]. When this and similar preparations failed to show consistent efficacy, efforts were directed towards other strategies, including cytokine modulation. The toxicity of LPS appears to be mediated by TNF-a. In endo- toxic and septic shock models, application of antibodies against TNF-a leads to increased survival [15,16]. Another study has shown that endotoxin tolerance, induced by repetitive i.p. injec- tions of low-dose LPS, prevents LPS-associated lethality and pro- tects against liver failure subsequent to i.v. LPS application [17]. This was associated with a decrease in TNF-a expression in the li- ver. Altogether these data suggest that a therapeutic approach for septic shock prevention should incorporate anti-inflammatory agents, and may focus on TNF-a, a prominent marker of sepsis [8,9,18], no matter the triggering pathogen. We have previously demonstrated that TNF-a mediates the del- eterious cardiac effects of venom from the venomous viper species, Vipera aspis (VAV) in a rat model of a snakebite [19,20]. In this model TNF-a levels were elevated in the serum of rats following administration of VAV. The cardiac toxicity induced by this venom, could be prevented by administration of either systemic anti-TNF- a antibodies and/or its soluble receptor [19,20]. 1043-4666/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2009.11.019 * Corresponding author. Address: Intensive Care Unit, Tel Aviv-Sourasky Medical Center, 6 Weizmann Street, Tel Aviv 64239, Israel, Tel.: +972 3 6973390; fax: +972 3 6973894. E-mail address: sorkine@tasmc.health.gov.il (P. Sorkine). 1 These authors equally contributed to this work. Cytokine 49 (2010) 319–324 Contents lists available at ScienceDirect Cytokine journal homepage: www.elsevier.com/locate/issn/10434666