- Email: [email protected]
The anti-inflammatory potency of dexamethasone is determined by the route of application in vivo
Immunology Letters 129 (2010) 50–52
Contents lists available at ScienceDirect
Immunology Letters journal homepage: www.elsevier.com/locate/
Letter to the Editor The anti-inflammatory potency of dexamethasone is determined by the route of application in vivo
a r t i c l e
i n f o
Keywords: Inflammation Dexamethasone Glucocorticoids Contact hypersensitivity Endotoxin shock
a b s t r a c t Glucocorticoids (GC) are highly potent anti-inflammatory agents frequently administered in clinical medicine. However, even for the most potent GC dexamethasone, only modest effects have been observed in several murine studies. Here we demonstrate that intraperitoneal administration of dexamethasone displays no anti-inflammatory activity in two different mouse models. Low doses of topically applied dexamethasone entirely prevented ear swelling in a contact hypersensitivity model in BALB/c mice, while intraperitoneally injected dexamethasone had no effect on disease progression. Moreover, subcutaneously administered dexamethasone completely inhibited lipopolysaccharide (LPS)-mediated lethality in C57BL/6 mice. In contrast, even ultra-high doses of intraperitoneally injected dexamethasone could not prevent endotoxin-induced death. In conclusion, these results demonstrate that intraperitoneal application of dexamethasone is ineffective in these models of inflammation, which has broad implications for mouse models evaluating the in vivo efficiency of GCs. © 2010 Elsevier B.V. All rights reserved.
Glucocorticoids (GC) are powerful drugs frequently used in a wide array of inflammatory disorders like allergies, asthma, sepsis, as well as autoimmune diseases such as rheumatoid arthritis, glomerulonephritis, multiple sclerosis, systemic lupus erythematosus, and psoriasis [1]. Dexamethasone is a synthetic GC member and its immunosuppressive potency is about 20–30 times that of hydrocortisone and 4–5 times of prednisone [2]. The potent anti-inflammatory properties of GCs are thought to result from inhibiting multiple immunocompetent cell types such as lymphocytes, neutrophils, eosinophils, mast cells, endothelial cells, monocytes/macrophages, and dendritic cells [1,3–6]. For in vivo studies in animal models GC are usually applied intraperitoneally, subcutaneously, or orally via the drinking water [3]. Interestingly, intraperitoneally applied dexamethasone was remarkably inefficient in preventing pathological changes in certain mouse models of human inflammatory diseases, even when doses up to 100 mg/kg were used [7–14]. It was therefore concluded that the glucocorticoid receptor is not an obligatory regulator of inflammation. While GC have been extensively studied in different murine models to elucidate their anti-inflammatory potencies and molecular mechanisms of action, their effects as they relate to the application route have not been thoroughly investigated. Here we describe two murine models of inflammation using BALB/c and C57BL/6 mice, where intraperitoneal injection of dexamethasone was surprisingly ineffective in the suppression of contact hypersensitivity (CHS) as well as in decreasing endotoxin-induced lethality. First, we tested to what extent the elicitation phase in a CHS model can be inhibited by dexamethasone. Hence, BALB/c mice were sensitized by painting oxazolone (Ox) on their shaved abdomens. Five days later the mice were injected intraperitoneally with 20 g dexamethasone or vehicle (ethanol) 30 min before challenge with Ox on the left ears. Surprisingly, we could not detect any 0165-2478/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2009.12.025
effect of dexamethasone on the recall reaction (Fig. 1A). Therefore, in a second setting we applied 30 g dexamethasone intraperitoneally 30 min before as well as 6 h after challenge. Although a slight decrease in ear swelling in dexamethasone-treated Oxsensitized mice was observed, this difference was not statistically significant (Fig. 1B). However, only 10 g of dexamethasone topically applied onto the left ear 30 min before as well as 6 h after challenge completely inhibited the inflammatory reaction (Fig. 1B). Moreover, this dose was efficient to induce systemic immunosuppression as simultaneous Ox-challenge of the untreated right ear did not provoke any swelling (Fig. 1B). These results indicate that dexamethasone is highly potent in inhibiting CHS when applied topically but not when the substance is injected intraperitoneally. To determine whether the ineffectiveness of intraperitoneally applied dexamethasone might be due to the genetic background of the BALB/c mouse or the CHS model itself, we next investigated the anti-inflammatory potency of dexamethasone in an endotoxin-induced lethality model using C57BL/6 mice. Intraperitoneal injection of 20 mg LPS per kg mouse weight led to lethality of all mice within 48 h (Fig. 2). 600 g (∼30 mg/kg) dexamethasone injected intraperitoneally 30 min before the LPS challenge did not significantly decrease lethality in this model (Fig. 2). In contrast, subcutaneously applied dexamethasone in doses as low as 60 g or even 6 g completely prevented LPS-induced death (Fig. 2). These results indicate that intraperitoneally applied dexamethasone is markedly ineffective in contrast to topical or subcutaneous application. In numerous studies intraperitoneal administration of dexamethasone even at extraordinary high concentrations was remarkably ineffective in preventing inflammation [3,7–15]. While the current literature does not hold an explanation for this phenomenon, we speculate that intraperitoneally applied dex-
Letter to the Editor / Immunology Letters 129 (2010) 50–52
51
Fig. 1. Contact hypersensitivity (CHS) was induced as follows: BALB/c mice (8–12 weeks; n = 5/group) were sensitized with 100 l of 2% oxazolone (Ox; Sigma) on their shaved abdomens on day 0. (A) On day 5, mice were treated intraperitoneally (i.p.) with 20 g dexamethasone (dex; Sigma D8893) dissolved in 10 l ethanol/90 l PBS or vehicle. 30 min thereafter mice were challenged with 10 l of 0.5% Ox or vehicle on the left ear. (B) Ox-sensitized mice were treated on day 5 intraperitoneally with 30 g dex, topically on the left ear with 10 g dex dissolved in 10 l ethanol or with vehicle (ethanol) 30 min before and 6 h after the Ox-challenge on both ears. Ear thickness was measured before and 24 h after challenge with a micrometer (Oditest® , Kroepelin, Schlüchtern, Germany). Bars represent means ± S.D. Statistical analysis was done using Student’s t-test.
Fig. 2. C57BL/6 mice (8–10 weeks; n = 5/group) were injected either intraperitoneally (i.p.) with 600 g dexamethasone (dex) or subcutaneously with 60 g dex or 6 g dex. Thirty minutes thereafter, 20 mg/kg of Escherichia coli LPS 055:B5 (Sigma) was injected and survival of animals was monitored for 5 days. Survival data is shown as Kaplan Meier plot.
amethasone might be degraded by members of the cytochrome P450 superfamily resulting in ineffective metabolites within the peritoneal cavity [16]. In addition, a distinct expression pattern of 11-hydroxysteroid dehydrogenases in the peritoneum might account for this effect [17,18]. In conclusion, we show that topical and subcutaneous but not intraperitoneal application of dexamethasone is highly effective in inhibiting inflammation in two mouse models. Our findings have important implications when assessing the anti-inflammatory features of this potent GC and may ultimately lead to a better understanding of the pharmacology of GCs. Acknowledgment TW and MDS are supported by the Else-Kröner Fresenius Stiftung. References [1] Rhen T, Cidlowski JA. Antiinflammatory action of glucocorticoids—new mechanisms for old drugs. N Engl J Med 2005;353:1711–23.
[2] Mager DE, Moledina N, Jusko WJ. Relative immunosuppressive potency of therapeutic corticosteroids measured by whole blood lymphocyte proliferation. J Pharm Sci 2003;92:1521–5. [3] Tuckermann JP, Kleiman A, Moriggl R, Spanbroek R, Neumann A, Illing A, et al. Macrophages and neutrophils are the targets for immune suppression by glucocorticoids in contact allergy. J Clin Invest 2007;117:1381–90. [4] Sternberg EM. Neural regulation of innate immunity: a coordinated nonspecific host response to pathogens. Nat Rev Immunol 2006;6:318–28. [5] Perretti M, D’Acquisto F. Annexin A1 and glucocorticoids as effectors of the resolution of inflammation. Nat Rev Immunol 2009;9:62–70. [6] Liberman AC, Druker J, Garcia FA, Holsboer F, Arzt E. Intracellular molecular signaling. Basis for specificity to glucocorticoid anti-inflammatory actions. Ann N Y Acad Sci 2009;1153:6–13. [7] Taube M, Carlsten H. Action of dexamethasone in the suppression of delayed-type hypersensitivity in reconstituted SCID mice. Inflamm Res 2000;49:548–52. [8] Chatterjee S, Premachandran S, Shukla J, Poduval TB. Synergistic therapeutic potential of dexamethasone and l-arginine in lipopolysaccharide-induced septic shock. J Surg Res 2007;140:99–108. [9] Xu T, Qiao J, Zhao L, He G, Li K, Wang J, et al. Effect of dexamethasone on acute respiratory distress syndrome induced by the H5N1 virus in mice. Eur Respir J 2009;33:852–60. [10] Liu ML, Lee YP, Wang YF, Lei HY, Liu CC, Wang SM, et al. Type I interferons protect mice against enterovirus 71 infection. J Gen Virol 2005;86:3263–9. [11] Yu Z, Ohtaki Y, Kai K, Sasano T, Shimauchi H, Yokochi T, et al. Critical roles of platelets in lipopolysaccharide-induced lethality: effects of glycyrrhizin and possible strategy for acute respiratory distress syndrome. Int Immunopharmacol 2005;5:571–80. [12] Sadikot RT, Jansen ED, Blackwell TR, Zoia O, Yull F, Christman JW, et al. High-dose dexamethasone accentuates nuclear factor-kappa b activation in endotoxin-treated mice. Am J Respir Crit Care Med 2001;164:873–8. [13] Frode-Saleh TS, Calixto JB. Synergistic antiinflammatory effect of NF-kappaB inhibitors and steroidal or non steroidal antiinflammatory drugs in the pleural inflammation induced by carrageenan in mice. Inflamm Res 2000;49:330–7. [14] Rabbani F, Myers A, Ramey E, Ramwell P, Penhos J. Acute protection against arachidonate toxicity by hydrocortisone and dexamethasone in mice. Prostaglandins 1981;21:699–704. [15] Han SJ, Choi JH, Ko HM, Yang HW, Choi IW, Lee HK, et al. Glucocorticoids prevent NF-kappaB activation by inhibiting the early release of platelet-activating factor in response to lipopolysaccharide. Eur J Immunol 1999;29:1334–41. [16] Gentile DM, Tomlinson ES, Maggs JL, Park BK, Back DJ. Dexamethasone metabolism by human liver in vitro. Metabolite identification and inhibition of 6-hydroxylation. J Pharmacol Exp Ther 1996;277:105–12. [17] Garbrecht MR, Schmidt TJ, Krozowski ZS, Snyder JM. 11Beta-hydroxysteroid dehydrogenase type 2 and the regulation of surfactant protein A by dexamethasone metabolites. Am J Physiol Endocrinol Metab 2006;290:E653–60. [18] Diederich S, Hanke B, Burkhardt P, Muller M, Schoneshofer M, Bahr V, et al. Metabolism of synthetic corticosteroids by 11 beta-hydroxysteroiddehydrogenases in man. Steroids 1998;63:271–7.
Thomas Weichhart ∗,1 Department of Internal Medicine III, Clinical Division of Nephrology and Dialysis, Medical University of
52
Letter to the Editor / Immunology Letters 129 (2010) 50–52
Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria Oliver Brandt 1 Department of Dermatology, Division of Immunology, Allergy and Infectious Diseases - DIAID, Medical University of Vienna, Austria Caroline Lassnig Mathias Müller Biomodels Austria VUW and Institute of Animal Breeding and Genetics, Veterinary University of Vienna, Veterinärplatz 1, A-1210 Vienna, Austria
Marcus D. Säemann Department of Internal Medicine III, Clinical Division of Nephrology and Dialysis, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria ∗ Corresponding
author. Tel.: +43 1 40400 5593; fax: +43 1 40400 4392. E-mail address: [email protected] (T. Weichhart) 1
Walter H. Hörl Department of Internal Medicine III, Clinical Division of Nephrology and Dialysis, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna,Austria Georg Stingl Department of Dermatology, Division of Immunology, Allergy and Infectious Diseases - DIAID, Medical University of Vienna, Austria
Both authors contributed equally. 30 December 2009 Available online 12 January 2010
Contents lists available at ScienceDirect
Immunology Letters journal homepage: www.elsevier.com/locate/
Letter to the Editor The anti-inflammatory potency of dexamethasone is determined by the route of application in vivo
a r t i c l e
i n f o
Keywords: Inflammation Dexamethasone Glucocorticoids Contact hypersensitivity Endotoxin shock
a b s t r a c t Glucocorticoids (GC) are highly potent anti-inflammatory agents frequently administered in clinical medicine. However, even for the most potent GC dexamethasone, only modest effects have been observed in several murine studies. Here we demonstrate that intraperitoneal administration of dexamethasone displays no anti-inflammatory activity in two different mouse models. Low doses of topically applied dexamethasone entirely prevented ear swelling in a contact hypersensitivity model in BALB/c mice, while intraperitoneally injected dexamethasone had no effect on disease progression. Moreover, subcutaneously administered dexamethasone completely inhibited lipopolysaccharide (LPS)-mediated lethality in C57BL/6 mice. In contrast, even ultra-high doses of intraperitoneally injected dexamethasone could not prevent endotoxin-induced death. In conclusion, these results demonstrate that intraperitoneal application of dexamethasone is ineffective in these models of inflammation, which has broad implications for mouse models evaluating the in vivo efficiency of GCs. © 2010 Elsevier B.V. All rights reserved.
Glucocorticoids (GC) are powerful drugs frequently used in a wide array of inflammatory disorders like allergies, asthma, sepsis, as well as autoimmune diseases such as rheumatoid arthritis, glomerulonephritis, multiple sclerosis, systemic lupus erythematosus, and psoriasis [1]. Dexamethasone is a synthetic GC member and its immunosuppressive potency is about 20–30 times that of hydrocortisone and 4–5 times of prednisone [2]. The potent anti-inflammatory properties of GCs are thought to result from inhibiting multiple immunocompetent cell types such as lymphocytes, neutrophils, eosinophils, mast cells, endothelial cells, monocytes/macrophages, and dendritic cells [1,3–6]. For in vivo studies in animal models GC are usually applied intraperitoneally, subcutaneously, or orally via the drinking water [3]. Interestingly, intraperitoneally applied dexamethasone was remarkably inefficient in preventing pathological changes in certain mouse models of human inflammatory diseases, even when doses up to 100 mg/kg were used [7–14]. It was therefore concluded that the glucocorticoid receptor is not an obligatory regulator of inflammation. While GC have been extensively studied in different murine models to elucidate their anti-inflammatory potencies and molecular mechanisms of action, their effects as they relate to the application route have not been thoroughly investigated. Here we describe two murine models of inflammation using BALB/c and C57BL/6 mice, where intraperitoneal injection of dexamethasone was surprisingly ineffective in the suppression of contact hypersensitivity (CHS) as well as in decreasing endotoxin-induced lethality. First, we tested to what extent the elicitation phase in a CHS model can be inhibited by dexamethasone. Hence, BALB/c mice were sensitized by painting oxazolone (Ox) on their shaved abdomens. Five days later the mice were injected intraperitoneally with 20 g dexamethasone or vehicle (ethanol) 30 min before challenge with Ox on the left ears. Surprisingly, we could not detect any 0165-2478/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2009.12.025
effect of dexamethasone on the recall reaction (Fig. 1A). Therefore, in a second setting we applied 30 g dexamethasone intraperitoneally 30 min before as well as 6 h after challenge. Although a slight decrease in ear swelling in dexamethasone-treated Oxsensitized mice was observed, this difference was not statistically significant (Fig. 1B). However, only 10 g of dexamethasone topically applied onto the left ear 30 min before as well as 6 h after challenge completely inhibited the inflammatory reaction (Fig. 1B). Moreover, this dose was efficient to induce systemic immunosuppression as simultaneous Ox-challenge of the untreated right ear did not provoke any swelling (Fig. 1B). These results indicate that dexamethasone is highly potent in inhibiting CHS when applied topically but not when the substance is injected intraperitoneally. To determine whether the ineffectiveness of intraperitoneally applied dexamethasone might be due to the genetic background of the BALB/c mouse or the CHS model itself, we next investigated the anti-inflammatory potency of dexamethasone in an endotoxin-induced lethality model using C57BL/6 mice. Intraperitoneal injection of 20 mg LPS per kg mouse weight led to lethality of all mice within 48 h (Fig. 2). 600 g (∼30 mg/kg) dexamethasone injected intraperitoneally 30 min before the LPS challenge did not significantly decrease lethality in this model (Fig. 2). In contrast, subcutaneously applied dexamethasone in doses as low as 60 g or even 6 g completely prevented LPS-induced death (Fig. 2). These results indicate that intraperitoneally applied dexamethasone is markedly ineffective in contrast to topical or subcutaneous application. In numerous studies intraperitoneal administration of dexamethasone even at extraordinary high concentrations was remarkably ineffective in preventing inflammation [3,7–15]. While the current literature does not hold an explanation for this phenomenon, we speculate that intraperitoneally applied dex-
Letter to the Editor / Immunology Letters 129 (2010) 50–52
51
Fig. 1. Contact hypersensitivity (CHS) was induced as follows: BALB/c mice (8–12 weeks; n = 5/group) were sensitized with 100 l of 2% oxazolone (Ox; Sigma) on their shaved abdomens on day 0. (A) On day 5, mice were treated intraperitoneally (i.p.) with 20 g dexamethasone (dex; Sigma D8893) dissolved in 10 l ethanol/90 l PBS or vehicle. 30 min thereafter mice were challenged with 10 l of 0.5% Ox or vehicle on the left ear. (B) Ox-sensitized mice were treated on day 5 intraperitoneally with 30 g dex, topically on the left ear with 10 g dex dissolved in 10 l ethanol or with vehicle (ethanol) 30 min before and 6 h after the Ox-challenge on both ears. Ear thickness was measured before and 24 h after challenge with a micrometer (Oditest® , Kroepelin, Schlüchtern, Germany). Bars represent means ± S.D. Statistical analysis was done using Student’s t-test.
Fig. 2. C57BL/6 mice (8–10 weeks; n = 5/group) were injected either intraperitoneally (i.p.) with 600 g dexamethasone (dex) or subcutaneously with 60 g dex or 6 g dex. Thirty minutes thereafter, 20 mg/kg of Escherichia coli LPS 055:B5 (Sigma) was injected and survival of animals was monitored for 5 days. Survival data is shown as Kaplan Meier plot.
amethasone might be degraded by members of the cytochrome P450 superfamily resulting in ineffective metabolites within the peritoneal cavity [16]. In addition, a distinct expression pattern of 11-hydroxysteroid dehydrogenases in the peritoneum might account for this effect [17,18]. In conclusion, we show that topical and subcutaneous but not intraperitoneal application of dexamethasone is highly effective in inhibiting inflammation in two mouse models. Our findings have important implications when assessing the anti-inflammatory features of this potent GC and may ultimately lead to a better understanding of the pharmacology of GCs. Acknowledgment TW and MDS are supported by the Else-Kröner Fresenius Stiftung. References [1] Rhen T, Cidlowski JA. Antiinflammatory action of glucocorticoids—new mechanisms for old drugs. N Engl J Med 2005;353:1711–23.
[2] Mager DE, Moledina N, Jusko WJ. Relative immunosuppressive potency of therapeutic corticosteroids measured by whole blood lymphocyte proliferation. J Pharm Sci 2003;92:1521–5. [3] Tuckermann JP, Kleiman A, Moriggl R, Spanbroek R, Neumann A, Illing A, et al. Macrophages and neutrophils are the targets for immune suppression by glucocorticoids in contact allergy. J Clin Invest 2007;117:1381–90. [4] Sternberg EM. Neural regulation of innate immunity: a coordinated nonspecific host response to pathogens. Nat Rev Immunol 2006;6:318–28. [5] Perretti M, D’Acquisto F. Annexin A1 and glucocorticoids as effectors of the resolution of inflammation. Nat Rev Immunol 2009;9:62–70. [6] Liberman AC, Druker J, Garcia FA, Holsboer F, Arzt E. Intracellular molecular signaling. Basis for specificity to glucocorticoid anti-inflammatory actions. Ann N Y Acad Sci 2009;1153:6–13. [7] Taube M, Carlsten H. Action of dexamethasone in the suppression of delayed-type hypersensitivity in reconstituted SCID mice. Inflamm Res 2000;49:548–52. [8] Chatterjee S, Premachandran S, Shukla J, Poduval TB. Synergistic therapeutic potential of dexamethasone and l-arginine in lipopolysaccharide-induced septic shock. J Surg Res 2007;140:99–108. [9] Xu T, Qiao J, Zhao L, He G, Li K, Wang J, et al. Effect of dexamethasone on acute respiratory distress syndrome induced by the H5N1 virus in mice. Eur Respir J 2009;33:852–60. [10] Liu ML, Lee YP, Wang YF, Lei HY, Liu CC, Wang SM, et al. Type I interferons protect mice against enterovirus 71 infection. J Gen Virol 2005;86:3263–9. [11] Yu Z, Ohtaki Y, Kai K, Sasano T, Shimauchi H, Yokochi T, et al. Critical roles of platelets in lipopolysaccharide-induced lethality: effects of glycyrrhizin and possible strategy for acute respiratory distress syndrome. Int Immunopharmacol 2005;5:571–80. [12] Sadikot RT, Jansen ED, Blackwell TR, Zoia O, Yull F, Christman JW, et al. High-dose dexamethasone accentuates nuclear factor-kappa b activation in endotoxin-treated mice. Am J Respir Crit Care Med 2001;164:873–8. [13] Frode-Saleh TS, Calixto JB. Synergistic antiinflammatory effect of NF-kappaB inhibitors and steroidal or non steroidal antiinflammatory drugs in the pleural inflammation induced by carrageenan in mice. Inflamm Res 2000;49:330–7. [14] Rabbani F, Myers A, Ramey E, Ramwell P, Penhos J. Acute protection against arachidonate toxicity by hydrocortisone and dexamethasone in mice. Prostaglandins 1981;21:699–704. [15] Han SJ, Choi JH, Ko HM, Yang HW, Choi IW, Lee HK, et al. Glucocorticoids prevent NF-kappaB activation by inhibiting the early release of platelet-activating factor in response to lipopolysaccharide. Eur J Immunol 1999;29:1334–41. [16] Gentile DM, Tomlinson ES, Maggs JL, Park BK, Back DJ. Dexamethasone metabolism by human liver in vitro. Metabolite identification and inhibition of 6-hydroxylation. J Pharmacol Exp Ther 1996;277:105–12. [17] Garbrecht MR, Schmidt TJ, Krozowski ZS, Snyder JM. 11Beta-hydroxysteroid dehydrogenase type 2 and the regulation of surfactant protein A by dexamethasone metabolites. Am J Physiol Endocrinol Metab 2006;290:E653–60. [18] Diederich S, Hanke B, Burkhardt P, Muller M, Schoneshofer M, Bahr V, et al. Metabolism of synthetic corticosteroids by 11 beta-hydroxysteroiddehydrogenases in man. Steroids 1998;63:271–7.
Thomas Weichhart ∗,1 Department of Internal Medicine III, Clinical Division of Nephrology and Dialysis, Medical University of
52
Letter to the Editor / Immunology Letters 129 (2010) 50–52
Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria Oliver Brandt 1 Department of Dermatology, Division of Immunology, Allergy and Infectious Diseases - DIAID, Medical University of Vienna, Austria Caroline Lassnig Mathias Müller Biomodels Austria VUW and Institute of Animal Breeding and Genetics, Veterinary University of Vienna, Veterinärplatz 1, A-1210 Vienna, Austria
Marcus D. Säemann Department of Internal Medicine III, Clinical Division of Nephrology and Dialysis, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria ∗ Corresponding
author. Tel.: +43 1 40400 5593; fax: +43 1 40400 4392. E-mail address: [email protected] (T. Weichhart) 1
Walter H. Hörl Department of Internal Medicine III, Clinical Division of Nephrology and Dialysis, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna,Austria Georg Stingl Department of Dermatology, Division of Immunology, Allergy and Infectious Diseases - DIAID, Medical University of Vienna, Austria
Both authors contributed equally. 30 December 2009 Available online 12 January 2010