Vascular allografts are resistant to methicillin-resistant Staphylococcus aureus through indoleamine 2,3-dioxygenase in a murine model A. Saito, MD,

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Vascular allografts are resistant to methicillin-resistant Staphylococcus aureus through indoleamine 2,3-dioxygenase in a murine model A. Saito, MD, PhD, N. Motomura, MD, PhD, K. Kakimi, MD, PhD, K. Narui, PhD, N. Noguchi, PhD, M. Sasatsu, PhD, K. Kubo, MSc, Y. Koezuka, PhD, D. Takai, MD, PhD, S. Ueha, PhD, S. Takamoto, MD, PhD The Journal of Thoracic and Cardiovascular Surgery Volume 136, Issue 1, Pages 159-167 (July 2008) DOI: 10.1016/j.jtcvs.2008.01.006 Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Figure 1 Antimicrobial activity of the allografts and control grafts against methicillin-resistant Staphylococcus aureus (MRSA) proliferation in vitro. The allografts were obtained from either the infrarenal or subcutaneous model at postoperative day 28. The explanted grafts and control grafts were submitted to Mueller–Hinton broth, respectively, and the suspension was applied to mannitol salt agar every hour to estimate the difference in MRSA proliferation. A, MRSA colony count is expressed in the graph by time course. MRSA proliferation was remarkable when cultured with control grafts (open circles), whereas allografts obtained by means of subdermal (open squares) or infrarenal (open diamonds) transplantation presented MRSA growth suppression. B, MRSA proliferation was visualized on the mannitol salt agar 8 hours (left) and 9 hours (right) after incubation. The MRSA proliferation was remarkably suppressed for allografts compared with that seen in control grafts. The Journal of Thoracic and Cardiovascular Surgery 2008 136, 159-167DOI: (10.1016/j.jtcvs.2008.01.006) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Figure 2 Results of mRNA expression for tumor necrosis factor (TNF) α (A), interferon (IFN) γ (B), and indoleamine-2,3-dioxygenase (IDO; C) in the explanted grafts by means of quantitative reverse transcriptase–polymerase chain reaction. The expression level was compared between control grafts (ctrl, small dot bar), allografts (closed bar), and isografts (open bar) at postoperative days (POD) 7 and 28, and the control grafts (without transplantation manipulation) were also examined. TNF-α and IFN-γ levels increased over time in the allografts, and the difference between the allografts and isografts was remarkable at POD28. IDO expression became apparent in the allografts at POD7, whereas almost no expression was observed in the isografts. ∗P < .05. The Journal of Thoracic and Cardiovascular Surgery 2008 136, 159-167DOI: (10.1016/j.jtcvs.2008.01.006) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Figure 3 Histologic detection of inflammatory changes and IDO expression in the allograft. A, Control; B, hematoxylin and eosin stain; C, immunohistochemical staining for indoleamine-2,3-dioxygenase (IDO). There was a remarkable inflammatory cell infiltration in the intima and adventitia (B), where IDO was widely expressed. Bars indicate 50 μm. DAB, Diaminobenzidine. The Journal of Thoracic and Cardiovascular Surgery 2008 136, 159-167DOI: (10.1016/j.jtcvs.2008.01.006) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Figure 4 Antimicrobial activity of tryptophan (Trp; Panel A) and its metabolite against methicillin-resistant Staphylococcus aureus (MRSA) at different concentrations (256 μg/mL, closed circles; 32 μg/mL, closed squares; 8 μg/mL, closed triangles). Bacterial growth was determined based on the optical density (O.D.) at 600 nm by a using microplate photometer. Among those substances, 3-hydroxykynurenine (3-HK) showed relatively strong suppression on MRSA growth at a concentration of 32 μg/mL (C), and 3-hydroxyanthoranilic acid (3-HAA; E) showed a very mild antimicrobial effect at 256 μg/mL. Kyn (Panel B), Kynurenine; 3-HK (Panel C), 3-Hydroxykynurenine; 3-HAA (Panel E), 3-Hydroxyanthoranilic acid; AA (Panel D), anthoranilic acid; QA (Panel F), quinolinic acid. The Journal of Thoracic and Cardiovascular Surgery 2008 136, 159-167DOI: (10.1016/j.jtcvs.2008.01.006) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Figure 5 Kynurenine (Kyn), 3-hydroxykynurenine (3-HK), and 3-hydroxyanthoranilic acid (3-HAA) content in the grafts by means of liquid chromatographic/tandem mass spectrometric (LC-MS/MS) analysis. The grafts, either explanted or control, were homogenized in distilled water, and the supernatant was submitted to LC-MS/MS. By using the standard curve, the content was quantified by measuring the identical peak area and calculating in accordance with the standard curve. Among tryptophan (Trp) and its metabolites, 3-HK was detected in the grafts, with a significant difference found between the allografts and the control grafts (see also Table 1). The Journal of Thoracic and Cardiovascular Surgery 2008 136, 159-167DOI: (10.1016/j.jtcvs.2008.01.006) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions