Local Gene Therapy with Indoleamine 2,3-Dioxygenase Protects Against Development of Transplant Vasculopathy in Chronic Kidney Transplant Dysfunction

ORIGINAL ARTICLE Local gene therapy with indoleamine 2,3-dioxygenase protects against development of transplant vasculopathy in chronic kidney transplant dysfunction

D Vavrincova-Yaghi1,2,3, LE Deelman1, H van Goor4, MA Seelen5, P Vavrinec1,2, IP Kema6, P Gomolcak7, A Benigni8, RH Henning1 and M Sandovici1,9

Chronic transplant dysfunction (CTD) is the primary cause of late allograft loss in kidney transplantation. Indoleamine 2,3- dioxygenase (IDO) is involved in fetomaternal tolerance and IDO gene therapy inhibits acute rejection following kidney transplantation. The aim of this study is to investigate whether gene therapy with IDO is able to attenuate CTD. Transplantation was performed in a rat Dark-Agouti to Wistar-Furth CTD model. Donor kidneys were incubated either with an adenovirus carrying IDO gene, a control adenovirus or saline. During the first 10 days recipients received low-dose cyclosporine. Body weight, blood pressure, serum creatinine and proteinuria were measured every 2 weeks. Rats were killed after 12 weeks. IDO had a striking beneficial effect on transplant vasculopathy at week 12. It also significantly improved body weight gain; it reduced blood pressure and decreased proteinuria during the follow-up. However, it did not affect the kidney function. In addition, IDO therapy significantly decreased the number of graft-infiltrating macrophages at week 12. The messenger RNA levels of forkhead box p3 and transforming grow factor-β were elevated in the IDO treated group at week 12. Here we show for first time a clear beneficial effect of local IDO gene therapy especially on transplant vasculopathy in a rat model of renal CTD.

Gene Therapy (2016) 23, 797–806; doi:10.1038/gt.2016.59

INTRODUCTION renal transplantation and the search for new targets of interven- Although the rate of acute graft loss has substantially been tion in CTD is still ongoing. reduced during the last decades due to novel (and combination Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme of) immunosuppressive medication together with better organ in the metabolism of tryptophan, whereby the essential amino preservation techniques, long-term graft survival has not acid L-tryptophan is converted into its catabolites, the 11 improved considerably.1,2 Consequently, the main cause of long- kynurenines. It is currently well known that IDO has a 12 term graft loss is the chronic transplant dysfunction (CTD).2 profound immunoregulatory activity, being involved not only 13 CTD is characterized by a progressive loss of renal function, in fetomaternal tolerance, but also in pathological processes proteinuria and de novo or worsening of pre-existing hyperten- such as tumor resistance, chronic infections and autoimmune – sion, coinciding with chronic histopathological lesions of trans- diseases.14 16 In organ transplantation, several studies have plant vasculopathy (TV) with neointima formation, interstitial confirmed the potential of IDO gene therapy in preventing acute fibrosis, tubular atrophy and glomerulosclerosis.3–5 The develop- rejection of skin,17 lungs,18 heart19 and pancreatic islets.20 More ment of CTD is a multifactorial process including both immune recently, it was found that inducible expression of IDO attenuates and nonimmune factors.6,7 Several approaches have been acute rejection of tissue-engineered lung allografts in rats.21 In employed to reduce the occurrence of CTD, including the use of addition, inhibitory effect of locally delivered CTLA4Ig was shown living donors, shortening of cold-ischemia time, the use of to be dependent on IDO activity within the allograft.22 We have machine-perfused instead of cold-stored renal grafts, reduction previously demonstrated protective effects of adenoviral gene of the incidence of acute rejection episodes and optimization of therapy with IDO against acute rejection following kidney chronic immunosuppressive medication.8–10 Despite these efforts, transplantation in rats.23 The evidence about the role of IDO there is still no prevention strategy as well as no effective and/or its therapeutic effect in CTD is however limited. In lungs, treatment available for CTD. Therefore, the improvement of long- sleeping beauty-based gene therapy with IDO inhibited allograft term graft survival remains the major challenge in the field of fibrosis.24 We have recently shown that early posttransplant

1Department of Clinical Pharmacy and Pharmacology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands; 2Faculty of Pharmacy, Department of Pharmacology and Toxicology, Comenius University in Bratislava, Bratislava, Slovakia; 3Graduate School for Drug Exploration - GUIDE, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands; 4Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands; 5Department of Internal Medicine, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands; 6Department of Laboratory Medicine, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands; 7Histopathology Laboratory, Faculty of Medicine, Slovak Medical University, Bratislava, Slovakia; 8Department of Molecular Medicine, IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Ranica, Bergamo, Italy and 9Department of Rheumatology and Clinical Immunology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands. Correspondence: Dr D Vavrincova-Yaghi, Department of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius University in Bratislava, Odbojarov 10, 832 32 Bratislava, Slovakia. E-mail: [email protected] Received 16 March 2016; revised 5 July 2016; accepted 18 July 2016; accepted article preview online 25 July 2016; advance online publication, 4 August 2016 IDO gene therapy of chronic transplant dysfunction D Vavrincova-Yaghi et al 798 tryptophan metabolism predicts long-term outcome of human clinical parameters such as body weight and blood pressure kidney transplantation.25 during the follow-up. Here we employ adenovirus-delivered IDO gene therapy of the renal transplant to study the therapeutic potency of IDO in a rat Urinary protein excretion, plasma creatinine and creatinine model of renal CTD. clearance Proteinuria, a hallmark of progressive renal injury, increased in the RESULTS saline and RGD-AdTL treated groups from, respectively, week 6 and 8 onwards. RGD-AdTIDO treatment significantly attenuated Successful gene delivery and plasma and urinary kyn/trp levels the increase of urinary protein excretion observed in the saline To demonstrate gene delivery in the kidney, four isotransplanta- group (Figure 2c). Plasma levels of creatinine and creatinine tions were performed in Dark Agouti (DA) rats transduced with clearance at the end of the study (12 weeks) were significant, fl arginine-glycine-aspartate (RGD)-AdTL expressing green uores- although mildly altered as compared with the baseline in all three cent protein (GFP). Ten days after transplantation, clear GFP groups. The values of plasma and urine creatinine, and the fi expression was documented mainly in broblast-like interstitial creatinine clearance at the end of the study are summarized in cells in the transduced kidney (Figure 1a), confirming our previous Table 1. RGD-AdTIDO group showed a trend toward increased 23,26 findings. However, no GFP expression was found anymore at levels of urinary creatinine and increased creatinine clearance at 12 weeks in the DA graft in both RGD-AdTL and RGD-AdTIDO the end of the follow-up, compared with the RGD-AdTL and saline group (Figure 1b). Moreover, no human IDO messenger RNA treatment, however, without reaching statistical significance. Thus, (mRNA) could be detected by real-time PCR in the DA grafts of the treatment with RGD-AdTIDO significantly decreased proteinuria in IDO group at 12 weeks. This is in line with our previous findings of time, without significantly affecting kidney function. transgene expression being limited to several weeks with this adenoviral vector. Development of TV, FGS, interstitial pre-fibrosis, interstitial Neither the serum levels of kynurenines and tryptophan, nor macrophage influx and C4d expression those of kyn/trp ratio were significantly different between groups at the end of the study (Table 1). Urinary kyn/trp ratio was slightly CTD in the rat is characterized morphologically by TV, mild glomerular basement membrane thickening and sclerosis, inter- higher in the IDO group at week 4, however, without reaching 2 fi stitial fibrosis and tubular atrophy. TV contributes to the graft statistical signi cance (data not shown). In conclusion, we found 27 GFP expression 10 days after transplantation, however neither GFP loss. The proportion of TV-affected vessels was dramatically staining nor human IDO mRNA was found at 12 weeks, suggesting lower in the IDO treated allografts compared with both RGD-AdTL transient expression of both GFP and IDO. and saline treated group (Figure 3a). When all TV-positive renal vessels were pooled per group, the intraluminal narrowing in the IDO treated allografts was significantly lower compared with both Clinical parameters RGD-AdTL and saline treated group (Figure 3b). IDO treatment had The treatment with RGD-AdTIDO significantly improved body thus a striking beneficial effect on the overall TV by lowering both weight gain of recipients in comparison with rats treated with the proportion of TV-affected vessels as well as the severity of the RGD-AdTL or saline, starting from week 6 after transplantation vasculopathy compared with the saline and control virus groups (Figure 2a). (Figure 3c). Furthermore, we found an abundant influx of Following transplantation, systolic blood pressure increased macrophages in the interstitium in both RGD-AdTL and saline gradually in the allograft recipients treated with saline and RGD- groups as quantified by ED-1-positive cells at 12 weeks. Treatment AdTL from a mean of 129.0 ± 2.5 mm Hg and 128.8 ± 1.8 mm Hg at with RGD-AdTIDO markedly reduced the influx of macrophages the beginning of the follow-up to 162.6 ± 7.7 mm Hg and into the graft (Figure 4). 154.1 ± 10.3 mm Hg at the end of the follow-up, respectively. In Periodic acid-Schiff staining revealed mild focal glomerulo- contrast, the RGD-AdTIDO treated group only showed a modest sclerosis (FGS). Expression of α-smooth muscle actin (SMA) by increase in systolic blood pressure from 128.6 ± 1.4 mm Hg at the immunohistochemistry was used as a marker of pre-fibrosis. RGD- beginning to 136.5 ± 1.8 mm Hg at the end of the follow-up. RGD- AdTIDO treated animals showed a trend toward decreased FGS AdTIDO treatment significantly prevented the rise of systolic blood and α-SMA score, although this did not reach statistical pressure compared with the RGD-AdTL and saline (Figure 2b). significance (Figures 5a and b). As expected, a positive correlation Thus, treatment with RGD-AdTIDO significantly improved relevant was found between the expression of α-SMA and ED-1-positive

Figure 1. GFP expression 10 days and 12 weeks after transplantation. Ten days after DA–DA isotransplantation (n = 4) clear GFP expression was documented in the transduced kidney (a). No GFP expression was found 12 weeks after DA-WF allotransplantation; representative picture from IDO group (b).

Table 1. Serum and urinary creatinine, creatinine clearance, serum and urinary kynurenine and tryptophan, serum and urinary ratio of kynurenine to tryptophan at the end of the study, week 12

Saline n = 5 RGD-AdTL n = 5 RGD-AdTIDO n =7

Serum creatinine (μmol l − 1) 45.00 ± 5.26 49.00 ± 7.38 40.00 ± 4.26 Urine creatinine (mmol l − 1) 6.12 ± 1.07 4.40 ± 0.40 7.27 ± 1.69 Cr. clerance (ml min − 1 kg − 1) 6.26 ± 1.21 3.01 ± 0.20 6.87 ± 0.74 Serum kynurenine (μmol l − 1) 1.70 ± 0.76 1.64 ± 0.20 1.76 ± 0.23 Serum tryptophan (mmol l − 1) 0.07 ± 0.01 0.08 ± 0.04 0.07 ± 0.00 Ratio kyn/trp 24.9 ± 2.6 21.0 ± 2.2 23.6 ± 2.7 Urinary kynurenine (μmol l − 1) 1.94 ± 0.39 1.11 ± 0.32 1.82 ± 0.28 Urinary tryptophan (mmol l − 1) 0.009 ± 0.005 0.004 ± 0.001 0.004 ± 0.001 Ratio kyn/trp 358.0 ± 92.9 320.7 ± 111.9 449.2 ± 47.3 Abbreviations: cr, creatinine; kyn, kynurenine; trp, tryptophan. There were no statistical differences between the groups. cells and between FGS and ED-1-positive cells across all three RGD-AdTIDO treatment decreased thus ACE mRNA, which groups (R = 0.587, Po0.05 and R2 = 0.558, Po0.05). might be a mechanism involved in the decreased blood pressure To address the issue of anti-donor immune responses, we in this group. performed an additional staining of C4d as a surrogate for antibody-mediated rejection. Antibody-mediated rejection is defined as a triad involving the presence of donor-specific DISCUSSION antibodies, positive C4d-peritubular staining on the biopsy and The major finding of the present study is that local adenovirus- histopathological evidence of antibody-mediated injury (glomer- mediated gene therapy with IDO clearly protects against ulitis, peritubular capillaritis and arteritis). In our study, interstitial development of TV, rise of systolic blood pressure and proteinuria C4d expression in RGD-AdTIDO group was not significantly lower in a rat model of renal CTD. as compared with the saline group (0.2 ± 0.18 vs 0.4 ± 0.24, TV leads to impaired perfusion of the graft and subsequent P = 0.72), although it was significantly different from the RGD-AdTL ischemic transplant dysfunction. Development of TV in CTD has group; 0.2 ± 0.18 vs 1.2 ± 0.3, Po0.05. We have not found other been associated with reduced graft survival both in humans and rats.2 Its development is thought to be the result of a local markers of antibody-mediated rejection (such as glomerulitis, fl arteritis or peritubular capillary multilamination). in ammatory response within the vessel wall, which triggers In conclusion, treatment with RGD-AdTIDO dramatically smooth muscle cell migration and proliferation. Endothelial decreased TV and significantly diminished the macrophage influx dysfunction and damage plays an important role in the initiation of TV, because endothelial cells respond to inflammatory in the renal graft. cytokines produced by activated T cells and/or macrophages and because endothelial cells are the first foreign cells encoun- β mRNA expression of foxp3, endogenous IDO and TGF- tered by the host immune system.30,31 In our study, IDO To establish whether IDO treatment affected the number of dramatically improved TV in addition to a reduction of f regulatory T cells (Treg) in the kidney, the expression level of its macrophage influx into the graft. The improvement of TV by marker forkhead box p3 (foxp3) was evaluated by real-time PCR. IDO might be a consequence of reduced initial immune response, RGD-AdTIDO treatment up-regulated the level of foxp3 mRNA together with reduced blood pressure (that is, less shear stress) significantly in comparison with the saline group (Figure 6a). and less influx of macrophages. TGF-β plays an important role in the generation of foxp3+ Reduced graft survival in CTD has also been associated with Treg28 and induction of tolerance.29 We observed a significant hypertension and proteinuria. Hypertension may promote athero- increase in the mRNA expression of TGF-β in IDO treated kidney, sclerosis within renal blood vessels or glomerular hypertension, compared with both saline and RGD-AdTL group (Figure 6b). which can increase glomerular permeability and consequently Finally, expression of renal rat IDO mRNA (rIDO) was quantified enhance protein leak.10 In our study, local gene therapy with IDO to assess whether transfection with human IDO influenced the significantly decreased systolic blood pressure and proteinuria. 32 expression of endogenous IDO. RGD-AdTIDO treatment up- Hofmann et al. showed that metabolization of tryptophan to regulated rIDO compared with the saline group (Figure 6c). There kynurenines by the IDO expressed in endothelial cells contributes was a positive correlation between rIDO and foxp3 mRNA to arterial vessel relaxation and thereby the control of blood (R = 0.556, Po0.05) and between rIDO and TGF-β mRNA pressure, during the inflammation and sepsis. This was mediated o by activation of the adenylate and soluble guanylate cyclase (R = 0.653 and P 0.05). Thus, RGD-AdTIDO treatment increased 32,33 the expression of tolerogenic markers at the end of the follow-up. pathways. Moreover, kynurenine administration decreases blood pressure in a dose-dependent manner in spontaneously hypertensive rats and it has been proposed as a novel Expression of renal RAAS components mRNA endothelium derived vascular relaxing factor.33 There is no As IDO gene therapy markedly attenuated the increase in systolic indication, however, that this mechanism is also involved in the blood pressure following transplantation, without indications for hypertension associated with CTD. In our study, the systemic increased systemic IDO activity, we evaluated the mRNA expres- levels of tryptophan and kynurenines were not different in the rats sion of renin, angiotensin II receptor type 1 (AT1R) and receiving local IDO gene therapy as compared with the controls. angiotensin converting enzyme (ACE) in the transplanted graft. As it is well known that blockade of the renin-angiotensin system Real-time PCR analysis showed no differences in mRNA expression with ACE inhibitors and angiotensin II receptor blockers slow of renin (Figure 7a) and AT1R (Figure 7b) between the down the progression of chronic allograft dysfunction,34 decrease experimental groups, whereas mRNA of ACE was slightly but blood pressure and proteinuria,35 we investigated the gene significantly down-regulated in the RGD-AdTIDO group in expression of renin-angiotensin system in the transplanted comparison with saline and RGD-AdTL groups (Figure 7c). kidneys. IDO did not influence the mRNA expression of renin

© 2016 Macmillan Publishers Limited, part of Springer Nature. Gene Therapy (2016) 797 – 806 IDO gene therapy of chronic transplant dysfunction D Vavrincova-Yaghi et al 800 be mediated by an up-regulation of TGF-β.36 Previously, an immunoregulatory mechanism involving TGF-β and Treg has been identified in mice recipients that had spontaneously accepted renal allografts.28 In those mice, early escape from cellular rejection was associated with regulation of graft-reactive T cells by a transient TGF-β-mediated mechanism. Accumulating data support the role of Treg in long-term allograft acceptance,37–39 while TGF-β appears to be an essential intermediary in this process.29,40 In the same model of spontaneous renal graft acceptance, long-term escape from rejection was associated with regulation of graft-reactive T cells by IDO, while TGF-β-mediated regulation was lost in time. The source of IDO in this model resided in the regulatory dendritic cells, which expressed IDO on stimulation by Treg. Conversely, it has also been shown that IDO+ dendritic cells are capable of stimulating the development of Treg.41,42 The most probable scenario in our study is that the initial expression of IDO in the renal graft led to the formation of Treg, with TGF-β tacking over the stimulation of Treg formation later in time. Another possibility is that the increased rat IDO is the direct or additional factor enhancing the foxp3 and TGF-β mRNA expression in the chronic phase after transplantation. In support to this hypothesis come the positive correlations we found between the rat IDO and both foxp3 and TGF-β mRNA levels. However, the magnitude of the changes in TGF-β and foxp3 cannot account for all the effects we documented. The most important risk factor for development of CTD is the incidence of acute rejection.10 Previously, we demonstrated beneficial effects of IDO gene therapy in protection against acute rejection of the renal graft.23 Therefore, another possible mechanism of the beneficial effects of IDO in CTD is the influence of IDO on acute, immune-mediated graft injury on top of the effect of the low-dose cyclosporine. However, the absence of acute rejection episodes does not preclude development of CTD,27 underlying additional mechanisms involved in CTD. Several gene therapy strategies have previously been employed to ameliorate renal CTD. Benigni et al.,43 showed that adeno- associated virus mediated CTLA4Ig gene transfer protected renal allografts from chronic rejection. Adenovirus-associated IL-10 gene therapy improved renal function and prolonged graft survival in a rat model of CTD.44 Gene therapy with plasmid encoding human hepatocyte growth factor diminished tubulo- interstitial damage, decreased inflammation and prevented late interstitial fibrosis.45 Furthermore, adenovirus-mediated antisense- ERK2 gene therapy attenuated glomerulosclerosis, proteinuria and inflammation in the renal graft in rats.46 Targeting of macrophage Figure 2. Body weight, blood pressure and proteinuria during the activity by adenovirus-mediated gene transfer of an immunor- follow-up. RGD-AdTIDO (n = 7) treatment significantly increased the egulatory cocktail ameliorated proteinuria and graft histology.47 body weight (a), decreased blood pressure (B) and decreased We used an Arg-Gly-Asp (RGD)-modified first generation adeno- proteinuria (c) during 12 week follow-up. *Po0.05 vs saline (n = 5) virus as vector for selective IDO gene delivery into the renal and RGD-AdTL (n = 5); #Po0.05 vs saline. kyn, kynurenine; trp, 23 ± graft. Theoretically, a prolonged expression of the therapeutic tryptophan; wk, week. data are presented as mean s.e.m. AUC gene would be required for long-term immunomodulation via were calculated and expressed as arbitrary units. Significance was tested with analysis of variance followed by a least significant gene therapy. We showed previously that, in the absence of any difference post hoc test. immunosuppression, the RGD-adenovirus-driven transgene expression lasted for only 2–3 weeks.26 Interestingly, when IDO was inserted into the adenovirus and delivered to the kidney and type 1 ATII receptor in the graft, however, it did significantly using the same technique, a clear increase of the GFP expression downregulate ACE mRNA. Thus, rather than a direct effect of in the RGD-AdTIDO compared with RGD-AdTL group at day 7 was kynurenines on vasodilatation, reduction of blood pressure in IDO found.23 This observation suggests that IDO-mediated inhibition treated animals may rely on reduced ACE expression. of T-cell infiltration or function prolongs the duration of human IDO gene therapy did not however significantly influence the IDO expression in the kidney by protection of the transduced cells kidney function throughout the study, although it did ameliorate against immune clearance. Cyclosporine may also prolong the the proteinuria. This apparent discrepancy may reflect ongoing duration of gene expression.48 However, no human IDO was found graft damage insufficient to alter the kidney function. at the end of the experiment. Previously, we have shown that local IDO is currently well known for its immunosuppressive effects, gene delivery with the RGD-modified adenovirus in the kidney one of which being related to generation of Treg. We documented induced selective expression of the transgene in the kidney.49 indeed an increased expression of the Treg marker foxp3 in the Accordingly, we find here no significant differences in systemic IDO group. This may be either a direct effect of IDO expressing (plasma) tryptophan and kynurenine levels, nor in kyn/trp ratio renal cells (in our case mostly the interstitial fibroblasts23) or may during the whole follow-up, suggesting effects limited to the renal

Figure 3. TV 3 months after transplantation. Treatment with RGD-AdTIDO (n = 7) significantly decreased the percentage of TV-affected vessels (presence of neointima formation) in comparison with RGD-AdTL (n = 5) and saline (n = 5) (a); *Po0.05 vs saline and RGD-AdTL. Treatment with RGD-AdTIDO significantly decreased the percentage of intraluminal narrowing when all measured TV-positive renal vessels were pooled per group in comparison with RGD-AdTL and saline (b). The number of TV-affected vessels x percentage intraluminal narrowing of TV-affected vessels (c), *Po0.05 vs saline and RGD-AdTL; representative pictures of saline (d), RGD-AdTL (e), and RGD-AdTIDO (f); data are presented as mean ± s.e.m. Significance was tested with analysis of variance followed by a least significant difference post hoc test.

Figure 4. The influx of macrophages into the interstitium. RGD-AdTIDO treatment (n = 7) significantly decreased the influx of macrophages into the graft (a). *Po0.05 vs saline (n = 5) and RGD-AdTL (n = 5); ED-1, marker for macrophages.; data are presented as mean ± s.e.m. Significance was tested with analysis of variance followed by a least significant difference post hoc test. Representative pictures of saline (b), RGD-AdTL (c) and RGD-AdTIDO (d).

Figure 5. FGS and α-SMA 12 weeks after transplantation. Treatment with RGD-AdTIDO (n = 7) did not inﬂuence signiﬁcantly the FGS (a) and the expression of α-SMA (b).

Figure 6. The effect of RGD-AdTIDO treatment on the mRNA levels of foxp3, TGF-β and rat IDO. RGD-AdTIDO (n = 7) treatment significantly increased the level of foxp3 (a), TGF-β (b) and rat IDO (c) mRNA. *Po0.05 vs saline and RGD-AdTL (n = 5); #Po0.05 vs saline. Values were standardized on GAPDH. Saline was expressed as 100%. Data are presented as mean ± s.e.m. Significance was tested with analysis of variance followed by a least significant difference post hoc test.

graft. This is obviously important in the light of possible systemic already documented protective effects of RGD-adenovirus-IDO in side-effects of any therapy for the transplanted kidney and should the current study. be taken into account when comparing local IDO gene therapy In conclusion, we show here for the first time that local and systemic treatment with tryptophan metabolites, which have adenoviral gene therapy with IDO has clear beneficial effects in a also shown promising results in models of skin, corneal50 and rat model of CTD, with striking protection against develop- liver51 transplantation. ment of TV. There are a number of limitations of the study. One limitation is the lack of an additional treatment arm with an IDO blocker. In addition, the first generation adenoviral vectors are not likely MATERIALS AND METHODS to be used in clinical transplantation. The main reason for Experimental design 2 choosing the first generation adenovirus as vector in our study Experiments were performed in a rat kidney transplant model for CTD, in was the significantly increased transduction potency of the which inbred male Dark Agouti rats (DA/OlaHsd, Haplotype RT1av1) were available Arg-Gly-Asp (RGD)-modified adenovirus for the kidney used as donors and male Wistar Furth (WF/NHsd, Haplotype RT1u) rats fi fi were used as recipients. Body weight of the animals at the time of the as compared with an unmodi ed rst generation and third transplantation was 210–250 g. The animals were housed in a light- and generation (gutless) adenovirus that we found in preliminary, temperature-controlled environment and fed standard rodent chow and short-term studies (refs 23,26 and unpublished data). This is due to water ad libitum. Experimental protocols were approved by the Animal an additional coxsackie adenovirus receptor-independent, RGD- Research Ethics Committee of the University of Groningen, The integrin-dependent cell entry pathway that ca be employed by Netherlands. the RGD-adenovirus as opposed to the unmodified adenovirus. Adenovirus-mediated gene delivery in donor kidneys followed by Moreover, our results suggest that sustained increase of IDO orthotropic kidney transplantation was performed as previously described.26 In brief, donor kidneys were infused with adenovirus solution expression in the kidney allograft may not be necessary to via the renal artery (4 × 1011 viral particles per animal) and incubated for promote long-term protection against graft failure. Nevertheless it 20 min/4 ºC. After perfusion with saline, kidneys were transplanted by an would be of interest to study in the future whether RGD-gutless- end-to-end anastomosis of the renal artery, vein and ureter. Warm adenovirus-mediated IDO gene therapy adds benefit onto the ischemia-time was 25 min. To prevent acute rejection, recipients received

Figure 7. The effect of RGD-AdTIDO treatment on the mRNA levels of renin, AT1R and ACE. RGD-AdTIDO (n = 7) did not affect the levels of renin mRNA (a), and AT1R (b), however, it did significantly downregulate the level of ACE mRNA (b). *Po0.05 vs saline (n = 5) and RGD-AdTL (n = 5); values were standardized on GAPDH; saline was expressed as 100%. Data are presented as mean ± s.e.m. Significance was tested with analysis of variance followed by a least significant difference post hoc test.

low-dose cyclosporine A (5 mg kg − 1 body weight; Sandimmune, Novartis, at 80 °C and further used for mRNA isolation. To document gene Arnhem, The Netherlands) subcutaneously during 10 days post transplan- expression in the renal graft, additional 4 DA to DA isotransplantations tation. The contralateral native kidney was removed after 10 days. were performed, whereby the transplanted kidney was transduced with Three groups of animals were included. In a first group, donor kidneys RGD-AdTL. These rats were killed 10 days thereafter and the transplanted were infused with an RGD-adenovirus carrying the reporter gene GFP and kidney was processed for paraffin embedding and further used for GFP the gene for human IDO (RGD-AdTIDO group). In a second group, an immunostaining. adenovirus carrying the reporter genes GFP and luciferase was used (RGD- AdTL group). An additional group receiving saline was included as control (saline group). Seven animals (3 from RGD-AdTL group, 1 from RGD- Adenoviral vectors AdTIDO group and 3 from saline group) in which surgical and urological RGD-modified adenoviruses52 were used as vectors for gene delivery. complications were observed were terminated at nephrectomy (day 10). A first generation recombinant adenovirus type 5 having an RGD sequence During the follow-up, one rat had to be killed prematurely (in week 8 after in the HI loop was kindly provided by Dr David T. Curiel (University of transplantation) in the RGD-AdTL treated group due to a deterioration of Alabama at Birmingham, Birmingham, AL, USA). This adenovirus contains its general condition, as indicated by piloerected fur and severe weight the genes for GFP (T) and firefly luciferase (L) under the control of a CMV loss (6% weight loss within 2 days). All rats in the RGD-AdTIDO and saline promoter, in the E1 region (RGD-AdTL). To construct the IDO vector, the group survived until the end of the experiment. The prematurely killed rat was excluded from all histological and biochemical analyses IDO gene was isolated from human placenta using PCR. Next, the complementary DNA of human IDO gene was cloned into the shuttle- described below. 53 For the long-term follow-up (12 weeks), the following groups were plasmid pAdTrack-CMV. Using homologous recombination in Escherichia studied: saline group (n = 5), RGD-AdTL group (n = 5) and RGD-AdTIDO coli BJ5183, the shuttle was integrated into the RGD-adenovirus plasmid 54 fi group (n = 7). Rats were housed in individually filtrated cages in ML2 unit. pVK503, resulting in a RGD-modi ed adenovirus genome with GFP and Blood pressure was measured every 2 weeks, blood samples and 24 h human IDO gene under the control of a CMV promoter cloned into the E1 urine were collected every 2 weeks. At the end of the study the rats were region. Viruses were propagated on HEK 293 cells and purified by double anesthetized with isoflurane and an aortic blood sample was collected. CsCl density centrifugation. The amount of viral particles was determined Plasma was isolated and stored at − 80 °C. The kidney was perfused with spectrophotometrically at 260 nm. The infectivity of the viruses was saline and removed. A midcoronal slice was fixed in 4% paraformaldehyde, determined by plaque assay on HEK 293 cells and expressed as plaque − processed for paraffin embedding and further used for (immuno) forming units per milliliter of virus stock (pfu ml 1). The viral particles/pfu histochemistry. A second slice was snap-frozen in liquid nitrogen, stored ratio of the virus stocks was 100:1 for both RGD-AdTL and RGD-AdTIDO.

Gene Therapy (2016) 797 – 806 © 2016 Macmillan Publishers Limited, part of Springer Nature. IDO gene therapy of chronic transplant dysfunction D Vavrincova-Yaghi et al 805 HPLC assay for IDO activity RNA isolation and real-time PCR IDO activity was defined as kynurenine/tryptophan ratio and it assessed Frozen kidney samples containing both the cortex and the medulla were both in plasma and urine at the end of the follow-up. The concentrations homogenized, and RNA was isolated using a Qiagen kit (Qiagen, Venlo, The of tryptophan and kynurenines were measured using a high-throughput Netherlands), which included a DNAse step. RNA (1 μg) was reverse- on-line solid-phase extraction-liquid chromatographic-tandem mass spec- transcribed, and complementary DNA was further used to analyze rat IDO, 55 β trometer (XLC-MS/MS) as described previously. Detection limit was 30 foxp3, human IDO gene, TGF- , renin and ACE gene expression using a − − 49 fi nmol l 1 for tryptophan and 1 nmol l 1 for kynurenines. real-time PCR protocol, as described previously. Sequence-speci c PCR primers were purchased from Biolegio (Nijmegen, The Netherlands). The sequences of the primers were as follows: rat IDO forward: 5′- Blood pressure measurement CTCCGAGAAGAAGTCGAGAA-3′, reverse 5′-TTCTCCAGACTGGCAGCTAT-3′; Before and every 2 weeks after transplantation, systolic blood pressure human IDO forward: 5′-TCATGGAGATGTCCGTAAGG-3′, reverse: 5′-GCC measurements were carried out in conscious animals with the tail-cuff AAGACACAGTCTGCATA-3′; foxp3 forward: 5′-CCACACCTCCTCTTCTTCCTT-3', ′ ′ β ′ plethysmography method (IITC Life Science, Woodland Hills, CA, USA). Rats reverse: 5 -TGACTAGGGGCACTGTAGGC-3 ; TGF- forward: 5 -ATACG ′ ′ ′ were trained to undergo blood pressure measurements 2 weeks before the CCTGAGTGGCTGTCT-3 , reverse: 5 -TGGGACTGATCCCATTGATT-3 ; renin fi forward: 5′-CTGTGCATACTGGCTCTCCA-3′,5′-reverse: GGCTTGGCCTAAA rst measurement. They were placed in restrainers while the temperature ′ ′ ′ – ACTAGGG-3 and ACE forward: 5 -GTGTTGTGGAACGAATACGC-3 , reverse: of the tail was maintained at 35 37 °C. For each rat, the value was ′ ′ fi 5 -CCTTCTTTATGATCCGCTTGA-3 . All values were expressed as saline = calculated as the mean of three to ve consecutive measurements. 100% and standardized on GAPDH.

Markers of the renal damage Statistical analysis Before and every 2 weeks after transplantation, the urinary protein Data are presented as mean ± s.e.m. AUC for blood pressure and excretion was determined by nephelometry (Dade Behring III, The proteinuria were calculated and expressed as arbitrary units (Sigma Plot, Netherlands) in 24 h urine. SPSS Inc., Chicago, IL, USA). Significance was tested with analysis of In addition, plasma and urine creatinine levels were measured using variance followed by a least significant difference post hoc test (SPSS Inc., i-STAT System (Abbott, Den Hague, The Netherlands) and DCA Vantage Chicago, IL, USA). The relationships between ED-1 and α-SMA/FGS and Analyzer+kit (Siemens Healthcare Diagnostics Inc., Deerfield, IL, USA) and between rIDO and foxp3/TGF-β were calculated using Spearman’s creatinine clearance was calculated using the formula creatinine clear- nonparametric correlation (SPSS Inc., Chicago, IL, USA). ance = (urine creatinine × urine flow)/(plasma creatinine × body weight). Difference was considered significant at Po0.05. To assess the degree of glomerular damage and TV, paraffin sections were stained with periodic acid-Schiff and Verhoeff, respectively. A qualified, independent pathologist semiquantitavely scored 50 glomeruli CONFLICT OF INTEREST on a scale of 0–4 by light microscopy in a blinded fashion. FGS was scored The authors declare no conflict of interest. as present when the collapse of capillary lumens, mesangial matrix expansion, hyalinosis and adhesion formation were present in the same quadrant. FGS was scored as 1, 2, 3 and 4 when 25%, 50%, 75% and 100% ACKNOWLEDGEMENTS of the glomerulus was affected, respectively. The total FGS score was This work was supported by a Dutch Kidney Foundation grant, the Netherlands and calculated by multiplying the score by the percentage of glomeruli with by a Science Grant Agency (VEGA 1/0667/14 and VEGA 1/0223/15), Slovak Republic. the same FGS score. The sum of these scores gives the total FGS score, ranging from 0 to 200. The presence of TV was assessed in all elastin- positive arteries with a diameter 4120 μm and a width to length ratio of at AUTHOR CONTRIBUTIONS least 1:3 (longitudinally cut arteries were excluded). The number of arteries Vavrincova-Yaghi wrote the paper, participated in research design, performance with TV as indicated by the presence of neointima formation was of the research and data analyses. Deelman participated in revising the paper, expressed as a percentage of the total amount of vessels that met the research design and data analyses. van Goor participated in revising the paper, aforementioned criteria. Moreover, the severity of intraluminal narrowing research design and immunohistology/histology analyses. Seelen participated was expressed as percentage occlusion of the lumen measured only in TV- in revising the paper, research design and data analyses. Vavrinec participated positive arteries in a blinded fashion.2 in performance of the research and data analyses. Kema participated in sample analyses and revising the paper. Gomolcak participated in immunohistology Immunohistochemistry analysis. Benigni participated in revising the paper. Henning participated in Immunohistochemistry was performed on 3 μm paraffin sections. Sections revising the paper, research design and data analyses. Sandovici participated in were stained for α-SMA (mouse monoclonal anti-α-SMA, 1:100 dilution of revising the paper, research design and data analyses. the 1:150 stock, Sigma Chemical Co, St Louis, MO, USA), GFP (rabbit polyclonal anti-GFP antibody, 1:1000 dilution, Molecular Probes, Leiden, the Netherlands), C4d (polyclonal anti-C4d antibody, Roche-Ventana, REFERENCES Tucson, AZ, USA) and macrophages (mouse monoclonal anti-ED-1, 1:500 1 Savikko J, Rintala JM, Rintala SE, Koskinen PK, von WE. Early short-term imatinib dilution, Serotec Ltd, Oxford, UK). Paraffin sections were dewaxed and treatment is sufficient to prevent the development of chronic allograft nephro- subjected to antigen retrieval by overnight incubation in 0.1 M Tris/HCl pathy. Nephrol Dial Transplant 2011; 26: 3026–3032. buffer, pH 9.0, at 80 °C. A two-step immunoperoxidase technique was used, 2 Waanders F, Rienstra H, Boer MW, Zandvoort A, Rozing J, Navis G et al. Spir- according to standard techniques. Peroxidase activity was developed using onolactone ameliorates transplant vasculopathy in renal chronic transplant 3.3′-diaminobenzidine tetrachloride and H2O2 or AEC chromogen dysfunction in rats. Am J Physiol Renal Physiol 2009; 296: F1072–F1079. substrate. C4d staining was performed on automated BenchMark Ultra 3 Chapman JR, O'Connell PJ, Nankivell BJ. Chronic renal allograft dysfunction. – slide staining system (Roche-Ventana, Tucson, AZ, USA). Tissue was treated J Am Soc Nephrol 2005; 16: 3015 3026. in CC2 solution for 44 min and incubated 12 min at 37 °C. The expression 4 Solez K, Colvin RB, Racusen LC, Sis B, Halloran PF, Birk PE et al. Banff '05 Meeting of ED-1 (CD68) and α-SMA by immunohistochemistry was measured using Report: differential diagnosis of chronic allograft injury and elimination of chronic allograft nephropathy ('CAN'). Am J Transplant 2007; 7:518–526. computer-assisted morphometry. A total of 40 (ED-1) or 30 (α-SMA) fields fi 5 Racusen LC, Solez K, Colvin RB, Bonsib SM, Castro MC, Cavallo T et al. The Banff 97 were evaluated per animal at a magni cation of × 200. For ED-1, the working classification of renal allograft pathology. Kidney Int 1999; 55:713–723. α number of positive cells per area was measured. For -SMA, the total 6 Halloran PF, Melk A, Barth C. Rethinking chronic allograft nephropathy: the staining (excluding glomeruli and arteries) was divided by the area concept of accelerated senescence. J Am Soc Nephrol 1999; 10:167–181. measured, and expressed as a percentage. An average score was 7 Paul LC. Chronic allograft nephropathy-a model of impaired repair from injury? calculated per section. The interstitial C4d staining was scored blindly by Nephrol Dial Transplant 2000; 15:149–151. independent pathologist (0 = none, 1 = weak, 2 = intermediate, 3 = severe 8 de Fijter JW. Rejection and function and chronic allograft dysfunction. Kidney Int and 4 = very severe). An average score was calculated per section. Suppl 2010; 119: S38–S41.

© 2016 Macmillan Publishers Limited, part of Springer Nature. Gene Therapy (2016) 797 – 806 IDO gene therapy of chronic transplant dysfunction D Vavrincova-Yaghi et al 806 9 Arend SM, Mallat MJ, Westendorp RJ, van der Woude FJ, van Es LA. Patient 35 Vavrinec P, van Dokkum RP, Goris M, Buikema H, Henning RH. Losartan protects survival after renal transplantation; more than 25 years follow-up. Nephrol Dial mesenteric arteries from ROS-associated decrease in myogenic constriction Transplant 1997; 12: 1672–1679. following 5/6 nephrectomy. J Renin Angiotensin Aldosterone Syst 2011; 12: 10 Joosten SA, Sijpkens YW, van KC, Paul LC. Chronic renal allograft rejection: 184–194. pathophysiologic considerations. Kidney Int 2005; 68:1–13. 36 Jalili RB, Forouzandeh F, Rezakhanlou AM, Hartwell R, Medina A, Warnock GL et al. 11 Brandacher G, Cakar F, Winkler C, Schneeberger S, Obrist P, Bosmuller C et al. Local expression of indoleamine 2,3 dioxygenase in syngeneic fibroblasts Non-invasive monitoring of kidney allograft rejection through IDO metabolism significantly prolongs survival of an engineered three-dimensional islet allograft. evaluation. Kidney Int 2007; 71:60–67. Diabetes 2010; 59: 2219–2227. 12 Mellor AL, Munn DH. IDO expression by dendritic cells: tolerance and tryptophan 37 Bickerstaff AA, Xia D, Pelletier RP, Orosz CG. Mechanisms of graft acceptance: catabolism. Nat Rev Immunol 2004; 4:762–774. evidence that plasminogen activator controls donor-reactive delayed-type 13 Munn DH, Zhou M, Attwood JT, Bondarev I, Conway SJ, Marshall B et al. Pre- hypersensitivity responses in cardiac allograft acceptor mice. J Immunol 2000; vention of allogeneic fetal rejection by tryptophan catabolism. Science 1998; 164:5132–5139. 281: 1191–1193. 38 Fantini MC, Becker C, Monteleone G, Pallone F, Galle PR, Neurath MF. Cutting 14 Uyttenhove C, Pilotte L, Theate I, Stroobant V, Colau D, Parmentier N et al. edge: TGF-beta induces a regulatory phenotype in CD4+. J Immunol 2004; Evidence for a tumoral immune resistance mechanism based on tryptophan 172:5149–5153. degradation by indoleamine 2,3-dioxygenase. Nat Med 2003; 9:1269–1274. 39 Graca L, Thompson S, Lin CY, Adams E, Cobbold SP, Waldmann H. Both CD4(+) 15 King NJ, Thomas SR. Molecules in focus: indoleamine 2,3-dioxygenase. Int J Bio- CD25(+) and CD4(+)CD25(-) regulatory cells mediate dominant transplantation chem Cell Biol 2007; 39: 2167–2172. tolerance. J Immunol 2002; 168: 5558–5565. 16 Huang L, Baban B, Johnson BA III, Mellor AL. Dendritic cells, indoleamine 2,3 40 Cobbold SP, Castejon R, Adams E, Zelenika D, Graca L, Humm S et al. Induction of dioxygenase and acquired immune privilege. Int Rev Immunol 2010; 29:133–155. foxP3+ regulatory T cells in the periphery of T cell receptor transgenic mice 17 Li Y, Tredget EE, Ghaffari A, Lin X, Kilani RT, Ghahary A. Local expression of tolerized to transplants. J Immunol 2004; 172: 6003–6010. indoleamine 2,3-dioxygenase protects engraftment of xenogeneic skin substitute. 41 Curti A, Pandolfi S, Valzasina B, Aluigi M, Isidori A, Ferri E et al. Modulation of J Invest Dermatol 2006; 126:128–136. tryptophan catabolism by human leukemic cells results in the conversion of. 18 Swanson KA, Zheng Y, Heidler KM, Mizobuchi T, Wilkes DS. CDllc+ cells modulate Blood 2007; 109: 2871–2877. pulmonary immune responses by production of indoleamine 2,3-dioxygenase. 42 Fallarino F, Grohmann U, You S, McGrath BC, Cavener DR, Vacca C et al. The Am J Respir Cell Mol Biol 2004; 30: 311–318. combined effects of tryptophan starvation and tryptophan catabolites down- 19 Li J, Meinhardt A, Roehrich ME, Golshayan D, Dudler J, Pagnotta M et al. Indo- regulate T cell receptor zeta-chain and induce a regulatory phenotype in naive leamine 2,3-dioxygenase gene transfer prolongs cardiac allograft survival. Am J T cells. J Immunol 2006; 176: 6752–6761. Physiol Heart Circ Physiol 2007; 293: H3415–H3423. 43 Benigni A, Tomasoni S, Turka LA, Longaretti L, Zentilin L, Mister M et al. 20 Alexander AM, Crawford M, Bertera S, Rudert WA, Takikawa O, Robbins PD et al. Adeno-associated virus-mediated CTLA4Ig gene transfer protects MHC- Indoleamine 2,3-dioxygenase expression in transplanted NOD Islets prolongs mismatched renal allografts from chronic rejection. J Am Soc Nephrol 2006; 17: graft survival after adoptive transfer of diabetogenic splenocytes. Diabetes 2002; 1665–1672. 51: 356–365. 44 Chen B, Kapturczak MH, Joseph R, George JF, Campbell-Thompson M, Wasserfall 21 Ebrahimi A, Kardar GA, Toolabi L, Ghanbari H, Sadroddiny E. Inducible expression CH et al. Adeno-associated viral vector-mediated interleukin-10 prolongs allograft of indoleamine 2,3-dioxygenase attenuates acute rejection of tissue-engineered survival in a rat kidney transplantation model. Am J Transplant 2007; 7: lung allografts in rats. Gene 2016; 576:412–420. 1112–1120. 22 Xiao B, Liu B, Song Y, Yu Z, Guo S. Local cytotoxic T-lymphocyte-associated 45 Herrero-Fresneda I, Torras J, Franquesa M, Vidal A, Cruzado JM, Lloberas N et al. antigen-4 immunoglobulin inhibition of rejection response is dependent on HGF gene therapy attenuates renal allograft scarring by preventing the indoleamine 2,3-dioxygenase activities in the allograft. Transplant Proc 2014; profibrotic inflammatory-induced mechanisms. Kidney Int 2006; 70:265–274. 46: 3637–3640. 46 Gong N, Dong C, Chen Z, Chen X, Guo H, Zeng Z et al. Adenovirus-mediated 23 Vavrincova-Yaghi D, Deelman LE, Goor H, Seelen M, Kema IP, Smit-van OA et al. antisense-ERK2 gene therapy attenuates chronic allograft nephropathy. Trans- Gene therapy with adenovirus-delivered indoleamine 2,3-dioxygenase improves plant Proc 2006; 38:3228–3230. renal function and morphology following allogeneic kidney transplantation in rat. 47 Yang J, Reutzel-Selke A, Steier C, Jurisch A, Tullius SG, Sawitzki B et al. Targeting of J Gene Med 2011; 13: 373–381. macrophage activity by adenovirus-mediated intragraft overexpression of 24 Liu H, Liu L, Fletcher BS, Visner GA. Sleeping Beauty-based gene therapy with TNFRp55-Ig, IL-12p40, and vIL-10 ameliorates adenovirus-mediated chronic graft indoleamine 2,3-dioxygenase inhibits lung allograft fibrosis. FASEB J 2006; injury, whereas stimulation of macrophages by overexpression of IFN-gamma 20: 2384–2386. accelerates chronic graft injury in a rat renal allograft model. J Am Soc Nephrol 25 Vavrincova-Yaghi D, Seelen MA, Kema IP, Deelman LE, van der Heuvel MC, 2003; 14: 214–225. Breukelman H et al. Early posttransplant tryptophan metabolism predicts long- 48 Cassivi SD, Liu M, Boehler A, Pierre A, Tanswell AK, O'Brodovich H et al. Transplant term outcome of human kidney transplantation. Transplantation 2015; immunosuppression increases and prolongs transgene expression following 99:e97–e104. adenoviral-mediated transfection of rat lungs. J Heart Lung Transplant 2000; 26 Sandovici M, Deelman LE, Smit-van OA, van GH, Rots MG, de ZD et al. Enhanced 19:984–994. transduction of fibroblasts in transplanted kidney with an adenovirus having an 49 Sandovici M, Deelman LE, van GH, Helfrich W, de ZD, Henning RH. Adenovirus- RGD motif in the HI loop. Kidney Int 2006; 69:45–52. mediated interleukin-13 gene therapy attenuates acute kidney allograft injury. 27 Kouwenhoven EA, Ijzermans JN, de Bruin RW. Etiology and pathophysiology of J Gene Med 2007; 9:1024–1032. chronic transplant dysfunction. Transpl Int 2000; 13:385–401. 50 Zaher SS, Coe D, Chai JG, Larkin DF, George AJ. Suppression of the allogeneic 28 Cook CH, Bickerstaff AA, Wang JJ, Nadasdy T, Della PP, Colvin RB et al. Sponta- response by the anti-allergy drug N-(3,4-dimethoxycinnamonyl) anthranilic acid neous renal allograft acceptance associated with "regulatory" dendritic cells results from T-cell cycle arrest. Immunology 2013; 138: 157–164. and IDO. J Immunol 2008; 180: 3103–3112. 51 Sun QF, Ding JG, Sheng JF, Zhu MH, Li JJ, Sheng ZK et al. Novel action of 29 Daley SR, Ma J, Adams E, Cobbold SP, Waldmann H. A key role for TGF-beta 3,4-DAA ameliorating acute liver allograft injury. Cell Biochem Funct 2011; 29: signaling to T cells in the long-term acceptance of allografts. J Immunol 2007; 673–678. 179: 3648–3654. 52 Dmitriev I, Krasnykh V, Miller CR, Wang M, Kashentseva E, Mikheeva G et al. An 30 Mitchell RN, Libby P. Vascular remodeling in transplant vasculopathy. Circ Res adenovirus vector with genetically modified fibers demonstrates expanded 2007; 100: 967–978. tropism via utilization of a coxsackievirus and adenovirus receptor-independent 31 Taflin C, Favier B, Baudhuin J, Savenay A, Hemon P, Bensussan A et al. Human cell entry mechanism. J Virol 1998; 72: 9706–9713. endothelial cells generate Th17 and regulatory T cells under inflammatory 53 He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B. A simplified system for conditions. Proc Natl Acad Sci USA 2011; 108: 2891–2896. generating recombinant adenoviruses. Proc Natl Acad Sci USA 1998; 32 Hofmann F. Ido brings down the pressure in systemic inflammation. Nat Med 95: 2509–2514. 2010; 16: 265–267. 54 Reynolds P, Dmitriev I, Curiel D. Insertion of an RGD motif into the HI loop of 33 Wang Y, Liu H, McKenzie G, Witting PK, Stasch JP, Hahn M et al. Kynurenine is an adenovirus fiber protein alters the distribution of transgene expression of the endothelium-derived relaxing factor produced during inflammation. Nat Med systemically administered vector. Gene Ther 1999; 6:1336–1339. 2010; 16: 279–285. 55 de Jong WH, Smit R, Bakker SJ, de Vries EG, Kema IP. Plasma tryptophan, 34 Rusai K, Schmaderer C, Hermans JJ, Lutz J, Heemann U, Baumann M. Direct renin kynurenine and 3-hydroxykynurenine measurement using automated on-line inhibition in a rat model of chronic allograft injury. Transplantation 2011; solid-phase extraction HPLC-tandem mass spectrometry. J Chromatogr B Anal 92: 999–1004. Technol Biomed Life Sci 2009; 877: 603–609.