Mathilde Tamain
email: mathilde.tamain@hotmail.fr

Johnny Sayegh

Arnaud Lionet

Philippe Grimbert

Carole Philipponnet

Marc Hazzan

Jean-François Augusto

Matthias Buchler

Etienne Merlin

George Kosmadakis

Mathias Büchler

Aurélien Tiple

Bruno Pereira

Cyril Garrouste

Anne- Elisabeth Heng

Philippe Lang

Anne-Elisabeth Heng

Extracorporeal photopheresis for the treatment of graft rejection in 33 adult kidney transplant recipients

Keywords: AAMR, acute antibody-mediated rejection ACR, acute cellular rejection AMR, antibody-mediated rejection Anti-HLA Abs, anti-human leukocyte antigen antibodies ASFA, American Society for Apheresis ATG, anti-thymocyte globulin CAMR, chronic antibody-mediated rejection CNIs, calcineurin inhibitors CMV, cytomegalovirus CTCL, cutaneous T cell lymphoma DSAs, donor-specific antibodies EBV, Epstein-Barr virus ECP, extracorporeal photopheresis ESKD, end-stage kidney disease KT, kidney transplantation MFI, mean-fluorescence intensity MMF, mycophenolate mofetil MP, methylprednisolone pulses NK, natural killer lymphocyte PBMCs, peripheral blood mononuclear cells

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Allograft rejection is a major issue in kidney transplantation (KT): episodes of acute allograft rejection were reported to be responsible for 12% of graft loss, with the highest risk in the first six months after transplantation. [START_REF] De | Rapport médical et scientifique de l'agence de la biomédecine[END_REF] Notably, antibody-mediated rejection (AMR) seems to be the leading cause of late graft failure. [START_REF] El-Zoghby | Identifying Specific Causes of Kidney Allograft Loss[END_REF][START_REF] Einecke | Antibody-Mediated Microcirculation Injury Is the Major Cause of Late Kidney Transplant Failure[END_REF] The current immunosuppressive therapies that are used for acute allograft rejection (intravenous steroids, plasmapheresis, rituximab, and intravenous immunoglobulins) are not always effective. [START_REF] Sautenet | One-year Results of the Effects of Rituximab on Acute Antibody-Mediated Rejection in Renal Transplantation: RITUX ERAH, a Multicenter Double-blind Randomized Placebo-controlled Trial[END_REF][START_REF] Fehr | Antibody-mediated kidney allograft rejection: therapeutic options and their experimental rationale: Therapeutic options in antibody-mediated rejection[END_REF] Moreover, these therapies have significant side effects, including direct toxicity and the risk of opportunistic infections and malignancies. [START_REF] Snanoudj | T-Cell-Depleting Antibodies and Risk of Cancer After Transplantation[END_REF][START_REF] Katabathina | Complications of Immunosuppressive Therapy in Solid Organ Transplantation[END_REF] In these situations, alternative methods are needed to optimize the graft outcome while minimizing the associated side effects. [START_REF] Tedla | Intravenous immunoglobulin in kidney transplantation[END_REF] Extracorporeal photopheresis (ECP) is an apheresis immunomodulatory treatment consisting of three stages: (1) the collection of peripheral blood mononuclear cells (PBMCs);

(2) the irradiation of PBMCs with ultraviolet A in the presence of psoralen, a photosensitizing agent; and (3) the reinfusion of treated cells to the patient. [START_REF] Heshmati | Mechanisms of action of extracorporeal photochemotherapy[END_REF] The mechanisms of action of ECP have not been completely elucidated. Ultraviolet A irradiation in the presence of psoralen provokes the cross-linking of deoxyribonucleic acid (DNA), triggering an apoptotic cascade in lymphocytes, especially activated lymphocytes. The proportion of apoptotic lymphocytes is insufficient to explain the ECP effects, but several studies have shown that the early injection of apoptotic cells induces tolerance. [START_REF] Maeda | Intravenous infusion of syngeneic apoptotic cells by photopheresis induces antigen-specific regulatory T cells[END_REF][START_REF] Xia | Extracorporeal photopheresisinduced immune tolerance: a focus on modulation of antigen-presenting cells and induction of regulatory T cells by apoptotic cells[END_REF] Indeed, the phagocytosis of apoptotic cells by antigen-presenting cells is responsible for a shift from a Th1 to a Th2 immune response; an increase in anti-inflammatory cytokine release (Interleukin-10 and TGF-β); a decrease in proinflammatory cytokine release (Interleukin-1α, Interleukin-1β, Interleukin-6 and TNF-α); a maintenance of the immature profile of dendritic cells; and the proliferation of regulatory cells. [START_REF] Heshmati | Updating ECP action mechanisms[END_REF] ECP was developed by Edelson et al. in the 1980s and was first used for the treatment of cutaneous T cell lymphoma (CTCL). [START_REF] Edelson | Treatment of cutaneous T-cell lymphoma by extracorporeal photochemotherapy. Preliminary results[END_REF] The American Society for Apheresis (ASFA) guidelines list ECP as a first-line therapy for CTCL and as a second-line therapy for cutaneous forms of graft-versus host-disease and for the prevention or treatment of rejection in heart and lungs transplant recipients. ,14 Indeed, two prospective studies have shown that ECP combined with standard immunosuppressive therapies as a prophylactic treatment may reduce the rejection rates and severity in heart transplantation. [START_REF] Barr | Prophylactic photopheresis and chronic rejection: effects on graft intimal hyperplasia in cardiac transplantation[END_REF][START_REF] Barr | Photopheresis for the prevention of rejection in cardiac transplantation[END_REF] In KT, several studies have been published on the use of ECP for the prevention or treatment of rejection. [START_REF] Baron | Immunomodulatory effect of extracorporeal photopheresis after successful treatment of resistant renal allograft rejection[END_REF][START_REF] Wolfe | Reversal of acute renal allograft rejection by extracorporeal photopheresis: a case presentation and review of the literature[END_REF][START_REF] Jardine | Photopheresis therapy for problematic renal allograft rejection[END_REF][START_REF] Horina | Photopheresis for renal allograft rejection[END_REF][START_REF] Sunder-Plassman | Renal allograft rejection controlled by photopheresis[END_REF][START_REF] Kusztal | Application of Extracorporeal Photopheresis in Kidney Transplant Recipients: Technical Considerations and Procedure Tolerance[END_REF][START_REF] Kusztal | Extracorporeal Photopheresis as an Antirejection Prophylaxis in Kidney Transplant Recipients: Preliminary Results[END_REF][START_REF] Genberg | Refractory Acute Renal Allograft Rejection Successfully Treated With Photopheresis[END_REF][START_REF] Fernández | Papel de la fotoaféresis en el tratamiento del rechazo celular agudo refractario en trasplante renal[END_REF][START_REF] Dall'amico | Successful treatment of recurrent rejection in renal transplant patients with photopheresis[END_REF][START_REF] Lamioni | Induction of regulatory T cells after prophylactic treatment with photopheresis in renal transplant recipients[END_REF] However, data from larger studies or prospective studies are limited or absent.

In the present study, we report the outcomes of 33 adult KT recipients with allograft rejection who were treated with ECP at 4 French Transplantation Centres. The aim of this study was to describe ECP (1) indications, (2) procedures and (3) results in kidney allograft rejection and to identify the factors influencing ECP efficacy.

Materials and methods

Study design and patients

We performed a multicentre retrospective study of 33 adult KT recipients with allograft rejection who were treated with ECP between 2001 and 2015 at 4 KT Centres: Angers (2 patients), Créteil (11 patients), Clermont-Ferrand (15 patients) and Lille (5 patients). We obtained the authorization of the local Ethics Committee (Comité de Protection des Personnes Sud-Est VI).

Data collection

Patient medical charts and demographic data were retrieved from the hospital registries. The following data were recorded: the age, gender, date of transplantation, postoperative immunosuppressive regimen, complications including cancers and infections, date of rejection, type of rejection with histologic data, rejection treatment and ECP-related data (date, indications, course and complications). We examined the KT outcomes of these patients, including the patient and graft survival, causes of graft loss, and patient death.

Rejection episodes were classified according to the Banff 2013 Classification. [START_REF] Haas | Meeting Report: Inclusion of C4d-Negative Antibody-Mediated Rejection and Antibody-Associated Arterial Lesions: Banff 2013 Meeting Report[END_REF] An acute antibody-mediated rejection (AAMR) diagnosis required (1) histologic lesions of acute or chronic tissue injury; (2) evidence of current/recent antibody interaction with the vascular endothelium (linear C4d staining in peritubular capillaries or at least moderate microvascular inflammation: g+ptc≥2); and (3) the presence of circulating DSAs. A chronic antibodymediated rejection (CAMR) diagnosis required (1) histologic lesions of chronic tissue injury;

(2) evidence of current/recent antibody interaction with the vascular endothelium; and (3) the presence of circulating DSAs. [START_REF] Haas | Meeting Report: Inclusion of C4d-Negative Antibody-Mediated Rejection and Antibody-Associated Arterial Lesions: Banff 2013 Meeting Report[END_REF] We extended these definitions to patients with at least 2 of the criteria. The composite scores of the Banff classification were used; acute composite score consisted of the sum of tubulitis (t), intimal arteritis (v), inflammation (i), glomerulitis (g), and peritubular capillaritis (ptc), whereas the chronic composite score included the sum of the chronic changes in the interstitium (ci), tubules (ct), glomerulus (cg), and vessels (cv). The acute microvascular-inflammatory score consisted of the sum of g and ptc. The acute composite AMR score consisted of the sum of g, ptc, and v. The acute composite ACR consisted of i, t, and v. [START_REF] Sadaka | A Banff Component Scoring-based Histologic Assessment of Bortezomib-based Antibody-mediated Rejection Therapy[END_REF] The stabilization of kidney function was defined as the absence or less than a 10% increase in the serum creatinine levels, whereas the worsening of kidney function was defined as the increase of serum creatinine levels of more than 10% of the baseline values.

Donor-Specific Antibodies (DSAs)

DSAs are expressed as the mean-fluorescence intensity (MFI). If the anti-human leukocyte antigen (anti-HLA) data were missing, old serum samples were used, if available, to perform a local identification of DSAs using a LABScreen® Single Antigen kit (One Lambda®). DSA specificity was defined as an MFI > 1000. A decrease in the DSA MFI > 25% was considered meaningful. [START_REF] Tambur | Sensitization in Transplantation: Assessment of Risk (STAR) 2017 Working Group Meeting Report[END_REF]

ECP procedure

During the procedure, patient blood was collected from a peripheral (n=4) or central venous access (n=4) or from an arteriovenous fistula (AVF) (n=25). The peripheral blood mononuclear cells (PBMCs) were separated by cytapheresis in parallel with the reinfusion of the processed blood to the patient. Then, the PBMCs were treated with methoxypsoralen (8-MOP®). The 8-MOP could be ingested 2 h before the procedure or a liquid form of 8-MOP could be added to the cytapheresis wells. After that, the PBMCs were exposed to ultraviolet A light with a 352 nm wavelength. Finally, the irradiated cells were reinfused into the patient.

Two kinds of techniques were available. The first one was an open system using two unconnected instruments: a flow-cell separator (Spectra Optia®, Cobe Spectra®, COM. TEC®, Amicus® or Haemonetics MCS Plus®) and another device for cell irradiation (PIT System®, Theraflex-ECP® or Macogenic®). The second was a closed system in which the separation, irradiation and reinfusion of the cells were fully integrated and performed automatically (Therakos® UVAR XTS and CELLEX® Photopheresis System). [START_REF] Perotti | A concise review on extracorporeal photochemotherapy: Where we began and where we are now and where are we going![END_REF] A treatment session was defined as one ECP procedure.

Statistical Analysis

Statistical analysis was performed using Stata software (StataCorp, College Station, US). All tests were two-sided, with a type I error of 0.05. Continuous data are expressed as the mean ± standard deviation or as the median [interquartile range], according to the statistical distribution (the assumption of normality was assessed using a Shapiro-Wilk test).

Continuous parameters were compared between independent groups using a Student's t-test or Mann-Whitney test if the assumptions of the t-test were not met ((1) normality and (2) homoscedasticity, as studied with a Fisher-Snedecor test). Concerning categorical parameters, the comparisons were performed using a chi-squared test or, when appropriate, Fisher's exact test. Due to the sample size, non-parametrical tests have often been preferred. Censored data were estimated by the Kaplan-Meier method (the Ying and Wei estimation of variance was used to take the centre effect into account) and were compared between groups by a marginal proportional hazards Cox model to consider the between-and within-centre variability.

These analyses were completed with multidimensional factorial analysis (factorial mixed data analysis, such as FMDA, to analyse the assets as the elements of qualitative and quantitative variables) to uncover the underlying relationships and structures (latent constructs) and to aggregate the subjects into clusters such that each cluster represented a topic to better describe the factors associated with the ECP results. [START_REF] Escoffier | Analyses Factorielles Simples et Multiples[END_REF] The parameters that were selected to be included in the FMDA were determined based on the univariate results and clinical relevance. The analytical study of the equations that were involved showed that the discriminant factors minimized the Mahalanobis distance of each group and maximized the distance between groups, providing compact groups that were spread as much as possible in the space. The usual methodology involves the transformation of the quantitative variables to categorical parameters, categorizing them into classes to ultimately use these new variables and the other variables for an exploratory multiple correspondence analysis (MCA). This methodology is relatively easy to implement and can be used when the sample size is sufficient (n > 100).

Otherwise, the MCA could give unstable and nonrobust results. Additionally, it is interesting to retain, through methods such as FMDA, the quantitative variables, particularly in 2 situations: when the number of categorical variables is very small compared with the number of the quantitative variables and when the sample size is low. 1 and2)

Results

Patient characteristics (Tables

The patient characteristics are summarized in Table 1. The ECP indications, courses and results are summarized in Table 2. All 33 patients had a diagnosed allograft rejection episode in a median time of 11 months [1-216] after transplantation. According to the Banff 2013 classification, the rejection episodes were AAMRs for 23 patients (70%), chronic antibody-mediated rejections (CAMRs) for 2 patients (6%), and acute cellular rejections (ACRs) for 8 patients (24%).

The ECP indications were kidney allograft rejections that were resistant to standard therapies (n=18); or kidney allograft rejections for patients for whom standard therapies were contraindicated due to the presence of active infections or cancers (n=15). Six patients (18%) had concomitant malignancy complications: 3 patients had cutaneous neoplasia (1 basocellular carcinoma, 1 cutaneous T-cell lymphoma and 1 Kaposi sarcoma), 2 patients had B-cell lymphoma and 1 patient had combined prostatic/lung tumours. Twenty-two patients (67%) had concomitant infectious complications, including one or more episodes of the following infections: 11 bacterial infections (4 septicaemia, 3 pneumonia, 2 pyelonephritis, 1 peritonitis and 1 hepatic abscess); 17 viral infections (13 Cytomegalovirus (CMV), 6 BK virus, 2 Epstein-Barr virus (EBV)) and 1 Pneumocystis jiroveci pneumonia.

Five patients (15%) received no additional treatment other than ECP. The other 28 patients received the associated treatments that are described in Table 2. The COBE® Spectra Apheresis System was used for 24 patients (73%), the THERAKOS® UVAR XTS® or CELLEX® Photopheresis System was used for 8 patients (24%), and both systems were consecutively used for 1 patient. The vascular access was an arterio-venous fistula in 25 patients (76%), tunnelled catheters in 4 patients (12%) and a peripheral venous catheter in 4 patients (12%).

ECP was initiated at a frequency of 2 sessions per week in 27 patients (82%) and one session per week in 6 patients (18%) for a period of one month. Thereafter, the session frequency was reduced during the maintenance period of the ECP treatment as follows: one session weekly, then one session every 2 weeks and finally one session monthly. Of the 6 patients who began ECP with one session a week, 5 belonged to the same centre. Patients underwent a median of 12.5 [3-60] ECP sessions over 3.5 months [0.5-67.5]. Twelve patients received less than 10 ECP procedures: 6 of these were dialysis-dependent in the month following the initiation of ECP, and 2 in the 3 months following the initiation of ECP; only one of these had a functional kidney at M12. The reported side effects were bacterial infections in 3 patients (1 catheter-related infection, 1 case of pneumonia, and 1 case of pyelonephritis), one episode of the dysfunction of the extracorporeal circuit with blood loss requiring no transfusion in 2 patients, isolated fever during an ECP session in 1 patient and venous-access dysfunction in 1 patient. Twentysix patients (79%) presented no side effects. 2 and3) Graft survival at 12 months after ECP initiation (M12) (Tables 2 and3; Figure 1) Twenty patients (61%) had a functional graft at M12. Patients with a functional graft at M12 underwent a significantly longer duration of ECP (4.5 months [1-67] versus 1.5 months [0.5-11] (p= 0.05)) and a higher number of ECP sessions (15 [7-60] versus 9 [3-29] (p=0.04)) compared to those of patients with a graft loss at M12. The acute composite AMR score was a risk factor for graft loss at M12 (HR: 1.44 [1.01-2.05]; p= 0.04) (Table 3; Figure 1B).

ECP results (Tables

Serum creatinine levels of more than 276 µmol/L at ECP initiation were associated with an increased probability of graft loss at M12, with an estimated sensitivity of 80% and an estimated specificity of 75%. Serum creatinine levels of more than 412 µmol/L at ECP initiation were associated with an increased probability of graft loss at M12, with an estimated sensitivity of 50% and an estimated specificity of 100%. The associated ROC curve is presented in Figure 2, with an area under the ROC curve of 0.82. None of the 3 patients who began ECP while they were dialysis-dependent recovered their renal function. Eight patients (24%) had a serum creatinine level higher than 412 µmol/L at ECP initiation. Among the 13 patients with graft loss at M12, 6 were dialysis-dependent in the month following ECP initiation and received less than 10 ECP procedures. Moreover, 4 patients were dialysisdependent in the month following ECP completion. 2 and3 versus 4 [0-6] (p=0.018)) compared to those of patients who did not experience a stabilization of kidney function (Table 2; Figure 1A).

Effects on the renal function at 12 months after ECP (M12) (Tables

Results of the Factorial Mixed Data Analysis (Figure 3)

The FMDA defined two clusters of patients. The cluster of patients with a functional graft at 12 months after ECP initiation tended to have cellular and/or chronic rejections and associated cancers. The cluster of patients with graft loss at 12 months after ECP tended to have acute and/or antibody-mediated rejections, higher serum creatinine levels, higher DSA levels, higher histologic scores of AMR and a longer delay between the rejection and ECP initiation compared to those in patients who did not experience graft loss.

Impact of ECP on the DSA MFI levels

There was no significant decrease in the immunodominant DSA MFI levels after ECP compared to those at baseline, but the pre-and post-ECP data were available for only 11 patients. Four patients presented a decrease of more than 25% of their immunodominant DSA MFI levels after ECP (14099 to 10715; 14117 to 9442; 7761 to 0 and 4000 to 1000, respectively).

Focus on ECP results in patients treated by ECP as a single therapy

Five patients (15%) were treated by ECP as a single therapy. Three of these patients had AAMR associated with either simultaneous lung and prostatic cancers, basocellular carcinoma or CMV infection. These patients all had a functional graft at M12. The total graft survival after ECP initiation was 24 and 72 months, respectively, in two patients. The other patient still had a functional graft at M24. All 3 of these patients had more than a 25% decrease in their immunodominant DSA MFI levels after ECP. One patient who was treated with ECP as a single therapy had CAMR associated with a CTCL and died with a functional graft at M22. The last patient had ACR associated with severe bacterial infections and began ECP while being dialysis-dependent and did not recover their kidney function.

Discussion

This multicentre retrospective study is the largest to report the use of ECP for kidney allograft rejection. Of the 33 patients with allograft rejections who were treated by ECP, 61% had a functional graft at M12, and 33% had a stabilization of kidney function. Interestingly, 3 patients with AAMR who were treated by ECP as a single treatment due to concomitant infections or cancers still had a functional graft at M12. Data from the literature about the use of ECP in KT are scarce and are conflicting regarding the treatment of rejection (Table 4). A case series reported 3 KT recipients with rejection episodes (2 chronic and 1 acute) who were unsuccessfully treated with ECP, which was performed on 2 consecutive days per month for 3 months. [START_REF] Horina | Photopheresis for renal allograft rejection[END_REF] Studies with more intensive ECP regimens seemed to report better results. Of 28 cases of KT rejections, ECP seemed to help to stabilize renal function in 14 patients (50%) and to improve renal function in 8 patients (29%). [START_REF] Baron | Immunomodulatory effect of extracorporeal photopheresis after successful treatment of resistant renal allograft rejection[END_REF][START_REF] Wolfe | Reversal of acute renal allograft rejection by extracorporeal photopheresis: a case presentation and review of the literature[END_REF][START_REF] Jardine | Photopheresis therapy for problematic renal allograft rejection[END_REF][START_REF] Horina | Photopheresis for renal allograft rejection[END_REF][START_REF] Sunder-Plassman | Renal allograft rejection controlled by photopheresis[END_REF][START_REF] Kusztal | Application of Extracorporeal Photopheresis in Kidney Transplant Recipients: Technical Considerations and Procedure Tolerance[END_REF][START_REF] Kusztal | Extracorporeal Photopheresis as an Antirejection Prophylaxis in Kidney Transplant Recipients: Preliminary Results[END_REF][START_REF] Genberg | Refractory Acute Renal Allograft Rejection Successfully Treated With Photopheresis[END_REF][START_REF] Fernández | Papel de la fotoaféresis en el tratamiento del rechazo celular agudo refractario en trasplante renal[END_REF][START_REF] Dall'amico | Successful treatment of recurrent rejection in renal transplant patients with photopheresis[END_REF][START_REF] Lamioni | Induction of regulatory T cells after prophylactic treatment with photopheresis in renal transplant recipients[END_REF] The « French Haute Autorité de Santé (HAS) » recommended the use of ECP in the prevention of rejection in cardiac transplantation. [START_REF]Photochimiothérapie extracorporelle : système fermé et système ouvert[END_REF] However, the evidence level was too weak to extend the recommendations to other solid organ transplantations. [START_REF]Photochimiothérapie extracorporelle : système fermé et système ouvert[END_REF] Nonetheless, the experts reported that ECP could be used in the prevention and/or treatment of refractory acute rejection, and in cases in which immunosuppressive treatment reduction was required because of infections post-transplantation lymphoproliferative disorders, as in most of the cases in our study. [START_REF]Photochimiothérapie extracorporelle : système fermé et système ouvert[END_REF] A large proportion of the patients in our study (82%) had a frequency of ECP sessions that were in accordance with the standard protocol used in France (2 sessions per week for 4 weeks, followed by 1 session per week for at least 3 to 6 months). [START_REF]Photochimiothérapie extracorporelle : système fermé et système ouvert[END_REF] This study is the first to identify predictive factors for response to ECP treatment. The FMDA highlighted that ECP could be particularly interesting in the treatment of cellular and/or chronic rejections and in the case of concomitant cancers. However, ECP had efficacy limitations. Indeed, ECP was systematically ineffective in patients with poor renal function at initiation (serum creatinine level > 412 µmol/L), which is consistent with the data in the literature. [START_REF] Clayton | Relationship between eGFR Decline and Hard Outcomes after Kidney Transplants[END_REF] Moreover, both univariate analysis and FMDA showed that ECP seemed to be less effective in patients with severe AAMR (high DSA levels and high histologic scores of AAMR) and in patients for whom there was a long delay between the rejection and ECP initiation. These results are consistent with the data in the literature about the efficacy of other treatments, such rituximab, plasmapheresis and intravenous immunoglobulins, in the treatment of AMR in KT, showing that the features of severe AMR are associated with graft loss. [START_REF] Loupy | Antibody-Mediated Rejection of Solid-Organ Allografts[END_REF][START_REF] Sautenet | One-year Results of the Effects of Rituximab on Acute Antibody-Mediated Rejection in Renal Transplantation: RITUX ERAH, a Multicenter Double-blind Randomized Placebo-controlled Trial[END_REF] In the present study, the ECP indications for the treatment of rejection were often related to the concomitant presence of cancers (18%) or infections (67%). This could be because such conditions usually lead to decreased immunosuppressive therapies, increasing the risk of allograft rejection and not allowing for additional potentially deleterious immunosuppressant therapies. [START_REF] Peraldi | Natural Killer Lymphocytes Are Dysfunctional in Kidney Transplant Recipients on Diagnosis of Cancer: Transplantation[END_REF][START_REF] Martin-Gandul | The Impact of Infection on Chronic Allograft Dysfunction and Allograft Survival After Solid Organ Transplantation: Infection and Chronic Allograft Dysfunction[END_REF] In such cases, ECP could be an interesting alternative treatment because of its immunomodulatory properties. [START_REF] Ebrahimi | Recent immunomodulatory strategies in transplantation[END_REF] A recent study showed that KT recipients with cancer had a higher incidence of acute rejection and presented a dysfunction of natural killer lymphocytes (NK). [START_REF] Peraldi | Natural Killer Lymphocytes Are Dysfunctional in Kidney Transplant Recipients on Diagnosis of Cancer: Transplantation[END_REF] Interestingly, ECP-treated patients have been reported to have a higher frequency of NK cells than that in controls. [START_REF] Zhu | The effect of extracorporeal photopheresis on T cell response in chronic graft-versus-host disease[END_REF] Furthermore, ECP has been demonstrated to promote Interleukin-1β production, which synergizes with IL-2 to induce tumour-specific cytotoxic CD8+ T cells and plays a critical role in the priming of CD8+ T cell-mediated antitumour immunity by dendritic cells. [START_REF] Yakut | Extracorporeal Photopheresis Promotes IL-1 Production[END_REF] Through these mechanisms, ECP may contribute to anti-tumoural immune responses. Limited data are available to support the role of ECP in immune responses to infections. However, in one study, ECP was used as a prophylactic treatment for allograft rejection in heart transplantation and was associated with a significant decrease in the plasma detection of CMV by PCR compared to that in the control group. [START_REF] Barr | Photopheresis for the prevention of rejection in cardiac transplantation[END_REF] Similar to the data in the literature, ECP was a relatively safe treatment in our study. [START_REF] Kusztal | Application of Extracorporeal Photopheresis in Kidney Transplant Recipients: Technical Considerations and Procedure Tolerance[END_REF] Indeed, 79% of the patients had no side effects, and the majority of the reported side effects were not life threatening. The main complication reported was a tunnelled catheter-related infection (patient 30), which led to the interruption of ECP at 6.5 months. The incidence of catheter-related infections was 2.2/1000 catheter days, which is higher than the other reported rate in the literature (0.82/1000 catheter days). [START_REF] Mandolfo | Hemodialysis tunneled central venous catheters: five-year outcome analysis[END_REF] Therefore, vascular access could be a technical limitation, and ECP could be more easily administered to patients with a functional arterio-venous fistula.

This study has several limitations. Due to its retrospective nature and its long period of inclusion, the indications and applications of ECP treatment were heterogeneous, and there was a lack of follow-up regarding the cancers and infections. In some cases, ECP failure may be explained by different factors. For instance, in the early 2000s, the Banff criteria were not discriminant for the AMR diagnosis, likely leading to insufficient rejection treatment and graft loss in 5 of the patients. Moreover, ECP was used as a rescue therapy, with a delayed initiation in a large proportion of the patients in our study: 24% of the patients began ECP with a serum creatinine level higher than the previously defined failure threshold of 412 µmol/L, 6 patients began ECP while they were dialysis-dependent, and the initial anatomopathological reports showed chronic damage. These factors could compromise the analysis of the impact of ECP. The anatomopathological data at ECP initiation were available for a large proportion of patients, and we showed that a high acute composite AMR score was a risk factor for graft loss. However, our sample size was too small to make any definitive conclusions about the other anatomopathological data, including chronic damage that is known to be predictive of graft loss. [START_REF] Nankivell | The causes, significance and consequences of inflammatory fibrosis in kidney transplantation: The Banff i-IFTA lesion[END_REF][START_REF] Bouatou | Response to treatment and long-term outcomes in kidney transplant recipients with acute T cell-mediated rejection[END_REF] Moreover, we did not have follow-up data regarding the renal allograft biopsies during ECP therapy to assess their impact. Finally, ECP was used as a single therapy in only 5 patients (15%). Thus, the role of ECP in comparison to the other anti-rejection treatments that were used remains unclear.

In conclusion, our analysis highlighted the potential role of ECP in the target patient population of kidney allograft recipients with cellular or moderate antibody-mediated kidney allograft rejections and concomitant opportunistic infections or malignancies. ECP should be initiated as soon as possible because it is inefficient in cases of highly impaired kidney function, most likely due to severe chronic histological damage. The results of renal allograft biopsy should be taken into consideration. Further prospective trials are needed to clarify the indications for ECP in the treatment of kidney allograft rejection. [START_REF] Genberg | Refractory Acute Renal Allograft Rejection Successfully Treated With Photopheresis[END_REF] Retrospective, monocentric 3 1 acute / 2 chronic rejections OKT3 2 cons s/mth during 3 mth 3 graft failures Baron ED, Photodermatol Photoimmunol Photomed, 2001 [START_REF] Wolfe | Reversal of acute renal allograft rejection by extracorporeal photopheresis: a case presentation and review of the literature[END_REF] Retrospective, monocentric 1 Acute rejection OKT3 2 cons s/wk during 3 wk Improvment . ELISPOT assay : increase of IL5-producing cells Genberg H et al, Transplant Proceed, 2005 [START_REF] Loupy | Antibody-Mediated Rejection of Solid-Organ Allografts[END_REF] Retrospective, monocentric 7 Acute rejections MP + ATG 2-3 s /wk with a total of 6-26s Follow up 12 mth : 5 improvments, 2 stabilizations At last follow-up (12-43 mth) 7 functional grafts Fernadez EJ, Nefrologia, 2015 [START_REF] Sautenet | One-year Results of the Effects of Rituximab on Acute Antibody-Mediated Rejection in Renal Transplantation: RITUX ERAH, a Multicenter Double-blind Randomized Placebo-controlled Trial[END_REF] Retrospective, monocentric 1 Acute rejection MP + ATG 1 s/wk for 14 s associated to IVIg and 1 s/wk for 6s 9 mth later Stabilization and decrease after the 1st treatment period, improvment after the 2nd treatment period Dall'Amico R, JASN, 1998 [START_REF] Peraldi | Natural Killer Lymphocytes Are Dysfunctional in Kidney Transplant Recipients on Diagnosis of Cancer: Transplantation[END_REF] Prospective, monocentric 3 Acute rejection MP + OKT3 2 cons s/wk during 1 mth, then 2 cons s/2 wk during 2 mth, then 1 s/mth during 3 mth 2 stabilizations, 1 graft loss Jardine MJ, Journal of Clinical Aphereis, 2009 [START_REF] Horina | Photopheresis for renal allograft rejection[END_REF] Prospective, monocentric 10 Acute rejection MP + ATG (9), OKT3 (1) 1 s/wk for a total of 5-12 s Follow up 65 mth : 9 stabilizations, 1 graft loss by initial disease recurrence Prophylactic treatment of transplant rejection Lamioni A, Transplantation, 20078) [START_REF] Sautenet | One-year Results of the Effects of Rituximab on Acute Antibody-Mediated Rejection in Renal Transplantation: RITUX ERAH, a Multicenter Double-blind Randomized Placebo-controlled Trial[END_REF] Prospective, monocentric

  ECP procedure ECP sessions were performed between March 2001 and May 2015, 18 months [2-221] after transplantation and 3 months [0.5-25] after the diagnosis of a rejection episode.

  ; Figure 1B) Eleven patients (33%) had a stabilization of kidney function at M12. These patients had a significantly lower Banff score of the acute microvascular inflammation (0 [0-3] versus 3.5 [0-6] (p = 0.018)) and a significantly lower Banff acute composite AMR score (2 [0-3]

Figure 1 .Figure 2 .

 12 Figure 1. Impact of the acute composite antibody-mediated rejection score (sum of glomerulitis, peritubular capillaritis and intimal arteritis). A. On kidney function at 12 months after extracorporeal photopheresis B. On graft survival at 12 months after extracorporeal photopheresis. Box height indicates the IQR with the lower and upper edges of the box representing the 25th and 75th percentiles, respectively. The horizontal line is the median. The lower whisker represents the 25th percentile minus 1.5 times the IQR and the upper whisker the 75th percentile plus 1.5 times the IQR. Values outside the whiskers are outliers.

  during the first 3 mth post transplantation At 6 mth : significantly better kidney function and increase in Treg. Side effects : hypotension (2), increase of body temperature > 37.5°C (4), red blood cell loss due to technical problems(3)

Table 4 . ECP treatment in kidney transplantation: a review of the literature Abbreviations: ATG: anti-thymocyte globulin, CNI: calcineurin inhibitors, cons: consecutive, MMF: mycophenolate mofetil, MP: methylprednisolone pulses, mth: months, OKT3: muromonab-CD3, s: sessions, wk: week

 4

	Study Reference	Study Design	Number of	ECP indications	Previous therapies	ECP schedules	Results on kidney function
			patients
	Transplant rejection treatment
	Sunder Plassman G et al, Lancet, 1995 )	Retrospective,	3	2 acute / 1 chronic	MP + ATG or OKT3 for acute	2s/2 wk during 4-8 mth	Follow-up 4-13 mth : 1 stabilization, 1 improvment for
	24	monocentric		rejections	rejections		acute rejections / 1 decrease for chronic rejection
	Horina JH et al, Lancet, 1995

interquartile range (A) and as hazard ratio (HR) and 95% confidence interval (B).

Abbreviations: btw: between, conc: concomitant, creat: creatinine D: dialysis, DFG: deceased with functional graft, FG : functional graft, f-up : follow-up, inf : infectious, IVig : intravenous immunoglobulines, MP : methylprednisolone pulses, N : no, ND : no dialysis, PEX : plasmapheresis

Table 3. Factors influencing extracorporeal photopheresis results at 12 months (M12) A. Factors influencing results on kidney function B. Factors influencing results on graft survival

A.

Stabilization of kidney function at M12

Worsening of kidney function at M12 n=11 (33%) n=22 (67%) Age at KT (years) 53.5 [25-70] 45 [20-


V Fretigny

P Barbay

Billoir

M Carrette

G Feugray

P Chamouni

V Le Cam Duchez

M Fretigny

V Barbay

Paul Billoir
email: paul.billoir@chu-rouen.fr

Prednisolone treatment induced temporary factor IX normalization in mild hemophilia B who required an epidural infiltration: A case report

Keywords: Hemophilia, Herniated Disc, Factor IX normalization, Epidural infiltration, Fresh Frozen Plasma

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Hemophilia B is an X-linked bleeding disorder due to F9 gene variations.

Hemorrhagic manifestations are the result of reduced levels, dysfunction or absence of liver synthesis of factor IX (FIX), whose function is to cleave and activate factor X (FX) [START_REF] Goodeve | Hemophilia B: molecular pathogenesis and mutation analysis[END_REF]. According to FIX plasma level, hemophilia B is classified as severe (FIX < 1%) moderate (1-5%) or mild (5-40%). Hemophilia B treatment mostly consists of replacement therapy with FIX concentrates which can be used on demand or in prophylaxis considering bleeding manifestation and joint status [START_REF] Dolan | Haemophilia B: Where are we now and what does the future hold?[END_REF].

We report a transitory factor IX increase in a patient with mild hemophilia B with low dose prednisolone treatment and an epidural infiltration without replacement therapy.

CASE REPORT

A 31-year-old man presented to the local Hemophilia Care Center for management of epidural infiltration. He had a personal and familiy history of mild hemophilia B due to c.1346G > A (p.Arg449Gln) variation in F9 gene exon 8 [START_REF] Chavali | Hemophilia B is a quasi-quantitative condition with certain mutations showing phenotypic plasticity[END_REF]. He was diagnosed at the age of 20 years after a diagnosis of carrier hemophilia in her sister. His mother and two sisters also carried the identified variation. No hemorrhagic manifestation was disclosed on clinical examination. The patient had a herniated disc and a history of oesophageal ulcers. His treatment consisted in prednisolone (30 mg/day, 0.37 mg/kg/day), tramadol, pregabalin, paracetamol and esomeprazole. Surprisingly, 13 days after the introduction of prednisolone for relieving back pain, level of FIX:C was 76% and APTT was 36.1 s. Biological assays did not reveal any anomaly other than neutrophilia (13.15 G/L) induced by corticosteroids. Liver function tests were normal and no clinical or biological inflammatory syndrome was found (CRP < 5 mg/l, fibrinogen= 2.57 g/l).

Eleven days later, FIX:C was 89% and APTT was 33.2 s. One more time, no clinical or biological inflammatory syndrome was found (fibrinogen= 2.72 g/l). The only change in therapy was a decrease in prednisolone dosage (10 mg/day; 0.12 mg/kg/day). New molecular analysis was performed, confirming a unique c.1346G > A variation. As FIX:C level was normal, no FIX concentrates was administered to perform the infiltration, which occurred without hemorrhagic complication.

FIX:C was tested 5 months later, in the absence of corticoid treatment. The level had dropped to 51% and inhibitor screen was negative (Figure 1).

DISCUSSION

We report the case of hemophilia B patient with transient normalization of factor IX, resulting in a temporary phenotypic recovery without administration of FIX concentrate.

To eliminate analytical interference, several control tests were performed on each sample. Each time, identical results were found. Also, expected low FIX:C levels plasma of other previously diagnosed hemophilia B patients were measured at the same time with no similar increase. A second assessment excluded an identity error.

Inflammation is a physical condition known to induce a global increase in coagulation factors. Indeed, FIX increase has been described in chronic inflammatory diseases such as metabolic syndrome [START_REF] Wannamethee | The metabolic syndrome and insulin resistance: relationship to haemostatic and inflammatory markers in older non-diabetic men[END_REF] or inflammatory bowel disease [START_REF] Kume | Activations of coagulation and fibrinolysis secondary to bowel inflammation in patients with ulcerative colitis[END_REF]. However, this hypothesis was rejected on both clinical and biological grounds in this patient who had normal inflammatory markers. After the elimination of these hypotheses, particular attention was given to patient treatments. The only differences between the three assessments were the variations in dosages of prednisolone (Figure 1). High factor levels coincided with use of corticoids whereas a decrease in factor levels was observed after the end of treatment. Several studies have demonstrated the implication of glucocorticoids in the increase of clotting factors. A study conducted by Brotman et al. showed a modest rise of FVII, FVIII and FXI in 24 healthy men with a 5-day glucocorticoid treatment [START_REF] Brotman | Effects of short-term glucocorticoids on hemostatic factors in healthy volunteers[END_REF]. Nevertheless, factor IX levels were not tested. The increase of clotting factors in Cushing syndrome has also been explored, along with the hypercoagulability state in this pathology. A review conducted by Coelho et al. compiled data on the rise of coagulation factors in hypercortisolism, including four studies on FIX [START_REF] Coelho | Adverse effects of glucocorticoids: coagulopathy[END_REF]. A study in Cushing syndrome demonstrated a higher mean FIX plasma level in patients compared [START_REF] Kastelan | Hypercoagulability in Cushing's syndrome: the role of specific haemostatic and fibrinolytic markers[END_REF] to healthy controls (147 % vs 104 %, p<0.001) and a non significant normalization after Cushing remission (171±47% vs 102±24%) [START_REF] Kastelan | Hypercoagulable state in Cushing's syndrome is reversible following remission[END_REF]. No mechanism of up-regulation in Cushing syndrome is described in literature. In our case, the only suspected underlying mechanism is corticoids-induced upregulation of mRNA transcription of factor IX.

FIX levels are also known to increase in hemophilia B Leyden, a rare subtype of hemophilia B in which phenotypic manifestations are observed during childhood [START_REF] Goodeve | Hemophilia B: molecular pathogenesis and mutation analysis[END_REF] [10]. Point mutation or deletion of the F9 promoter region reduces transcription factor binding, inducing low FIX levels with variable degrees of severity. The proximal promoter mutation does not affect the androgen response element (ARE). Their ARE is intact and responsive to testosterone. Factor IX level increases with production of growth hormone and testosterone increases during puberty. To our knowledge, the c.1346G > A point mutation identified in our patient has never been previously described in Hemophilia B Leyden patient [START_REF] Rallapalli | An interactive mutation database for human coagulation factor IX provides novel insights into the phenotypes and genetics of hemophilia B[END_REF]. This mutation induces a catalytic modification, without FIX increase. Moreover, the progressive FIX level rising during childhood is lacking in our observation making this diagnosis unlikely. Furthermore, no study has demonstrated FIX normalization in hemophilia B with c.1346G > A variation yet.

Finally, based on this case report, diagnosis of mild hemophilia B should be performed with perfect knowledge of patient's treatment to avoid misdiagnosis. A temporary normal hemostasis permit to perform epidural infiltration without replacement therapy.

Disclosure

The authors stated that they had no interests which might be perceived as posing a conflict or bias

  Factor IX coagulant activity (FIX:C) was measured (Coagulation Factor IX Deficient plasma, Siemens Healthcare, Marburg) and fluctuated between 35 and 46% without inhibitor (normal level of FIX: >70 %). An activated partial thrombin time (APTT) (PTT automate®, Diagnostica Stago, Asnières sur Seine, France) was performed and measured at 43.95 and 40.3 s. (control: 32.8 s). This patient had previously received FIX concentrate for a tooth extraction.


Francine Behar-Cohen
email: francine.behar@gmail.com

Marilyn Dernigoghossian

Charlotte Andrieu- Soler

Rinath Levy

Raphaël Cohen

Min Zhao

Potential antiedematous effects of intravitreous anti-VEGF, unrelated to VEGF neutralization

Keywords: proteins, vitreous, osmotic pressure, retina, anti-VEGF, macular edema, eye, AMD, gene

The intravitreous injection of therapeutic proteins that neutralize vascular endothelial growth factor (VEGF) family members is efficient to reduce macular edema associated with wet age-related macular degeneration (AMD), retinal vein occlusion (RVO) and diabetic retinopathy (DR). It has revolutionized the visual prognosis of patients with macular edema. The antiedematous effect is dependent on an intravitreous dose of drug, which varies between patients and requires frequent and repeated injections to maintain its effects. At the time when optimizing the duration of anti-VEGF effects is a major challenge, understanding how anti-VEGF reduces macular edema is crucial. We discuss herein how anti-VEGF exerts antiedematous effects and raise the hypothesis that mechanisms, unrelated to VEGF neutralization, might have been underestimated.

Arguments in favor of additional antiedematous mechanisms, unrelated to VEGF neutralization

Bevacizumab does not reach the choroid but still reduces macular edema in wet AMD

Introduction

More than 20 years after VEGF was recognized as a major actor in retinal and choroidal neovascularization, anti-VEGFs have revolutionized ophthalmological practice and the visual prognosis of the major retinal blinding diseases. The natural evolution of wet AMD has radically changed after the pivotal Marina study reported in 2006 that ~90% of patients treated with monthly injections of ranibizumab maintained their vision (<15 letters loss) at 1 and 2 years, compared with 60% in the untreated control group. More impressively, ~30% of patients gained 15 letters in the treated group compared with only 5% in the untreated group at 1 year [START_REF] Rosenfeld | Ranibizumab for neovascular age-related macular degeneration[END_REF]. Twelve years later, several trials have confirmed the efficacy and safety of different proteins designed to neutralize VEGF (ranibizumab, bevacizumab, and aflibercept) in the treatment of wet AMD [START_REF] Solomon | Anti-vascular endothelial growth factor for neovascular age-related macular degeneration[END_REF]. Anti-VEGF also efficiently reduced macular edema associated with DR [START_REF] Virgili | Anti-vascular endothelial growth factor for diabetic macular oedema: a network meta-analysis[END_REF] and RVO [START_REF] Sangroongruangsri | Comparative efficacy of bevacizumab, ranibizumab, and aflibercept for treatment of macular edema secondary to retinal vein occlusion: a systematic review and network meta-analysis[END_REF]. Although the clinical benefit of anti-VEGF-neutralizing proteins is undeniable, the exact mechanisms of their antiedematous effects in various forms of macular edema are not fully understood. A deeper clarification of these mechanisms could help identify novel molecular downstream targets, potentially accessible to therapeutic modulation. Here, we discuss these mechanisms and suggest that part of the antiedematous effects of intravitreously injected anti-VEGF proteins could be unrelated to VEGF neutralization.

Anti-VEGF drugs reduce vascular permeability

Most of the antiedematous effects of anti-VEGF in the retina are thought to result from a decrease in VEGFinduced vascular permeability, which, by reducing fluid entry in the retina, subsequently reduces macular edema. Two of the three anti-VEGF agents commonly used for intravitreous injections are approved for ophthalmic indications, ranibizumab and aflibercept, whereas bevacizumab is used off-label to reduce macular edema of various origins, but more commonly associated with choroidal and retinal neovascularization, DR, and RVO. Bevacizumab is a humanized monoclonal 150 kDa antibody that binds to all isoforms of VEGF-A. Ranibizumab is a 48-kDa fab fragment with a 10-to 100-fold increased VEGF-binding affinity compared with bevacizumab. Aflibercept is a 115-kDa soluble decoy receptor comprising an all-human amino acid sequence of the second immunoglobulin (Ig) domain of human VEGFR1 and the third Ig domain of human VEGFR2 fused with the constant region (Fc) of human IgG1. Similar to bevacizumab and ranibizumab, aflibercept binds all isoforms of VEGF-A and, in addition, binds placental growth factor (PlGF) and VEGF-B [START_REF] Stewart | Pharmacokinetics, pharmacodynamics and pre-clinical characteristics of ophthalmic drugs that bind VEGF[END_REF]. All anti-VEGFs neutralize the propermeating effects of VEGF on endothelial cells. Indeed, upon binding to VEGFR2, VEGF-A destabilizes the tight junctions through the phosphorylation of occludin via PKC beta activation, through the downregulation of occludin expression by free cytosolic beta catenin increase and, through the internalization of occludin via SRC family kinase activation. VEGF also enhances the trans-endothelial permeability through endothelial nitric oxide synthase (eNOS)|-dependent caveola formations [START_REF] Daruich | Mechanisms of macular edema: beyond the surface[END_REF].

In retinal diseases, including DR or RVO, anti-VEGFs target retinal vasculopathy in the inner retinal layers, which are accessible to drugs injected into the vitreous. In wet AMD, choroidal neovascularization (CNV) grows from the choroidal vessels located under the retinal pigment epithelium (RPE). To target CNV, the drug injected into the vitreous should be able to cross all retinal layers to reach the site of pathologic angiogenesis. For this purpose, Genentech developed ranibizumab (48 kDa) based on the observation that, after intravitreous injection in the monkey eye, a full-length150-kDa anti-VEGF antibody poorly penetrated the RPE and the choroid [START_REF] Mordenti | Comparisons of the intraocular tissue distribution, pharmacokinetics, and safety of 125I-labeled full-length and Fab antibodies in rhesus monkeys following intravitreal administration[END_REF]. Not only the inner limiting membrane, but also the external limiting membrane was a barrier to the antibody penetration; the latter membrane is also a filter to macromolecular diffusion across the retina, limiting further the diffusion of large stoke radius proteins [START_REF] Daruich | Mechanisms of macular edema: beyond the surface[END_REF]. Although bevacizumab does not reach its target site, it does reduce macular edema resulting from CNV and both ranibizumab and bevacizumab are currently used for intravitreous injections in the treatment of wet AMD with similar efficacy [START_REF]Ranibizumab and bevacizumab for neovascular age-related macular degeneration[END_REF].

Anti-VEGF drugs do not act primarily as anti-angiogenic agents in wet AMD

All anti-VEGF drugs bind VEGF165 with high affinity and prevent VEGF proliferating activity on endothelial cells with IC50 values of 0.088±0.032, 0.090±0.009, and 0.500±0.091 nM for ranibizumab, aflibercept, and bevacizumab, respectively [START_REF] Yang | Comparison of binding characteristics and in vitro activities of three inhibitors of vascular endothelial growth factor A[END_REF]. In vitro, anti-VEGF molecules not only prevent endothelial cell proliferation [START_REF] Yang | Comparison of binding characteristics and in vitro activities of three inhibitors of vascular endothelial growth factor A[END_REF] and vessel growth, but also reverse the angiogenic processes towards normal vasculature [START_REF] Sitohy | Early actions of anti-vascular endothelial growth factor/vascular endothelial growth factor receptor drugs on angiogenic blood vessels[END_REF].

However, in AMD, despite repeated anti-VEGF injections, 80% of type 1 CNV (located under the RPE) continue to grow when evaluated by optical coherence tomography angiography (OCT-A) [START_REF] Xu | Long-term progression of type 1 neovascularization in age-related macular degeneration using optical coherence tomography angiography[END_REF], although CNV lesion remodeling occurs shorty after intravitreous injections of anti-VEGF. Given that anti-VEGF agents do not eliminate the CNV despite years of intravitreous anti-VEGF injections, maintenance of treatment is required to reduce macular edema.

Correlation between anti-VEGF 'drying' effects and VEGF levels is variable.

In a pure vascular disease, such as branch RVO (BRVO), VEGF vitreous levels poorly correlate with macular thickness and ~30% of patients do not have increased VEGF levels [START_REF] Shimura | Visual prognosis and vitreous cytokine levels after arteriovenous sheathotomy in branch retinal vein occlusion associated with macular oedema[END_REF], although VEGF neutralization efficiently reduces macular edema associated with BRVO in >80% of cases [START_REF] Sangroongruangsri | Comparative efficacy of bevacizumab, ranibizumab, and aflibercept for treatment of macular edema secondary to retinal vein occlusion: a systematic review and network meta-analysis[END_REF]. In patients with diabetic macular edema, various studies have shown that intravitreous injections of anti-VEGF drugs efficiently neutralize VEGF and also significantly reduce other proedematous cytokines [intercellular adhesion molecule 1 (ICAM-1), interleukin (IL)6, and IL8] [START_REF] Hillier | Aqueous humor cytokine levels as biomarkers of disease severity in diabetic macular edema[END_REF]. The decrease of such cytokines appears important because glucocorticoids that have limited or no effect on VEGF-A levels [START_REF] Campochiaro | Pro-permeability factors in diabetic macular edema; the diabetic macular edema treated with ozurdex trial[END_REF] significantly reduce proinflammatory and proedematous cytokines and chemokines through their transcriptional regulation of nuclear factor (NF)-κB, and have stronger 'drying' effects than anti-VEGFs in diabetic macular edema.

Systemic and intravitreous antiedematous effects of bevacizumab in wet AMD

The systemic injection of bevacizumab, which directly targets CNV, takes longer to reduce macular edema compared with the direct intravitreous injection of bevacizumab [START_REF] Geitzenauer | Early effects of systemic and intravitreal bevacizumab (avastin) therapy for neovascular age-related macular degeneration[END_REF], which was shown to have poor penetration in the retina and limited access to the CNV. This suggests that the route of administration impacts the effect of bevacizumab.

Although bevacizumab does not neutralize rodent VEGF, its injection in rodent vitreous exerts ocular effects

Various biological effects of bevacizumab are constantly reported in rodent models of retinal diseases. Two recent publications reported antiangiogenic effects of intravitreous bevacizumab in the rat eye [START_REF] Lv | Intravitreal bevacizumab injection attenuates diabetic retinopathy in adult rats with experimentally induced diabetes in the early stage[END_REF][START_REF] Mu | Multivesicular liposomes for sustained release of bevacizumab in treating laser-induced choroidal neovascularization[END_REF] although bevacizumab does not bind to rat VEGF and does not neutralize rat VEGF [START_REF] Fuh | Structure-function studies of two synthetic anti-vascular endothelial growth factor Fabs and comparison with the avastin Fab[END_REF]. suggests that non-VEGF-mediated effects result from the intravitreous injection of bevacizumab.

Proposed mechanisms

Change in oncotic pressure gradients VEGF controls the fenestration of choroidal endothelial cells and anti-VEGF drastically reduces the number of fenestrations [START_REF] Peters | Ultrastructural findings in the primate eye after intravitreal injection of bevacizumab[END_REF]. Given that fenestrations regulate the passage of proteins into the choroidal stroma [START_REF] Chang | Histologic localization of indocyanine green dye in aging primate and human ocular tissues with clinical angiographic correlation[END_REF], anti-VEGFs might reduce the protein concentration in the choroidal stroma and, thus, reduce its interstitial pressure and, subsequently, the volume of water in the choroid. This is evidenced by the reduction in choroidal thickness after anti-VEGF treatments.

By contrast, the vitreous is a low-protein-concentration media (~0.5 mg/ml) and injection of therapeutic proteins in the vitreous, from 0.3 mg for ranibizumab and up to 2 mg for aflibercept, creates a significant increase in the vitreous oncotic pressure. Given that the retina is permeable to water [START_REF] Marmor | Barriers to fluorescein and protein movement[END_REF], it is expected that water accumulated in the retina might follow protein gradients, as in any other biological environment. Thus, the modification of protein gradients might have impact retinal thickness. This could explain the rapid change in retinal thickness and recurrence of macular edema when the concentration of therapeutic proteins in the vitreous decreases below a threshold, which can vary between different individuals, and with the underlying disease mechanisms.

Intravitreous injection of proteins induces rapid transcriptional regulations in the retina

In clinical studies, for obvious ethical reasons, subjects included in the control group are sham injected (they do not receive any injection). However, in all preclinical studies, control animals are treated with the injection of a 'vehicle' solution and also do not receive a neutralized form of the protein or an isotype control antibody or a control fab. Thus, the effect of a 'control' protein is never tested. Yet, important changes might be induced by the injection of a macromolecule into the vitreous, the physiological composition of which is low in protein. To explore this question, the effects of ranibizumab (Lucentis® 0.5 mg/m, Novartis, Switzerland) were compared with the same dose of a control fab (50 kDa, OAMA04119, Aviva Systems Biology) in 0.01% polysorbate 20 (to match the concentration of polysorbate 20 used in Lucentis ®) or to water at 24 h after injection in the vitreous of 8-week-old male Lewis rats. The volumes of injections were calculated to match the volume injected in a human vitreous, resulting in a final vitreous concentration of 0.1 mg/ml. A full pan-transcriptomic analysis of retinal transcripts was performed after 24 h, a time that is too short to observe a potential immune reaction of the rat to a human protein. All experiments were performed in accordance with the European Communities Council Directive 86/609/EEC and approved by local ethical committees. Total neuroretina RNA was extracted using the RNeasyMini Kit (Qiagen) including DNase I treatment. RNA integrity was checked on the Agilent 2100 Bioanalyser. At least three independent biological replicate samples were sequenced and used for downstream analysis. RNA sequencing was performed on Illumina HiSeq2000 platform. The average number of reads per sample was 27 M. Reads from each sample were processed as follows. First, reads were trimmed using an in-house Perl script with a minimum phred quality of 20 per vase and a minimum read length of 30 base pairs. On average, 24% of reads per sample were discarded. The resulting reads were later aligned to the Rattus norvegicus genome assembly 3.4 (from Ensembl) using Tophat version 2.0.10. At least 12 million reads were aligned to the genome for each sample. Gene expression was then quanitifed to obtain read count and FPKM values. The nonadjusted read counts for each gene were used for statistical calculation of global differential expression using DESeq2. Differentially expressed genes were selected at an adjusted P values of ≤0.05 and fold changes >1.5 [22].

At 24 h, 139 genes were significantly regulated by ranibizumab (six downregulated and 133 upregulated) and 225 genes were significantly regulated by the control fab (20 downregulated and 205 upregulated) [22]. As expected, 88% of the genes (122/139) regulated by ranibizumab were also regulated by the fab, given that ranibizumab does not neutralize rat VEGF. The genes regulated in the neuroretina were unrelated to VEGF neutralization. Among the regulated genes, 4% have known roles in angiogenesis (Figure 1). Angptl4, encoding angiopoietin-like 4, was downregulated. Its aqueous level was recently shown to correlate with the response to ranibizumab [START_REF] Choudhary | Therapeutic targets of renin-angiotensin system in ocular disorders[END_REF]. The gene encoding angiotensin, an important player in retinal diseases [START_REF] Uno | Impaired expression of thrombospondin-1 in eyes with age related macular degeneration[END_REF], was also downregulated. By contrast, Thbs1, encoding thrombospondin 1, was upregulated by the fab; this is an important antiangiogenic factor in retinal diseases, and is decreased in eyes with wet AMD [START_REF] Tauseef | Transient receptor potential channel 1 maintains adherens junction plasticity by suppressing sphingosine kinase 1 expression to induce endothelial hyperpermeability[END_REF]. Several genes involved in the maintenance of tight junctions were upregulated, such as Sphk1, encoding sphingosine phosphate kinase 1 [START_REF] Zhang | The emerging roles of clusterin on reduction of both blood retina barrier breakdown and neural retina damage in diabetic retinopathy[END_REF], and the gene encoding clusterin [START_REF] Li | Caveolin-1 increases proinflammatory chemoattractants and blood-retinal barrier breakdown but decreases leukocyte recruitment in inflammation[END_REF]. Expression of Cav1, encoding caveolin 1, was also downregulated, which could protect the blood-retinal barrier [START_REF] Visanji | Lymphatic vasculature in human dural superior sagittal sinus: Implications for neurodegenerative proteinopathies[END_REF]. Another important finding was that injection of proteins in the vitreous induced the upregulation of podoplanin, a known marker of lymphatic vessels [START_REF] Wahl | Osteopontin inhibits osmotic swelling of retinal glial (Müller) cells by inducing release of VEGF[END_REF]. Osteopontin, which inhibits the osmotic swelling of retinal muller glia [30], was upregulated, which could directly influence the retinal thickness (Figure 1).

There are obviously many limitations to this experiment that should be interpreted with caution. The rat retina differs from the human retina because it lacks a macula, and the different formulations were tested on the short term on a normal eye and not on an eye with a retinal disease. Nevertheless, these simple experiments show that the expression of numerous genes, known to have significant effects on macular edema in humans, is regulated shortly after injection into the rat eye of a protein that does not neutralize rat VEGF (ranibizumab or the control fab). Such gene regulations were measured in the neuroretina, meaning that a drug that would not penetrate deeply in the outer retina could exert such effects.

Concluding remarks and perspectives

At a time when, to prolong the duration of their antiedematous effects, higher doses of anti-VEGF proteins are injected in the vitreous, it is important to understand the biological consequences of increasing protein concentrations in the vitreous. Similar to any other compartment in the body, the concentration of proteins in the different compartments of the eye is highly regulated and governs the metabolism of adjacent tissues. A better understanding of the exact mechanisms of the antiedematous effects of anti-VEGF therapeutic proteins, directly related to neutralization of VEGF or to other properties of the physicochemical structure of the molecules, could help design optimized compounds with enhanced antiedematous effects [22]. The retinal transcriptional regulation of ranibizumab (Lucentis®) and of the control Fab +0.01% polysorbate 20 was compared with water at 24 h after injection (0.1 mg/ml final vitreous concentration). The list of genes significantly regulated by ranibizumab was then compared with the list of genes regulated by the control fab + 0.01% polysorbate 20. Green: number of genes downregulated, red: number of genes upregulated. (b) Group of genes regulated by the fab+polysorbate 20, classified by their biological functions. (c) Restricted list of the most regulated genes, by fab or ranibizumab, known to have a role in macular edema, retinal angiogenesis, or the control of the blood-retinal barrier. Abbreviations: ang, angiotensin; Angptl4, angiopoietin-like 4; cav1, caveolin 1; clu, clusterin; pdpn, podoplanin; sphk1, sphingosine kinase 1; SPP1, osteopontin; thbs1, thrombospodin 1.

Figure 1 .

 1 Figure 1. Transcriptional effects of ranibizumab or control fab on the rat neuroretina at 24 h after intravitreous injection. (a) Representation of the experimental scheme.The retinal transcriptional regulation of ranibizumab (Lucentis®) and of the control Fab +0.01% polysorbate 20 was compared with water at 24 h after injection (0.1 mg/ml final vitreous concentration). The list of genes significantly regulated by ranibizumab was then compared with the list of genes regulated by the control fab + 0.01% polysorbate 20. Green: number of genes downregulated, red: number of genes upregulated. (b) Group of genes regulated by the fab+polysorbate 20, classified by their biological functions. (c) Restricted list of the most regulated genes, by fab or ranibizumab, known to have a role in macular edema, retinal angiogenesis, or the control of the blood-retinal barrier. Abbreviations: ang, angiotensin; Angptl4, angiopoietin-like 4; cav1, caveolin 1; clu, clusterin; pdpn, podoplanin; sphk1, sphingosine kinase 1; SPP1, osteopontin; thbs1, thrombospodin 1.


MD Christophe Trésallet
email: christophe.tresallet@aphp.fr

J L Cardin

J Belghiti

A Cortes

M Richard

E Martinod

Medical expert witness testimony in France and in Europe

Summary

Health care professionals are usually at a loss when it comes to medical "complaints", essentially because they lack knowledge with regard to existing litigation procedures.

After a short reminder of the different rights of appeal in France, we describe how medical appeals function in other European countries.

Next, we give the details of how the evaluation of claims of bodily damage works, a process in which every physician may be called upon to participate several times in a career), either as the defendant, or as a medical counselor, or as an expert. The goal of this update is to understand the different compensation appeal circuits available to patients and help the surgeon demystify and dedramatize the situation while preparing for the medical expert witness testimony. All such testimony reports, via whatever appeal circuit, follow a similar procedure, even if they are not exactly identical.

1-Introduction

When a patient, or his or her beneficiary, believes that he or she has been a victim « following an action of prevention, diagnosis or care » (article L. 1142-28 of the French public health code (PHC)), they can initiate a process to determine the responsibility of the health care professional, or public or private health care facility, and claim a compensation for the damage incurred. The French law n° 2002-303 (March 4, 2002) concerning the « rights of patients and quality of health care » (the so-called Kouchner Law) allows patients to initiate a claim up to 10 years after the date of consolidation. Health care professionals or facilities will be considered responsible for the damage to patients only if a fault (negligence or error) can be proven (article L 1411-2 of the PHC).

In order to place the study of medical accidents under the prism of common law and responsibility, the causality between the fault and the damage alleged by the patient must be analyzed. In this respect the patient has several options to obtain some form of financial compensation for the damage to which he or she has been exposed.

While the patient has the choice of which action to take, the burden lies with the plaintiff to prove that the health care professional(s) have committed a fault that engages their responsibility (according to the responsibility system, which is based on a proven fault). For this, the patient has the right to access his or her medical record according to the articles L1111-7 and L.1110-4 of the PHC. The patient consequently has the right to ask for an out-of-court settlement (direct negotiation with the physician's insurance company), but also to refer the case to an arbitration and compensation board (ACB) or a law court to solicit a counter-testimony from a medical expert witness.

In certain cases, however, the health care professional must actively prove that correct and adequate information was provided to the patient (regime called « reversal of the burden of proof » often invoked by patients, as based on article L.1111-2 of the PHC).

2-

The different circuits for adjudication of claims (Figure 1) [START_REF] Grare | Présentation générale du droit positif: les expertises en responsabilité médicale[END_REF] 2.1 Out-of-court settlements This circuit is strongly encouraged by judges and insurance companies because of the rapidity of settlement, much more expeditious than when the procedure goes to court or through an ACB (especially the ACB of the Greater Paris region).

Following this procedural stream, the patient refers the case directly to the physician or the health care facility that he or she believes is responsible for the damages, by registered mail with acknowledgement of receipt. The goal is to obtain an out-of-court settlement, initiated by either the patient him-or herself, or the juridical insurance protection of the victim. This settlement approach should be preferred whenever the fault is unquestionable, and a compromise solution can be easily found.

Accordingly, a medical examination of the plaintiff should be performed by the medical officer (most often designated by the patient's insurance company) who then addresses a medical expert witness testimony to the defendant. Based on this report, the judicial protection system of the defendant can propose either an out-of-court medical counter-medical expert witness testimony to identify and evaluate the damages, or propose a compensation, or even, contest that any damage occurred.

If the patient accepts the compensation, the settlement procedure is terminated by a compromise proceeding protocol that forbids any further contention for the same damage (articles 2044 and following of the civil code), EXCEPT in case of aggravation.

If the out-of-court solution fails, the patient still has the right to present his or her case to an ACB or refer the matter to a court of law.

Out-of-court circuit after referral to an ACB [2]

The ACB, instituted by the French law of March 4, 2002, can be consulted directly by the patient, without any cost to the patient, with or without a lawyer (there is a standardized blank form letter that the patient must fill in, and send via registered mail and acknowledgement of receipt).

The claim is usually seen and examined fairly quickly (quicker than the normal judiciary route even though sometimes, the delays can be almost the same). As indicated by its name, the ACB has a double function: arbitration and compensation, but only for damages that attain a certain level of severity as fixed by law (articles L1142-1 through L.1142-3 and 1142-3 of the PHC) :

-permanent functional deficiency (PFD) >24 % -temporary sick leave > 6 consecutive months or 6 non-consecutive months over a period of one year _disability considered to be particularly severe for activities of daily living -definitive incapacity to exercise one's previous profession -temporary functional deficiency ≥ 50% for 6 consecutive months or 6 non-consecutive months over a period of one year.

If the claim is considered acceptable, the counter-medical expert witness testimony is set up, usually with a single expert. This person should be on the official list of the National commission for medical accidents (Commission Nationale des Accidents Médicaux (CNAmed)). The expert's report should be sent to the ACB within three months of the consultation with the patient.

After reception of the observations from the parties on the expert's report (the claim statements), the commission meets, interviews each of the parties, and pronounces either a notice of rejection, or a notice of responsibility fixing the different points of damage requiring compensation. It is then the responsibility of the company that insures the professional or health care facility to either make an offer or refuse to do so, within a delay of four months.

If the claim is rejected by the ACB, the plaintiff can still bring the matter to a court of law (administrative or high court, cf infra) or else, the National Office of Compensation of Medical Accidents (Office National d'Indemnisation des Accidents Médicaux (ONIAM)).

The ONIAM replaces of the dependant's insurance company, by initiating a recusal of the defaulting insurance company of the health care professional or facility (with the risk of a 15% penalty).

The judicial circuit: High Court or Administrative Court

The victim has the possibility of bringing the case to court: either directly from the start, or after failure to come to an out-of-court settlement (cf supra). The litigation will then be treated according to jurisdictions of common law where the presence of a lawyer is mandatory. The financial burden of the procedure and medical expert witness testimony must be beared by the plaintiff.

Litigations between the patient and a physician in private practice

or/working in a private institution are judged in Supreme Court (or in a district court if the damages are < €10,000). For physicians who work in the public sector, litigations are handled by an Administrative Court.

Once the case is registered and deemed receivable, the judge designates an expert from an official list. The expert is expected to organize a counter-medical expert witness testimony and provide a report with substantiating reasons (cf. infra). This report with substantiating reasons provides the grounds for debate to determine the responsibility with the administrative judge either after a judiciary medical expert witness testimony has taken place or after rejection of the previous mandatory request.

A judgement is usually made within one year after the final decision has been taken.

Referral to the Board of Medicine

Although there is no possibility of counter-testimony by a medical expert witness through this circuit, alerting the Departmental Board of Medicine concerning a conflict is being used increasingly today when physicians are involved in law suits. Of note, the debate within the Departmental Board is centered on the deontological aspect of the management offered to the patient, not the quality of care (medical expert witness testimony). Patients and their counsellors may not be aware of this aspect, thinking that once again, the goal of this referral is to determine the responsibility of the physician but this is not the role of the Board.

The Departmental Board is obliged to organize an arbitration once it has been solicited according to the article L.4124-2 of the PHC (relative to hospital practitioners) or the article 4123-2 of the PHC [START_REF] Senat | l'indemnisation des victimes d'accidents thérapeutiques[END_REF].

3-Comparison with other countries in Europe

In France, victim compensation for therapeutic accidents reposes on the common law principles of medical responsibility. Responsibility, however, differs according to where the health care professional works (public or private sector) and the nature of the medical procedures.

In the first scenario, the physician is not held responsible personally for the damage he or she causes during his or her professional activity, unless it is proven that the fault was done outside of any professional norms. In principle, the responsibility falls upon the hospital, and since 1992, according to administrative justice, this is considered a simple negligence by act of the State council decision of 10 April 1992, by which the High French court over-ruled the necessity of a serious offense to engage the responsibility for a procedure performed in a public hospital.

Moreover, since 1993, French justice acknowledges that hospitals are accountable for "therapeutic hazards" or "random therapeutic events", according to the ruling of State Counsel BIANCHI (April 9,1993).

According to this decision, the high administrative jurisdiction laid down the principle according to which hospitals are accountable for any procedure leading to severe damage, regardless of any relationship to the patient's initial status, even in the absence of any negligence or error, as long as there was no particular reason to think that a given patient was particularly exposed.

Conversely, physicians exercising in private practice are personally responsible, but generally the judge establishes the medical responsibility in the common law scheme of a proven error. This means that the physician can be held responsible only if the patient can prove and/or demonstrate that an error was committed.

The principal characteristics of compensation systems in our European neighbors (Germany, Denmark, Italy, United Kingdom, Sweden, and Switzerland) vary somewhat, but, excepting Italy, these countries have specific procedures that allow for patient compensation without having to go to court.

In any event, in both of these countries, even if the parties choose to have follow an extra-judiciary process, they are still free to bring the case to court, since the conclusions of the mediation panels are not legally binding.

-in the United-Kingdom, the 1999 reform of the civil procedure established a "pre-judicial protocol" to resolve medical conflicts. This protocol invites both parties, first, to search for an out-of-court solution before taking any legal action and also to respect a very precise calendar of events. Non-adherence to this protocol can give rise to severe penalties, as the judge can consequently refer the case to civil jurisdiction if the out-of-court solution fails.

-in Denmark and Sweden, the law exempts the victim of a therapeutic accident from having to prove that there was negligence or error.

Sweden was the first European country to separate the notion of "medical responsibility" and "right of compensation". Starting in 1975, the county councils, who manage the hospitals, and the insurance companies, agreed on a compensation protocol for therapeutic accidents providing the victims with compensation even in the absence of a negligence or error. With this as an example, the Danish government adopted a similar law in May 1991, effective July 1, 1992, by which most of the victims of medical mishaps could be compensated in the absence of any negligence or error or notion of liability. All that is necessary is to establish that the prejudice sustained by the patient could have been avoided (if the physician had clearly adhered to secundum Artem, in the absence of equipment failure or if other methods had been established) or that the damage is the consequence of a true therapeutic hazard (that is, a very rare complication or one much more severe than could be expected).

All these requests for compensation are treated by the Patients' Association for Insurance that includes all the insurance companies of health care givers as covered by law. This association checks that all requests are receivable, and evaluates the damages. An ad hoc commission examines any recourse made against the decisions of the Association. In its turn, Sweden followed Denmark and replaced the compensation protocol of therapeutic accidents by the law of 1996 relative to patient insurance, which came into effect Jan 1, 1997. The Swedish law foresees a similar system, even if the criteria of eligibility are not exactly the same.

Conclusion:

Management of medical misadventures is common topic in all countries in Europe and has privileged out-of-court settlements in case of conflicts between patients and health care professionals. In all countries studied, except Italy, the medical contention is not (either partly or completely) under the jurisdiction of common law rights. The solutions, although quite variable, have some common characteristics: a concern to compensate victims by a simple and inexpensive procedure and speedy settlements [START_REF] Senat | l'indemnisation des victimes d'accidents thérapeutiques[END_REF]. However, as there are always exceptions to the rule in the assessment of therapeutic misadventures that can lead to compensation, the French system probably is one of the most accomplished and complete that exists today.

Figure 1 :

 1 Figure 1: Possible actions after a medical accident

Generally speaking:

-in Italy, compensation of therapeutic accidents continues to be ruled by common law courts. The judges determine the amount of damages based on medical experts reports.

-In Germany and in Switzerland, physicians have set up an extrajudiciary system of mediation. In Germany, the regional medical boards Patients can bring their case to these panels if they think they have been a victim of a medical error. This leads to an evaluation and decision with regard to the existence or not of a medical error. In Germany, where the statute of mediation panels varies from one region to another, some even reach decisions on the amount of compensation to be awarded. In Switzerland, however, the panels of extra-judiciary mediation never evaluate the amount of damages, but their conclusions can be used for the out-of-court settlements between the parties.

In both countries, the systems are practically cost-free for the patients.

Although the German panels have been criticized, in particular because some of them do not involve the patients in their procedures, they seem to function satisfactorily, since attribution of civil liability to physicians has practically disappeared. Conversely, in Switzerland, the activity of the extra-judiciary panels is more restricted.