Extracorporeal photopheresis after heart transplantation.

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Extracorporeal photopheresis after heart transplantation

The addition of extracorporeal photopheresis (ECP) to a standard immunosuppressive drug therapy after heart transplantation in clinical studies has shown to be beneficial, for example, by reducing acute rejection, allograft vasculopathy or CMV infection. However, the protocols varied considerably, have a predetermined finite number of ECP treatments and adjuvant immunosuppressive regimens used in combination with ECP have differed significantly. Furthermore, there are scarce data to guide which patients should be treated with ECP and when or who would benefit further if ECP were to be continued long term to increase the safety by reducing immunosuppressive drug toxicities without losing efficacy. The knowledge of the tolerance-inducing effects of ECP-like upregulation of regulatory T cells and of dendritic cells may allow to develop a strategy to monitor immunomodulation effects of ECP to further identify ECP responders, the optimal individual ECP schedule and whether ECP therapy can replace or reduce immunosuppressive drug therapy.

Markus J Barten*,1 & Maja-Theresa Dieterlen2 1 University Heart Center Hamburg, Department of Cardiovascular Surgery, Hamburg, Germany 2 University Hospital Leipzig, Heart Center, Department of Cardiac Surgery, Leipzig, Germany *Author for correspondence: Tel.: +49 407 4100 Fax: +49 407 4100 m.barten@ uke.de

Keywords: dendritic cells • heart transplantation • immunomodulation • photopheresis • regulatory T cells

Transplant rejection is one of the main causes of death following heart transplantation (HTx). Approximately 25% of HTx recipients develop a rejection within the first year after transplantation. Acute rejection of cardiac transplants can be divided into two different types: acute cellular rejection (ACR) and antibody-mediated rejection (AMR). Alloreactive CD8 + cytotoxic T- cells are known to mediate ACR by direct killing of the graft cells. In addition, activated CD4 + helper and CD8 + cytotoxic Tcells produce cytokines that lead to recruitment and activation of inflammatory cells, which also injure the graft [1] . AMR is mediated by alloantibodies that are produced by B- lymphocytes. These antibodies bind to alloantigens on vascular endothelial cells which leads to endothelial injury and intravascular thrombosis that results in graft destruction [1] . While acute rejection occurs in the first months after HTx, chronic rejection in the

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form of cardiac allograft vasculopathy (CAV) can develop months to years post-transplantation. CAV is characterized by a chronic inflammatory reaction in blood vessel walls that results in intimal smooth muscle cell proliferation and vessel occlusion [1] . Clinically, chronic rejection in cardiac transplantation has been associated with the development of donor-specific human leukocyte antigen (HLA) antibodies [2] . Patient survival is diminished significantly after the detection of CAV, and CAV and graft failure (most likely undetected CAV) are, in addition to malignancy, the most important causes of death in patients who survive the first year after transplantation [3] . Although immunosuppressive treatments have been advanced over recent years, rejection episodes still threaten the survival of HTx patients. Due to their small therapeutic range immunosuppressants like calcineurin- and mammalian target of rapamycin-inhibitors (mTORI)

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Review Barten & Dieterlen are dosed on the basis of regular measures of drug levels in the blood, while mycophenolic acid, azathioprine and steroids are given with a fixed dosed. Anyway, the occurrence of rejection and infections (e.g., cytomegalovirus, [CMV]) within individuals cannot be predicted. Therefore, management of immunosuppressant treatments invariably results in a complex trade-off between efficacy and long-term toxicity. As such, patient followup with physicians is performed frequently in order to allow treatment strategies to be continually re-evaluated and modified, especially as the risk of acute rejection and importance of long-term side effects temporally change. Long-term administration of immunosuppressants may lead to adverse toxic reactions, including renal dysfunction, increased risk of cardio-vascular disease (e.g., new onset or exaggerated existing diabetes mellitus, hypertension, hyperlipidemia) and bone marrow depression. Furthermore, side effects such as alterations of the skin and the mucous membrane, peripheral edema, malignancies and increased risk of infections including bacterial (e.g., pneumonia), fungal and viral such as CMV may develop. Some of these long-term side effects require treatment with additional medication, and recurrent stays in the hospital are often necessary. Novel, less-toxic immunologic procedures are desperately needed to accomplish the desired transplant tolerance, but without the undesirable side effects. Interestingly, extracorporeal photopheresis (ECP) has been shown to benefit patients after organ transplantation. ECP therapy The idea of using the plant compounds psoralens, which are activated by sunlight, topically to treat skin diseases goes back to ancient Egyptian times. In the 1970s, psoralens after systemic administration, so-called psoralen plus ultraviolet ‘A’ (PUVA) light, were established originally to cure patients suffering from psoriasis [4] . The original term ‘extracorporeal photochemotherapy’, or later named ‘extracorporeal photopheresis’ (ECP) was derived from PUVA, and was found to be effective for the treatment of advanced stages of cutaneous T-cell lymphoma [5] , in patients with graft-versus-host disease (GvHD) [6,7] or following solid organ transplantation [8–10] . The first reports about the application of ECP after HTx were published in 1992 [11,12] . ECP is an apheresis technique that requires collecting a portion of the patient’s venous whole blood in a medical device located outside the patient’s body (extracorporeal). The blood is subsequently separated into its components through centrifugation within the device, with red blood cells (erythrocytes) and most blood plasma immediately returned into the patient’s circulation (Figure 1) . The separated fraction mainly comprises white blood cells (lymphocytes, monocytes and

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granulocytes), which are mixed with the photosensitizing drug methoxsalen. After activation through ultraviolet-A (UV-A) light, the methoxsalen/UV-A-treated cells are unable to proliferate and are primed to undergo apoptosis [13] . The treated mononuclear cells are then returned to the patient’s circulation. A photopheresis cycle comprises two of these photopheresis sessions over a period of 1–3 h, depending on the venous access, each on two consecutive days. The frequency of ECP therapy after HTx depends on the indication and the success of the therapy (Figure 1) [14] . In addition to the standard ECP treatment, modified protocols of ECP were developed that include the overnight incubation of white blood cells prior to returning cells into the patient. This modification which is also called transimmunzation, modifies the cellular and cytokine components of the ECP reinfusate and may have the potential to enhance the clinical efficacy of this successful immunotherapy [15] . ECP-induced effects Apoptosis

The detailed mechanism of action of ECP treatments has not been elucidated to date, but apoptosis of the irradiated mononuclear blood cells is suggested to play an important role in the process. The loss of the proliferation potential of mononuclear cells and their subsequent apoptosis after UV-A irradiation was reported in vitro [16–18] and in vivo [19] . Furthermore, Schuerfeld et al. [20] showed in a small study with six HTx patients suffering from recurrent acute rejection, that ECP induced apoptosis in lymphocytic cells of rejection infiltrates obtained from endomyocardial biopsies. In that study, the highest number of apoptotic cells was found in the first 4 days after ECP treatment. The number of apoptotic cells in lymphocyte infiltrates in the first 60 days after ECP treatment was greater than before ECP. The data presented in this study supported the hypothesis that ECP in HTx recipients may function via the induction of apoptosis of inflammatory cells [20] . After re-infusion, the apoptotic lymphocytes are phagocytized by immature DCs, which further mature and present peptides as antigens. Through this process, mature DCs subsequently stimulate Tregs [21] . Interestingly, Zheng et al. [22] reported that ECP after HTx generates apoptotic lymphocytes of both, recipient origin and of donor origin, which in turn have the potential to induce antigen-specific regulatory T-cells (Tregs) in transplant recipients. Nevertheless, apoptosis alone is not the sole cause for the effects observed in ECP-treated patients [23] . Studies with the incentive to investigate the mechanism of action of ECP have shown that the effect is primarily attributable to immunomodulatory processes [24] .

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Photoactivated WBCs return to patient

Photoactivation with UV-A light

8-MOP

WBCs are treated with 8-MOP and exposed to UV-A light

ECP system draws blood from patient

Blood separation by centrifugation into cellular components and plasma (= buffy coat); RBCs return to patient

Figure 1. Mechanism of action of extracorporeal photopheresis. Blood is removed from the patient, and the RBCs and WBCs are separated; RBCs are immediately returned to the patient, whereas WBCs are treated with MOP and UV-A radiation to photoactivate the drug; photoactivated WBCs are then returned to the patient. ECP: Extracorporeal photopheresis; MOP: 8– methoxypsoralen; RBC: Red blood cell; UV-A: Ultraviolet- A; WBC: White blood cell. Adapted with permission from [14] .

Post-transplant immunomodulation

The primary inactivation of mononuclear cells, especially of lymphocytes through photopheresis, has secondary effects within the network of the immune system. The interaction (or uptake) of autologous lymphocytes treated with 8-methoxypsoralen and UV-A light by recipient dendritic cells (DCs) causes several immunological changes, which include: the activation of DCs [25] ; the induction of Tregs [26,27] ; changes in the interleukin expression profile and a shifting of the immune balance [26,28,29] . DCs and Tregs play an important role in the development of transplant tolerance. Recently, a new possible mechanism of ECP on DC stimulation has been studied, for example, the role of platelet immobilization and activation [30] , and the role of plasma protein binding integrins and plasma proteins [31] . ECP initiates cellular differentiation and


subsequently cytokine secretion, for example, TNF-α and IL-6, which in turn activate CD36-positive macrophages [32] . More recent work on cellular and cytokine changes after ECP demonstrated an increase of IL-4- and IL-10-producing T helper cells type 2 (Th2) [33] , a decrease of IFN-γ- and IL-2-producing T helper cells type 1 (Th1) [33,34] , a Th1/Th2 balance restoration toward Th2 [33,35,36] and the induction of a Treg subset synthesizing IL-10 [37] . Results of our study in HTx recipients showed increased levels for Th1, Th2 and T helper cells 17 (Th17) cytokines in patients treated with ECP [38] . Within the ECP-treated patients, differences were observed between the prophylactic treated and the patients with ACR: a shift toward increased Th2 cytokine levels were observed for the prophylactic ECP treatment group, while the ACR group showed the ten-

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Review Barten & Dieterlen dency for increased Th1 cytokine levels during ECP treatment. Furthermore, increased Th17 levels were observed for 22% of the patients from the prophylactic treatment group and for 56% of the patients from the ACR group [38] . In summary, these studies reported that ECP treatment is leading to induction and secretion of different cytokines, whereupon the immune balance status is changed. Although, the reports about cytokine changes following ECP differ substantially and seem to be dependent on the indication. Tolerance induction

The long-term outcome of HTx remains relatively disappointing because of an increasing incidence of cardiac allograft vasculopathy CAV, malignancies or adverse effects of immunosuppressants. The development and clinical introduction of tolerance-inducing therapies may have the potential to replace immunosuppressive treatment. Tolerance-inducing strategies target on the elimination of allogeneic responses against the graft and the simultaneous maintenance of normal immune function. In general, two different cell types were identified to be involved in tolerance induction after transplantation: Tregs and DCs [39,40] . Tregs are a subset of T lymphocytes and act multi-directionally by preventing migration of effector T- cells, natural killer cells or B cells to target organs, resulting in their inability to cooperate with APCs and thus inducing anergy [41] . DCs modulate the differentiation of naive T-cells into T helper cells, and thus are a major component in the regulation of the T-cell response [42] . Plasmacytoid dendritic cells (pDCs) are one of two identified subsets of DCs which are involved in tolerance induction after transplantation [43] . A possible mechanism for tolerance induction through pDCs was identified in an experimental model which showed that pDCs acquire alloantigens in the allograft, then migrate to lymphoid tissue and induce the generation of Tregs [44] . The second DC subset is myeloid DCs (mDCs), a cell subset that can secrete large amounts of IL-12, which is a potent driver of T helper cells of type 1 (Th1) [45] . These cells were identified as the dominant type of DCs with the capacity to induce tolerance [46] , but the detailed mechanisms are not fully understood to date. Both DC subtypes, pDCs and mDCs, were reported to be mediators of ECP effects [47] . Beside inducing apoptosis of lymphocytes, ECP treatment also recruits mDCs and stimulates Th1-driven immune responses in patients with leukemic cutaneous T-cell lymphoma [48] . In GvHD, ECP recruits more pDCs that promote the expansion of Th2/Tregs cells, which could lead to T-cell anergy or tolerance induction [47] .

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The mechanism by which ECP exerts its protective effects may be attributed to the induction of Tregs and DCs. The first studies on lung transplanted recipients [49] and patients with GvHD [50,51] reported increased levels for Tregs after ECP treatment. Furthermore, Lamioni et al. [27] described the induction of tolerogenic DCs and an increase of Tregs after ECP treatment in pediatric patients with heart transplant rejection. Recent works on identification of gene markers for tolerance reported about a ‘tolerance gene signature’ in peripheral blood of liver and kidney transplant recipients, whereat, there is only a minimal overlap between the gene signatures of tolerant kidney and liver transplant recipients [52] . These data indicate a possible organ specificity of tolerance biomarkers. Clinical application of ECP after heart transplantation

ECP has become an integral part of immunomodulation at many transplant centers after bone marrow or lung transplantation to prevent and treat GvHD [53] or lung transplantation [54] , respectively. Apart from HTx, ECP is already being used in pioneering centers in the area of prophylaxis and anti-rejection therapy. We found 16 published reports from the last two decades about the application of ECP treatment after HTx that described the treatment of 180 HTx patients (Table 1) . The efficacy of ECP therapy to avoid acute rejection episodes after HTx has been shown in several studies. Thus, the comparability of ECP to conventional corticoid therapy has been demonstrated [55] , and prophylactic ECP treatment reduced the number of acute rejection episodes compared with triple-drug immunosuppressive regimen alone [56–59] . Several studies have been performed without control groups and with very low patient numbers [11,20,60–63] . Two studies reported reduced panel-reactive antibodies (PRA) level after ECP treatment [12,59] , which might be an important aspect for the risk evaluation for AMRs and the development of CAV, because development of AMR and CAV is known to be associated with high PRA levels. One retrospective study comprised a larger number of HTx patients, included a control group and showed a reduced risk for rejection after 3 months of ECP therapy [64] . A recent study investigated the changes of the immunological profile during ECP treatment in HTx patients, and showed that eight of nine HTx patients with biopsy-proven acute rejection (BPAR) did not show rejection grade ≥ 1B (ISHLT 1990) after three ECP treatments [38] . One important study investigated the effects of different ECP treatment frequencies, comparing the single-day and the more intense ECP treatment over two consecutive days in HTx patients [57] .


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[12]

Rejection reversal eight of nine for ECP and seven of seven for corticosteroids ECP may be as effective as corticosteroids for treating ISHLT rejection grades 2, 3A and 3B No adverse effects

Reduced PRA levels after ECP Cardiac function remained class I (New York Heart Association Criteria) at 13 to 24 months after initiation of ECP Patients suffered few rejection episodes and no infectious complications

ECP reversed eight of nine and corticosteroids seven of seven rejection episodes Complete rejection reversal occurred at a median of 25 days after ECP and of 17 days after corticosteroid therapy Function of heart allograft remained normal in both treatment groups No adverse effects observed with ECP

Approximately two whole blood volumes were processed A minimum of 5 × 109 mononuclear cells were treated in each ECP treatment A second ECP treatment was repeated after 48 h if less than 5× 109 mononuclear cells were treated in the first treatment ECP started 2 weeks to 6 months after HTx Two consecutive days at 3 weeks intervals for 1 year After 1 year, the treatments were tapered to an 8-week schedule

A minimum of 5 × 109 mononuclear cells were treated in each ECP treatment A second ECP treatment was repeated after 24 h if less than 5 × 109 mononuclear cells were treated in the first treatment The original treatment was repeated if EMB performed 7 days later showed unchanged ISHLT rejection grade

Sixteen HTx patients with ISHLT rejection grades 2, 3A and 3B randomized to ECP or corticosteroid therapy

Two multiple transplant patients with high PRA levels and 2 multiparous women with high risk of sensitization (all HTx) Patients were treated with ECP in addition to conventional immunosuppression Sixteen HTx patients with ISHLT grades 2–3 rejection not associated with hemodynamic compromise were randomized to receive high-dose corticosteroids (n = 7) or ECP (n = 9)

CostanzoResults on Nordin et al. rejection prospective study randomized

Results on rejection prospective study nonrandomized (no control group)

Rose et al.

CostanzoResults on Nordin et al. rejection prospective study randomized

ECP: Extracorporal photopheresis; EMB: Endomyocardial biopsy; HTx: Heart transplantation; ISHLT: International Society for Heart and Lung Transplantation; pDC: Plasmacytoid dendritic cell; PRA: Panel reactive antibody; Treg: Regulatory T cell.

[55]

[89]

CostanzoResults on Seven HTx patients on triple Nordin et al. rejection drug regimen with moderate prospective study rejection nonrandomized (no control group)

Ref.

Eight of nine rejections were reversed as assessed by EMB 7 days after ECP No adverse effects

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Results

Study population A minimum of 5 × 10 mononuclear cells were treated in each ECP treatment A second ECP treatment was repeated after 48 h if less than 5 × 109 mononuclear cells were treated in the first treatment

Study type

Schedule of ECP treatment

Author

Table 1. Overview of reported clinical applications of extracorporeal photopheresis therapy after heart transplantation.



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Results on Eight HTx patients with rejection recurrent rejection prospective study nonrandomized (no control group)

Results on prophylaxis prospective study randomized

Dall’Amico et al.

Barr et al.

ECP treatments at two consecutive days for a total of 24 ECP procedures per patient: day 1/2, 5/6, 10/11, 17/18, 27/28 during month 1 after HTx; every 2 weeks during months 2 and 3; every 4 weeks for months 4, 5 and 6

Weekly intervals during the first month, 2-week intervals during the second and third months, and monthly for another 3 months

ECP was performed weekly for 4 weeks and then monthly for 5 months

[61]

[58]

ECP reduced cardiac rejection episodes No significant differences in the rates and types of infection Eight protocol violations and cases of noncompliance including one patient with an adverse event (bacteremia)

[90]

[57]

[56]

Ref.

Seven of eight patients with reduction of the number and severity of rejection episodes ECP allowed reduction of daily immunsuppressive therapy No major side effects

Response to ECP with rapid loss of fever, improvement of exercise tolerance, normalization of cardiac hemodynamics and improvement in EMBs

More impressive decrease of acute rejection episodes by double ECP (group III) than by single ECP (group II) in comparison to control within the first 4 weeks after HTx Both ECP schedules (group II and III) reduced acute rejection episodes in the first 10 months after starting ECP compared to control ECP-treated patients with fewer infections

No adverse effects due to ECP treatment Acute rejection episodes occurred in all patients of all groups One patient (control group) died due to acute severe rejection 5 weeks after HTx; one patient died in group III Total number of rejection was decreased in the ECP groups compared to control

Results


Sixty HTx patients randomly assigned to triple-drug immunosuppressive therapy alone (n = 27) or in conjunction with ECP (n = 33)

Results on Two HTx patients with rejection multiple episodes of rejection Prospective study nonrandomized (no control group)

Wieland et al.

Fifteen HTx patients in three Group I: control group without ECP study groups (five patients per Group II: ECP on days 1, 3, 7, 14, 28 and group) subsequently every 4 weeks for 5 months (10 ECP courses) Group III: ECP on two consecutive days, starting on days 1 + 2, continuing on days 5 + 6, 10 + 11, 17 + 18 and 27 + 28 and monthly for the next 5 months (20 ECP courses)


Meiser et al. Results on prophylaxis Prospective study nonrandomized

Study population Fifteen HTx patients in three Group I: control group without ECP study groups (five patients per Group II: ECP on days 1, 3, 7, 14, 28 and group) subsequently every 4 weeks for 5 months (10 ECP courses) Group III: ECP on two consecutive days, starting on days 1 + 2, continuing on days 5 + 6, 10 + 11, 17 + 18 and 27 + 28 and monthly for the next 5 months (20 ECP courses)

Study type

Meiser et al. Results on prophylaxis Prospective study nonrandomized

Author

Table 1. Overview of reported clinical applications of extracorporeal photopheresis therapy after heart transplantation (cont.).

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Results on Eleven HTx patients with rejection recurrent rejection prospective study nonrandomized (no control group)

Dall’Amico et al.

Weekly intervals during the first month, 2-week intervals during the second and third months, and monthly for another 3 months

Prophylactic ECP was performed at two consecutive days every 4 weeks during the first year, every 6 weeks during the first half of the second year and every 8 weeks during the last half of the second year

Fraction of EMBs with grade 0/1A increased during ECP Fraction of EMBs with grade 3A/3B decreased Six rejection relapses observed in 60-month follow-up Four rejection relapses reversed by ECP Mean doses of immunosuppressive drugs were lower after 6 months of ECP

No difference between both groups regarding infection or acute rejection incidence ECP group with reduced PRA levels within the first 6 months ECP group showed reduced coronary artery intimal thickness at the first and second year


Twenty three HTx patients randomly assigned to tripledrug immunosuppressive therapy alone (n = 13) or in conjunction with ECP (n = 10)

Results on prophylaxis prospective study randomized

Barr et al.

[62]

[59]

[20]

Two consecutive days twice a week in the first month, weekly intervals in the second month, 2-week intervals in the following 2 months and at monthly intervals in the last 2 months totaling 20 ECP treatments per patient over 6 months

ECP allowed the reduction of conventional immunosuppressive therapy during the treatment in all patients Two of six patients with a single episode of rejection grade 3A after 3 months with a low dosage of immunosuppressive drugs

Results on Six HTx patients with recurrent rejection acute rejection Prospective study nonrandomized (no control group)

Schürfeld et al.

[63]

Ref.

Giunti et al.

Results

Reduction of acute rejection episodes Results on Six HTx patients with recurrent Two consecutive days twice a week in along with concomitant reduction of rejection acute rejection the first month, weekly intervals in the immunosuppressive therapy in all patients prospective study second month, 2-week intervals in the Moderate acute rejection episodes nonrandomized following 2 months, and at monthly (no control group) intervals in the last 2 months totaling 20 decreased from 0.4 to 0.07 rejections per ECP treatments per patient over 6 months month per patient Two of six patients with a single episode of ISHLT grade IIIA rejection No deaths occurred during ECP treatment No side effects


Study type

Author

Study population




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Results on prophylaxis and rejection prospective study nonrandomized

Dieterlen et al.

Eighteen HTx patients with ECP (n = 9 with prophylactic ECP treatment, n = 9 with ECP after cellular rejection). Seven HTx patients without rejection episodes and without ECP served as control group

Two-day treatment every 4 weeks for 3 months

Two-day treatment every 3 to 6 weeks Three hundred and forty – for a target of 18 months three HTx patients with ECP (n = 36) or without ECP (n = 307) ECP indication: episodes of rejection with hemodynamic compromise (n = 12), recurrent (n = 9) or persistent (n = 11) rejection or as prophylaxis in the presence of anti-donor antibodies (n = 4)

Eighty percent of the HTx patients responded to ECP treatment with an increase of Tregs and pDCs Increased CD39+ Tregs and higher levels for Th1, Th2 and Th17 cytokines during ECP treatment compared to control group

Reduced rejection risk after 3 months of ECP ECP reduced the risk for subsequent hemodynamic compromise rejection and/or death from rejection in patients with increased risk for rejection

Case I: ISHLT grade IA or B for 4 months Case II: ISHLT grade 0 or IB for the next 9 weeks; patient died at POD-147 Case III: ISHLT grade 0 or IA for 6 years Case IV: ISHLT grade 0, IA and II; 10 months after completion of ECP patient died (cardiac arrest) with ISHLT grade IIIA cellular rejection

Results


Results on rejection retrospective study nonrandomized

Kirklin et al.

Results on Four HTx patients with cardiac Three weekly sessions of two consecutive rejection allograft rejection (ISHLT ECP treatments prospective study grade 3A or 4) nonrandomized (no control group)


Lehrer et al.

Study population

Study type

Author


[38]

[64]

[91]

Ref.




The authors could show that a more impressive decrease of acute rejections episodes was achieved by ‘double’ ECP than by single ECP treatment in the first 4 weeks after HTx. All studies published after 1994 used ECP therapies at two consecutive days. To date, the most common regimen comprises one cycle on two daily consecutive sessions a week or every other week [65] . Another ECP component also changed over time: until the middle of the 1990s patients received methoxsalens orally before beginning mononuclear cell collection. Newer ECP devices facilitate the direct addition of methoxsalens to mononuclear cells in the devices, outside the patients’ body. Clinical trials

ECP after HTx has been in clinical use for more than 20 years, but the number of large clinical trials is very low. Hence, we assorted all published reports about ECP application after HTx with more than 10 patients. One of the first studies included 16 HTx patients with moderate BPAR with rejection grades 2, 3A and 3B (ISHLT 1990) [60] . Patients were randomized to receive either ECP or pulse corticoidsteroid therapy. The number of ECP treatments was complied with the number of treated mononuclear cells: if less than 5 x 109 mononuclear cells were treated in the first ECP treatment, a second ECP treatment was performed after 48 h. Rejection reversal was obtained for eight of nine ECP-treated and for seven of seven corticosteroidtreated patients. Later on, changes in the number and immunophenotypes of heart allograft infiltrating cells were compared and attempted to relate these changes to the functional status of heart allograft infiltrating cells in the two treatment groups [55] . The complete rejection reversal occurred at a median of 25 days after ECP and of 17 days after corticosteroid therapy showing a comparable efficacy of ECP as corticosteroids for treating BPAR with a histological rejection grade higher than 2 ISHLT (1990). Another clinical trial with 15 HTx patients in three study groups investigated the dose effect of ECP after HTx [56,57] . ECP was applied in five single-day treatments: in the first month and subsequent 5 months; or on two consecutive days five-times in the first month and then monthly thereafter for 5 months. Additionally, a control group without ECP treatment was included. Within the first four weeks after HTx the number of ACR episodes was three in patients with an extensive ECP therapy compared with five or six ACR episodes in patients with single or no ECP therapy, respectively. Over the total observation time (around 10 months), however, both ECP schedules had nine ACR episodes compared with 20 ACR episodes in the control group.


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In the largest randomized clinical trial to date, 60 HTx patients were divided into two groups [58] . Both groups received a standard combination of cyclosporine A, azathioprine and prednisone. The patients of the trial group additionally received 24 sessions of ECP over a period of 6 months. The analysis showed a significantly smaller amount of rejections per patient in the ECP group (0.9 vs 1.4 in the comparison group). Moreover, the percentage of patients with two or more rejection episodes was significantly lower in patients treated with ECP (18%) than in patients without ECP treatment (48%). An infection with CMV was significantly less detected in the ECP group than in the control group with standard immunosuppression alone. All other infections, however, occurred in both groups similarly [58] . A randomized clinical trial examined the indication of ECP in the long-term progress after HTx, based on the occurrence of CAV that was ascertained using intravascular ultrasound (IVUS). In the first as well as in the second year after HTx, patients with additional photopheresis developed significantly less thickening of coronaries than the control group who received only standard immunosuppressive drug therapy. The infections after long-term ECP-treated patients did not increase compared with those in the control patients [59] . Results of a case study with 11 HTx patients showed a decrease of severe rejections (grade 3A/B; ISHLT 1990), from 42 to 18% after a 6-month therapy with ECP [62] . In another retrospective trial, the data of 36 patients with severe rejections after HTx, who were treated with ECP, were compared with a control group of 307 patients receiving only immunosuppressive therapy comprising a calcineurin inhibitor (cyclosporine A or tacrolimus) plus a proliferation inhibitor (azathioprine or mycophenolic acid) and steroids. After only 3 months of treatment with ECP, the patients’ risk of suffering from another severe rejection was already reduced by approximately 70% [64] . The most recent study included 25 HTx patients in two ECP treatment groups (n = 9 each) and one control group including patients without ACR and without ECP treatment (n = 7) [38] . ECP was performed for prophylaxis (n = 9) or for secondary prevention of BPAR (n = 9). Eight of nine patients (89%) with BPAR responded to three ECP treatments as the histopathological evidence of the first endomyocardial biopsies after three ECP treatments did not show a rejection grade ≥1B (ISHLT 1990). Indications and contraindications of ECP

Two different indications for ECP after HTx have been investigated so far: ECP for prevention of rejec-


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Review Barten & Dieterlen tion (category I according to the American Society for Apharesis) and ECP for secondary prevention of rejection (category II according to the American Society for Apharesis) [24] . Contraindications to ECP therapy are allergy against methoxsalens. Women of child-bearing potential should be counseled before embarking on a course of ECP, because methoxsalens may cause fetal harm [66] . Furthermore, it has been demonstrated that methoxsalens/psoralens show reproductive toxicity in animal studies [67] , which indicates that there is a potential risk for effects in humans. Anyway, the dose of methoxsalen in modern ECP therapy with a UV-A radiation setting is very low [66] , and the appropriate risks are considered to be improbable. Additionally, the presence or history of basal cell carcinoma is a specific caution and requires close observation and treatment [66] . Adverse effects of ECP

ECP is regarded as a safe and well-tolerated treatment. The incidence of reported side effects is extremely low at less than 0.003% [68] . In general, adverse reactions of ECP are of a temporary nature and mostly associated with the catheterization of the vessel (e.g., hematoma, infection). The most common complication is transient hypotension due to volume shifts. Since platelet losses can occur in the machine, a starting platelet count for at least 20,000 is required [66] . Patients suffering from thrombocytopenia or patients with relative contraindications to heparin use should be anticoagulated for ECP treatment by acid citrate dextrose-A [69] . In contrast to conventional immunosuppressive therapies, there is no evidence that ECP increases the incidence for infections, relapses and secondary malignant tumors [14] . The major part of clinical studies on HTx patients reported that neither side effects nor adverse reactions were observed in the context of ECP treatment. In addition to treatment course and possible adverse reactions, patients should be informed about the increased light sensitivity up to 24 h after ECP therapy, and should avoid exposure to sunlight or wear sun protection (e.g., UV-protective glasses, sunscreen with a high sun protection factor) during that time. Monitoring strategies of ECP effects

Up to date, beside the clinical response no monitoring strategy is available for the ECP treatment after solid organ transplantation, and especially after HTx. The monitoring of immunological processes and immunomodulatory effects caused by ECP therapy is of great clinical relevance and may help to evaluate the individual success of this intervention. As a medical device, ECP does not have to pass through the more

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rigorous criteria which apply to the marketing authorization process of medical products. Accordingly, there are no reliable data on a hypothetic dose-effect, optimal rhythm of administration, drug interactions or the pharmacokinetics and the pharmacodynamics of this treatment [65] . However, suitable biomarkers are necessary for measuring and quantification of these parameters. In the last decade, research work focused on the elucidation of the immunological processes that are responsible for ECP efficiency. Several components and cell subsets of the immune system could be linked to the effects of ECP therapy. For example, the percentage of circulating functional Tregs correlated with the duration after ECP treatment, and varied according to the graft function [49,51] . To use Tregs for monitoring ECP effects, it might be helpful to closely analyze subsets of Tregs such as CD62L- or CD39-positive Tregs, [70,71] , activated memory-like Tregs (TREM) [71] and forkhead box P3-positive (FoxP3 +) Tregs [72] . However, it has to be mentioned that Treg levels before ECP treatment are often highly variable. Therefore, it seems to be reasonable that the ECP treatment effect of the Treg population is used for analysis. A recent study on HTx patients analyzed Tregs and their different subsets after prophylactic ECP treatment and in ECP-treated patients with BPAR, and analyzed the ECP treatment effects [38] . The authors demonstrated that most of the patients showed an increase of Tregs and pDCs after ECP treatment. Furthermore, CD39-, CD62L-, CD120b- and CD147positive Tregs subsets were analyzed, and it has been shown that CD39-positive Tregs, which characterize a Treg subset with high suppressive potential as well as activated memory-like Tregs (TREM), increased during ECP therapy [38] . The effect of ECP on B-lymphocyte function was not considerably investigated and mainly focused on studies with patients suffering from GvHD. Kuzmina et al. reported that the relative amount of immature CD19 + CD21- B-lymphocytes represents a useful cellular biomarker to predict the clinical response to ECP in GvHD patients [73] . Another subpopulation of B- lymphocytes was analyzed by Papp et al. [74] : B-lymphocytes that expressed the Fas receptor (CD95), which is a key molecule for the induction of apoptosis, were similar in ECP-treated and -untreated patients and did not change significantly during ECP treatment of patients with systemic sclerosis. Beside T and B- lymphocytes, DCs are promising candidates as biomarkers for in vivo ECP effects. In the early 1990s, the changes of DC subsets after ECP therapy in patients with bone marrow transplantation were reported [33,75] . The results of both studies



showed that mDC population decreased while the pDCs increased after ECP therapy [33,75] . More than 10 years later, Dieterlen et al. [38] confirmed these results in ECP-treated HTx patients and proposed classification criteria based on individual courses of Tregs and pDCs to discriminate between patients with positive ECP effect and patients without an effect to ECP therapy. Another important aspect for the development of an ECP monitoring strategy are measures of distinct cytokines or the immune balance status. Compared to conventional immunosuppressive drugs, ECP causes specific modulation of the immune response and restores the immunological balance [76] . A promising candidate of the tumor necrosis factor family, the B-cell activating factor (BAFF), represents a potentially useful biomarker in the prediction of ECP treatment outcome in GvHD patients during a period of stable immunosuppression. BAFF is produced by APCs and is involved in immature B-lymphocyte survival and promotes the production of autoantibodies [77] . A comprehensive monitoring panel for research use was developed for HTx patients and includes measures of Tregs, pDCs and the immune balance status determined on the basis of serum levels of IL-2, IL-4, IL-10, IL-17 and IFN-γ [38] . Despite the fact that a number of cellular parameters are identified as potential biomarkers for monitoring ECP effects, none of these markers have been validated for clinical use or introduced in the clinical practice. Additionally, it has to be mentioned that the research in the field of biomarker identification for ECP has been performed in studies with different ECP indications (e.g., GvHD, post-HTx), and that the currently available data are limited. Because ECP has immunomodulatory effects, the immunological findings could highly depend on the patients’ baseline prior to the start of ECP. Furthermore, the immunological findings for different indications, for example, for the case of treatment of ACR compared with chronic rejection after HTx may differ and require further excellent research studies. Up to date, an ongoing interventional efficacy study investigates subsets of Tregs as a predictor of response in ECP-treated patients after bone marrow transplantation [92] . The knowledge of the immunological profile before, during and after ECP treatment may help: to assess the efficacy and the efficiency of ECP for an indication (e.g., after HTx) or even individually for the patient; to define the startand end- point of ECP therapy; to evaluate the frequency of ECP therapy and to reduce the exposure of immunosuppressive drugs.


Review

Conclusion & future perspective In conclusion, based on the current data it can be said that ECP is a safe method with high effectiveness and few adverse reactions used to prevent or successfully treat ACR after HTx. An increased susceptibility for opportunistic infections after ECP has not been determined to date. First clinical results show a positive trend in the prevention of CAV and CMV infection. Up to date ECP following HTx is not introduced in the clinical routine. Only a few centers are using ECP as an adjunctive to standard immunosuppressive drug therapy, especially to treat steroid-resistant ACR or AMR. Clear guidelines or recommendations for using ECP after HTx do not exist, because the current study results are not useful to solve multiple problems. First, the variation of the study protocols about the timing for ECP (when to start and to stop). Second, at present there is no information available on how to identify ECP responders which is in contrast to the use of ECP after lung transplantation [54] . Third, the variation of the adjuvant immunosuppressive drug protocols used in the former studies makes the interpretation about the efficacy of ECP even more difficult. Last, there is no strategy available on how to monitor ECP effects or if ECP has the potential to replace immunosuppressive drugs. On the other hand, there is still a medical need due to problems in both the short term and the long term after HTx, which require to be solved to increase graft and patient survival in the long term. Immediately post-HTx, the emphasis is more focused on efficacy, with taking more of a ‘back seat’ to preventing acute rejection (AR). Over the course of the months as the risk of ACR reduces, the focus shifts to preventing long-term graft failure and drug toxicity. AR after HTx is the cause of 12% of deaths between 30 days and 1 year after transplantation [3] and caused by cellular- and antibody-mediated processes [78] . ACR affects 30–50% of patients in the first year, mostly without clinical evidence (subclinical) and comprises T-cell-induced inflammatory response that leads to lymphocytic infiltration into the myocardium. In contrast to ACR, AMR affects 3–15% of HTx recipients and is mediated by B-lymphocytes. Clinically, AMR is often suspected when there is hemodynamic instability. Thus, its diagnosis may be solely based on decreased cardiac ejection fraction even in the absence of histologic or immunologic findings [79] . Furthermore, AMR has become a more striking event in the last decade because of the broad diagnostic use of the multiplex technology to detect donor specific and nonspecific antibodies against HLA or antibod-


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Study design ECP HTx Stage 1: CNI-minimization 12 months treatment

Stage 2: CNI-free 12 months treatment

Randomization

HTx Control group: CNIstd + MPA + steroids

ECP group: CNI + MPA + steroids ECP weekly

1. CNI red

ECP group: mTORI + MPA + steroids 2. CNI red

ECP bi-weekly ECP monthly

Time-points I Mo 1

II Mo 2-3

III Mo 4-6

3. CNI red

IV Mo 7-12

ECP bi-monthly

V Mo 13-17

Mo 18

VI Mo 19-24

Figure 2. Study design for prospective, randomized, controlled, parallel group, open-label study in heart transplant recipients. Screening will be before HTx and randomization to the two treatment groups at month 1 (1:1 ratio). During the first month post-HTx patients are randomized according to center standards receiving an immunosuppressive therapy with CNI, MPA drug and steroids. After randomization, patients in the study group are treated with ECP till the end of study time: weekly till the end of month 3, biweekly till month 6, monthly till month 12, and bi-monthly from month 13 to study end at month 24 post-HTx. In the study group, the study comprises two stages with CNI-minimization in stage 1 till month 12 post-HTx and a CNI-free regimen thereafter till study end at month 24 post-HTx. Stage 1: CNI blood concentration will be reduced three-times at months 3, 6 and 12, respectively. At month 11, run-in phase with additional mTORI will be started. Stage 2: drug regimen comprises mTORI, MPA and steroids. In the control group, the patients will receive an immunosuppressive drug regimen with CNI (std, blood concentration), MPA and steroids for the whole study period. For both groups, steroids will be tapered according to center care. At all study time points, the following assessments will be done: endomyocardial biopsy, renal function, safety assessment, immune cell monitoring (Tregs, pDCs, Th1/Th2 cytokine panel), antibody HLA-class I and II as well as non-HLA. At study time points IV and VI, additional cardiac angiography will be performed. CNI: Calcineurin-inhibitor; ECP; Extracorporeal photophoresis; HTx; Heart transplantation; mTORI: Mammalian target of Rapamycin-inhibitors; MPA: Mycophenolate acid-based; pDC: Plasmacytoid dendritic cell; Std: Standard.

ies against non-HLA. Moreover, the number of recipients with existing HLA and non-HLA antibodies is increasing as the number of ventricular assist devices (VADs) implantation is rapidly increasing worldwide to treat heart failure due to the lack of organs. suitable grafts as bridge to HTx. VAD implantation is associated with an increased rate of antibody immunization against HLA and non-HLA [80] . In the long term, outcome of HTx remains problematic because of an increasing incidence of CAV over time post-HTx or of toxic effects of immunosuppressive drugs. CAV is a major risk factor for morbidity and mortality after HTx and occurs in 8–10%, 18–19% and 32–50% within the first, third and fifth year, respectively [81] . As a multifactorial disease, CAV is induced

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by nonimmunologic factors, for example, donor age, hyperlipidemia, hypertension, CMV infection or donor age or immunologic risk factors, for example, ACR and humoral rejection [82] . A CNI-based regimen is the cornerstone of immunosuppressive therapy after HTx, because CNIs have increased survival by reducing acute rejection. However, the dose- and time-dependent nephrotoxic effects of CNIs can limit long-term survival. Chronic renal failure is a major cause of morbidity and mortality in long-term heart transplant patients. Thus, there is a medical need to avoid CNI-related toxicity as early as possible. At present, a CNI-free regimen containing of mTOR-inhibitor, mycophenolate acid and steroids often fails in the early phase after HTx due to the high



incidence of ACR [83] . Additionally, treatment of other drug toxicities like the increase of cardiovascular risk factors, for example, diabetes mellitus, hypertension, lipid disturbance and the high risk of malignancies not only requires prescription of costly co-medications but often lengthy hospital stays are inevitable. In deep consideration of the aforementioned medical need for an improved graft and patient survival together with the availability of the new ECP device generation and current knowledge about the immunomodulatory effects of ECP, leaves the door open to establish ECP therapy after HTx in the clinical routine. Thus, we propose a three-step application of future prospective, controlled, randomized studies to explore ECP therapy after HTx by using well-defined study protocols. Regarding the blinding of the study, it should be deliberated about whether a ‘sham’ photopheresis like it is described for ECP studies in systemic sclerosis [84] or type 1 diabetes [85] has to be included for the control group. First application, therefore, could be to evaluate effectiveness of ECP in the treatment of BPAR of both types,

Review

ACR and AMR, to assess proportion of patients with resolved rejection episodes and proportion of patients with reduced histological grade of severity of ACR. Similar to treat acute GvHD after bone marrow transplantation [86] , a schedule for an ECP therapy could be an intense weekly therapy on two consecutive days for 1 month. In patients without hemodynamic compromise, ECP could be the first-line treatment and, on the other hand, in case of hemodynamic compromise ECP could be combined with steroids or anti-thymocyte-globulin. A second ECP application could be to treat patients with first diagnosis of CAV. Again a schedule for ECP could be similar as already used after bone marrow transplantation [87] . ECP therapy could be performed weekly or biweekly on two consecutive days for 12 weeks, and, thereafter, biweekly or monthly for another 3 months. The third and the most striking application of ECP would be to evaluate if the immunomodulation potential of ECP could replace immunosuppression after HTx would be a CNI-minimization and/or CNI-free drug regimen.

Long-term

Short-term

Minimize acute rejection

Controlling CAV risk

Low drug toxicity

Establish graft function

Immunomodulation ECP Immunosuppression

Reduce risk of infection

Protect renal function

Avoid future drug toxic effects

Good renal function

Less hospitalization

Increased graft and patient survival

Figure 3. Different medical needs in the short- and long-term after heart transplantation, which might be solved with the immunomodulation effects of extracorporeal photophoresis (ECP) resulting in a reduction of immunosuppression. CAV: Cardiac allograft vasculopathy.



939

Review Barten & Dieterlen In such a study, the novel and well-defined knowledge of the first and second application of ECP will be used. In Figure 2, we propose a controlled study where HTx patients are either randomized to receive standard

triple-drug immunosuppressive therapy alone (CNI standard blood concentrations, mycophenolate acid, MPA-based drug and steroids) or standard triple-drug therapy plus ECP. Randomization will be conducted

Executive summary ECP-induced effects Apoptosis • In heart transplantation (HTx) patients with recurrent acute rejection extracorporeal photopheresis (ECP) induced apoptosis in lymphocytic cells of rejection infiltrates until 10–60 days after treatment. • Hypothesis: ECP in HTx recipients may function via the induction of apoptosis.

Post-transplant immunmodulation • Interaction or uptake of donor lymphocytes treated with 8-methoxypsoralen and UV-A light by recipient dendritic cells (DCs) causes immunological changes: activation of DCs, induction of Tregs, changes in the cytokine expression and a shift of the Th1/Th2 cytokine profile.

Tolerance induction • Tolerance-inducing strategies target on the elimination of allogenic responses against the graft and the simultaneous maintenance of normal immune function. • ECP-treated HTx recipients with conventional immunosuppressive drug regimen exhibited a higher frequency of peripheral T cells with Treg phenotype and function. • Plasmacytoid dendritic cells (pDCs) acquire alloantigens in the allograft, then migrate to lymphoid tissue and induce the generation of Tregs.

Clinical trials

• ECP therapy is effective to avoid acute cellular rejection episodes after HTx. • Prophylactic ECP treatment reduced the number of acute rejection episodes compared with triple-drug immunosuppressive regimen alone. • Reduction of panel of reactive antibodies, incidence of cardiac allograft vasculopathy measured with intravascular ultrasound and incidence of CMV infection. • Resolved steroid-resistant acute cellular rejection after 3 months of therapy. • Successful treatment of low histological grade of acute cellular rejection with monthly ECP therapy for 12 weeks. • One cycle on two daily consecutive sessions has shown to be favorable compared with a single ECP therapy in the first 4 weeks.

Indications and contraindications of ECP • Indications for ECP after HTx have been investigated so far; prevention of: –– Acute cellular rejection (category I according to the American Society for Apharesis), and –– Secondary prevention of acute cellular rejection (category II according to the American Society for Apharesis). • Contraindications to ECP therapy as follows: –– Allergy against methoxsalens/psoralen; methoxsalens may cause fetal harm; presence or history of skin carcinoma requires close observation and treatment.

Adverse effects of ECP • Temporary and mostly associated with the catheterization of the vessel (e.g., hematoma, infection). • Most common complication is transient hypotension due to volume shifts. • Possible platelet losses in the machine requires a starting platelet count for at least 20,000. • Methoxsalen may cause a higher sensitivity to light for up to 24 h after therapy, so avoiding sunlight exposure or wearing sun protection is recommended.

Monitoring strategies of ECP treatment may allow to • Determine the onset, and duration of ECP therapy. • Differentiate between responders and nonresponders. • Tailor further treatment intervals to each patient’s needs.

Conclusion • Proof of principle of ECP therapy after HTx has been accomplished. • ECP not established in clinical routine. • Medical need to resolve problems in the short and long term after HTx. • Prospective, controlled and randomized clinical trials are necessary. • Future studies should use a three-step approach to introduce ECP in the clinical routine.

940




during the first month after HTx. ECP will be performed always on two subsequent days every other week until week 12, bi-weekly till week 24, monthly till month 12 and bi-monthly till month 24 post-transplantation. CNI target blood concentrations will be reduced stepwise at month 3 and 6 until withdrawal at month 12 in the study group receiving ECP. At month 11, a run-in phase with additional mTORI will be started, so that the drug regimen comprises mTORI, MPA and steroids at month 13 till study end. In the control group, the patients will receive CNI standard treatment over the whole study time. The follow-up should be till 2 years post-HTx. Assessments of clinical events like BPAR will be performed per protocol and when ACR is suspected. Furthermore, CAV will be detected with angiography and renal function by measuring creatinine clearance and calculated glomerular filtration rate. Besides safety assessments (including the incidence of CMV infection), PRA levels (HLA class I and II) and the incidence of non HLA-antibodies would be monitored. Because immune responses are orchestrated by many factors and the balanced, orchestrated interplay between these regulates the actions of the immune system, the trend goes to a global immune monitoring in clinical applications that includes a number of different cellular and molecular markers. Current clinical

measures are insufficient for the description of the complex interplay between cells and molecules accounting for the various immune-mediated processes occurring in patients [88] . Research studies identified a number of promising immunological markers that need to be validated in large clinical ECP studies. Helpful markers or a useful panel of markers are necessary to encompass the complexity of the immunological processes caused by ECP. The gained knowledge of the study results after such a three-step approach of ECP treatment has the potential to start ECP in the short-term and to continue in the long-term after HTx to control AR, chronic graft failure and reduce drug toxicity to result in an increased long-term graft and patient survival (Figure 3) . Financial & competing interests disclosure MJ Barten received honorarium from Therakos, Inc. MT Dieterlen did not have any conflicts to declare. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. Writing assistance was utilized in the production of this manuscript. A freelance writer edited the draft paper which the author prepared. Writing support was funded by Therakos, Inc. of severe acute and chronic graft-versus-host disease. Blood 92(9), 3098–3104 (1998).

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Single-center experience with extracorporeal photopheresis in pediatric heart transplantation.

Lung transplantation and extracorporeal photopheresis: The answer to bronchiolitis obliterans?

Percutaneous extracorporeal membrane oxygenation for graft dysfunction after heart transplantation.

Extracorporeal membrane oxygenation support after pediatric orthotopic heart transplantation.

Extracorporeal photopheresis: Focus on therapeutic immunomodulation.

Novel Application of Extracorporeal Photopheresis as Treatment of Graft-versus-Host Disease Following Liver Transplantation.

Extracorporeal photopheresis promotes IL-1β production.

Extracorporeal photopheresis versus standard treatment for acute graft-versus-host disease after haematopoietic stem cell transplantation in paediatric patients.

Extracorporeal photopheresis versus alternative treatment for chronic graft-versus-host disease after haematopoietic stem cell transplantation in paediatric patients.

Extracorporeal photopheresis in acute and chronic graft-versus-host disease.

Quilty effect after extracorporeal photopheresis in a patient with severe refractory cardiac allograft rejection.

Extracorporeal photopheresis in prevention and treatment of acute GVHD.

Extracorporeal photopheresis for the treatment of Crohn's disease.

Extracorporeal photopheresis for the treatment of cutaneous T-cell lymphoma.

Graft failure after infant heart transplantation: impact of extracorporeal membrane oxygenation on outcomes.

Predictors and Outcome of Extracorporeal Life Support After Pediatric Heart Transplantation.

Effect of extracorporeal photopheresis on lung function decline for severe bronchiolitis obliterans syndrome following allogeneic stem cell transplantation.

Extracorporeal photopheresis combined with pentostatin in the conditioning regimen for canine hematopoietic cell transplantation does not prevent GVHD.

Heart transplantation after ten years.

Ischemic Stroke after Heart Transplantation.

Abdominal aortic aneurysm repair after renal transplantation with extracorporeal bypass.

Extracorporeal membrane oxygenation for cardiac failure after congenital heart operation.

Extracorporeal photopheresis increases neutrophilic myeloid-derived suppressor cells in patients with GvHD.

Successful heart transplantation after prolonged cardiac arrest and extracorporeal life support in organ donor-a case report.