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ARTICLE
Intraoperative Tissue-Immunosuppressive Therapy Reduces Rejection Episodes in Heart Transplant Recipients

Objectives: Our study was conducted to determine the effects of intraoperative antithymocyte globulin administration on donor hearts procured after cardiocirculatory death. We evaluated the impact of antithymocyte globulin on graft function and related parameters during isothermic blood cardioplegia.

Materials and Methods: In this prospective and randomized single center study, 30 patients with orthotropic heart transplant were divided into 2 groups: group 1 included 15 patients who received retrograde antithymocyte globulin infusion via coronary sinus intraoperatively and immediately after organ procurement and group 2 included 15 patients who received traditional antithymocyte globulin infusion after implantation.

Results: Study patients had a mean age of 33.8 years (range, 15-56 y). All patients had panel reactive antibody less than 10% except for 3 patients. The cluster of differentiation 3-positive cell count decrease was more than 20%. The inotropic therapy dose required and the myocardial pressure (stiffness) were less for group 1 patients. These patients had less acute rejection episodes than group 2 (0% vs 13.3%; P < .05).

Conclusions: Favorable clinical outcomes were observed in terms of less acute rejection episodes and better graft function at least during the early posttransplant period. Intraoperative antithymocyte globulin treatment may have a preventive effect for acute cellular rejection in heart transplant patients.


Key words : Antithymocyte globulin, CD3, Immuno­suppression

Introduction

Allograft rejection is one of the crucial dilemmas and concerns of early mortality after heart transplant.1 The main purposes of any immunosuppressive protocol are adapting the host’s immune reaction by minimizing non-self recognition and intervention against foreign antigen structures; the long-term survival of patients with full functioning graft is the final aim of organ and tissue transplant. With the reduction of rejection risk, immunosuppressive therapy has made organ transplant feasible.2

The inclusion of cytolytic antibodies as immuno­suppression induction treatment, such as anti-T-cell globulins, in the prophylactic therapy protocol has increased success with solid-organ transplant.3 Prevention of steroid-resistant rejection was the initial target in treatment and has led to the development of polyclonal antibodies. The use of antilymphocyte drugs has enhanced immuno­suppression and optimized outcomes in allograft recipients. Antileukocyte serum was first defined by the Russian immunologist Élie Metchnikoff in 1899.4

Induction treatment is a “supplemental pro­phylactic treatment strategy” that decreases the hazard of acute rejection in the early posttransplant period. Although the mechanisms of action of anti-immune serum have not yet been fully explained, without a doubt there is now evidence that antithymocyte globulins (ATG) are useful agents for managing these clinical circumstances. However, current immunosuppressive regimens are still suboptimal in graft failure rates and have adverse effects, and a general consensus for the best treatment protocol has not been reached.5

Antibody therapy is usually applied at a fixed dose for 1 or 2 weeks posttransplant. This regimen has been successfully modified for renal transplant by substitution of a perioperative single high dose(9 mg/kg) of ATG in nonsensitized patients.6 In general, ATG has a strong suppressive impact on activated lymphocytes, a fact that endorses its use in clinical situations where activated lymphocytes play a predominant role, as in the case of T-cell-mediated graft rejection.3 When high-dose intraoperative ATG is used, the introduction of calcineurin inhibitors can be postponed, thereby protecting cardiac function and completely avoiding use of corticosteroids in the primary immunosuppression regimen.7

In cardiac transplant, however, “intraoperative continuous infusion” at the start of surgery, as successfully used in renal and renal-pancreatic transplant, is not preferred as a protocol. To assess the clinical impact and efficacy of intraoperative continuous ATG infusion versus the established conventional prophylactic ATG regimen, we conducted a prospective randomized study. This investigation will allow us to elucidate whether this new protocol is associated with efficacy and safety similar to the conservative therapy.

Materials and Methods

Patients
Our study was performed at a tertiary reference cardiac surgery hospital and approved by our local ethics committee. This was a prospectively designed study that included 32 consecutive patients who had orthotropic heart transplant at our center between July 1, 2011 and July 30, 2014. Patients were randomized into 2 groups based on the infusion timing of ATG (ATG-Fresenius S; Fresenius Biotech GmbH, Gräfelfing, Germany). Group 1 initially included 17 patients who had ATG infused via coronary sinus both intraoperatively and post­operatively. Group 2 included 15 patients who had only postoperative ATG infusion (conventional treatment group). To allow concise and balanced progression of the study, the randomization of the 2 groups was done consecutively, with group 1 patients being the last 17 patients of the study period. Patients were informed about the procedure and gave informed consent preoperatively. Patients with preoperative thrombocytopenia, leukopenia, or active bacterial, fungal, or viral infections were not included in this study. Two patients were excluded from study results due to death from a surgical complication (bleeding) and death due to renal failure (Table 1).

Surgical procedure
Under general anesthesia with full monitorization of all hemodynamic parameters, total cardiopulmonary bypass was initiated. Isothermic blood cardioplegia was applied immediately after aortic cross-clamping. All anastomosis procedures were made under cross-clamping. All bicaval orthotropic heart transplants were performed by the same surgical team.

Study design
This study was conducted in donor hearts obtained after cardiocirculatory death (category III and category IV according to modified Maastricht classification)8 to determine the effects of intraoperative ATG administration applied during isothermic blood cardioplegia on graft function and related parameters. Retrograde isothermic blood cardioplegia infusion with the ATG solution via the coronary sinus was started as soon as the donor heart arrived to the surgical department.

Total doses were the same for both groups, with the only difference being that 7 to 10 mg of the total dose were infused intraoperatively for group 1 patients. For this procedure, 7 to 10 mg of ATG were infused intraoperatively via cardioplegia cannulas for group 1 patients until the end of surgery. Postoperatively, group 1 patients received 3 mg/kg ATG for 7 days and group 2 patients (control group) received standard ATG therapy consisting of 7 daily doses of ATG beginning immediately after termi­nation of surgery and on arrival to the intensive care unit. Intraoperatively, all patients received a single intravenous bolus of 1000 mg methylprednisolone immediately before removal of the aortic cross-clamp.

Blood samples were taken from the aortic ostium at the beginning of the infusion, before the anas­tomosis, and after the anastomosis. Total lymphocyte count, platelet counts, and granulocyte numbers were determined just before heart transplant and every 24 hours thereafter for 30 days. The diagnosis of acute cardiac allograft rejection was made according to the criteria of the International Society for Heart and Lung Transplantation.9

Postoperative period
All patients had the same follow-up protocol posttransplant. Immediate posttransplant prophylaxis for infections included short-term antibiotic agents such as ceftazidime and piperacillin and oral nystatin. Patients who were negative for cytomegalovirus (CMV) infection but had donors who were CMV positive were given acyclovir at 5 mg/kg/day.

Efficacy parameters included vital signs and the need for postoperative transfusions. Acute rejection was not considered as an adverse event but as treatment failure. Safety analysis was based on adverse events directly noted by the medical staff or explained by the patients.

Statistical analyses
All patient data were recorded at a computerized database system from laboratory findings and hard copies of medical records. All statistical analyses were performed using commercially available SPSS statistical software (version 16.0.1, SPSS Inc., Chicago, IL, USA). Continuous variables are expressed as means and standard deviation (SD). For categorical data, absolute and relative frequencies were considered and categorical variables were compared using chi-square test and Fisher exact test where appropriate. In addition, cluster of differentiation 3 (CD3) count was analyzed in group 1 patients using chi-square test.

Continuous data, including mean results and SD, median, quartiles, minimum, maximum, and number of valid values, were compared using independent sample t tests or Mann-Whitney U tests. P < .05 was considered to be statistically significant. All P values reported were 2-sided and intended to be descriptive.

Results

Patient characteristics
Our study population included 25 men (84.5%) and 5 women (16.6%). Mean age (SD) was 33.8 (6.3) years (range, 15 to 56 y). Preoperative causes of heart failure were chronic ischemic heart disease in 20 patients (66.6%), primary cardiomyopathy in 8 (26.6%), and other causes in 2 (7%). Eight of 15 patients (53.3%) in group 1 (continuous infusion) and 9 of 15 patients (60%) in group 2 (conventional infusion) had at least 1 attendant disease at orthotopic heart transplant. Most patients were classified as III or IV according to the New York Heart Association criteria. At the time of enrollment, all patients were nonsmokers except for one. No significant differences were shown between the 2 groups regarding mean age, sex, indication for cardiac transplant, and cardiac functional class. Mean age of donors was 44 years (range, 23-61 y). No significant differences were shown among donors regarding age, sex, cause of death, total cold ischemia time, and other surgical parameters during transplant (Table 1).

Early survival information
No perioperative deaths were reported except for the 2 excluded patients. Rejection was monitored during hospital stay. There were no deaths in the final patient groups. No differences were observed between the 2 groups with regard to clinical performance or electrocardiographic assessment.

Group 1 patients showed less echocardiographic abnormalities, insofar as left ventricular shortening fraction and expected wall thickness alterations, after transplant than group 2 patients. In addition, the doses needed for inotropic therapy and the myocardial pressure (stiffness) measurements were less for group 1 patients than for group 2 patients. At last follow-up before hospital discharge, patients had New York Heart Association grade I/II disease.

Laboratory findings
Immediately after the first ATG administration, total lymphocyte counts quickly and significantly decreased in group 1 patients; however, a similar T-lymphocyte depletion affecting CD3 and CD8 subpopulations was shown for both groups (Table 2) but was only significant for group 1.

Both groups showed thrombocyte count decreases at the beginning of induction therapy. However, on the day of transplant, thrombocyte counts were particularly lower in group 2 patients (Table 2). Marked increases in thrombocyte counts were shown over the subsequent days in both groups, although results were only significant for group 1. No cases of severe thrombocytopenia were shown in any of the patients that required discontinuation of ATG treatment (< 50 × 103/μL). Moderate leukocytopenia was reported in 4 patients in group 1 and 6 patients in group 2; however, no patients required discontinuation of ATG infusion.

All patients in group 1 except for 3 had panel reactive antibody (PRA) less than 10%. CD3-positive cell count was decreased by more than 20% for group 1 patients (Table 3). Unfortunately, group 2 patients did not undergo CD3 cell analyses. Although this was a study limitation, we were able to compare total lymphocyte counts between the 2 groups (P < .03).

Group 1 patients had no acute rejection episodes (0%) versus 2 of 15 patients (13.3%) in group 2 (P < .05). The 2 patients underwent endomyocardial biopsies, which showed acute rejection according to early postoperative clinical and echocardiographic findings, and received antirejection therapy.

Discussion

When risk for allograft rejection is increased during the initial posttransplant period, nearly all immuno­suppressive therapies for solid-organ transplant recipients follow the use of additional strength treatment. CD4-positive T cells play a primary role in acute cardiac allograft rejection, and acute CD4-positive T-cell-mediated rejection requires major histocompatibility complex (MHC) II expression in the allograft, pointing to the importance of direct graft recognition. A key point for acute cellular CD4-mediated rejection is whether the rejection happens via direct MHC class II presentation by the graft and/or via indirect donor antigen presentation by host MHC class II-bearing antigen-presenting cells.10

Specific antibody induction agents or high-dose intravenous corticosteroids or combinations of both can be used to allow immunosuppressive efficacy at this early stage. Implementation of induction treat­ment during the immediate postoperative period or peri­operatively has become useful in cardiac transplant. This approach can improve graft survival after transplant, with a noteworthy reduction in calcineurin inhibitor dosage and reduction of associated adverse events without compromising allograft survival.

The use of ATG to prevent rejections has shown promise in solid-organ transplant,11,12 including cardiac transplant.13-15 After rabbit ATG as a prophylaxis treatment was first defined in 1987 by Kawaguchi and associates,16 antilymphocyte antibo­dies are now administered in almost 22% of transplant recipients.17 Currently, ATG is the only induction drug approved for cardiac transplant.

The immunosuppressant ATG is a polyclonal anti-T-lymphocyte immunoglobulin product procured from rabbits immunized with human Jurkat cells.18 Its mechanisms of action on T cells include com­plement-mediated cytolysis of T-cells, stimulation of apoptosis, blocking of signal transduction pathways, opsonization of activated cells, and suppression of adhesion.19

Antithymocyte globulin can reduce levels of lymphocyte subsets expressing surface proteins. It temporarily decreases leukocyte and platelet num­bers, which return to normal levels after application. Specific changes in lymphocyte action can occur that block these surface molecules called clusters of dif­ferentiation (CD).10,20 This efficacy of ATG is likely to be a result of the complement binding to the cell-ATG composite, and reduced CD2, CD3, CD4, and CD8 counts have been noted. Indeed, a single high-dose implementation may affect cytokine levels, decreasing interleukin 1β, tumor necrosis factor-α, and interferon-γ levels, suddenly increasing interleukin 10, but not affecting interleukin 12-p70.10

Antithymocyte globulin can have a better effect than activated T lymphocytes by blocking mech­anisms like antigen recognition (MHC classes I and II) and complement-dependent cytolysis of activated lymphocytes, blocking signal transduction pathways accountable for activation of T cells, inhibiting leukocyte adhesion on endothelial cells and subsequent extravasation21 and/or opsonization of activated cells, and stimulating Fas-mediated apoptosis of activated lymphocytes.5

In our study, we analyzed the efficacy and safety of 2 induction therapy regimens in patients early after heart transplant (standard therapy with rabbit ATG for 7 days versus ATG infusion started immediately at transplant followed by low-dose therapy). Effectiveness of treatment was assessed by improved graft survival. The rationale of an initial intraoperative retrograde infusion approach was whether, in addition to T-cell depletion, ATG could more efficaciously avoid initial damage.

The above-mentioned mechanisms of ATG appear to work together to protect the allograft from initial inflammatory harm and to allow allorecognition processes and occurrence of an immune response. In general, initiation of ATG infusion at an early phase may decrease the strength of the initial immune reaction. Many experimental studies have reported that ATG preparations may have a useful impact against ischemia-reperfusion injury in nontransplant models.2,6 Abudher and associates22 found that high-dose intraoperative ATG may enhance cyclosporine sensitivity. In a study of 30 cardiac transplant recipients, patients given ATG for 7 days tended to have less cardiac allograft vasculopathy than those who received ATG for 3 days (28% vs 50%; P = .05).23 Martins and associates reported that the accumulative dose of rabbit ATG may also be related to better renal graft and subsequent patient survival rates.24

Both of the regimens demonstrated comparable efficacy results, with a low incidence of acute rejection and without graft loss despite low cyclosporine concentrations. This could explain the relatively low incidence of calcineurin inhibitor-related adverse events. Particularly notable was that there was no mortality among patients in the high-dose group.

In addition, intraoperative application of ATG was not related with hemodynamic complications and was well tolerated. For both patient groups, severe adverse effects related to ATG use were not detected.

Faggian and associates, in a study that compared a single high intraoperative dose of rabbit ATG followed by 1.5 mg/kg for 5 days (n = 14) versus standard 7-day therapy (n = 16), noted similar efficacy between regimens. Early high-dose ATG may preserve effectiveness, but a fixed therapy of 1.5 mg/kg for 5 days alone may be a less effective rejection prophylaxis than 7-day therapy.23 Krasinskas and associates reported that monitoring ATG based on peripheral T-lymphocyte counts in cardiac transplant recipients led to a decrease in the total ATG dose from 10 to 15 mg/kg to 1 to 5 mg/kg without an increase in acute rejection.25 In our study, we have applied the same dose of ATG for both groups postoperatively. Koch and colleagues confirmed this finding that lymphocyte-adapted monitoring can preserve efficacy with a significant reduction in cumulative rabbit ATG dose compared with a conventional fixed-dose regimen.26

The impact of ATG therapy on peripheral blood lymphocyte subsets was analyzed by flow cytometry. A higher percentage of patients in group 1 had transient leukopenia; however, despite the existence of comorbidity, no differences between the 2 groups were detected. Intraoperative ATG infusion decreased total lymphocyte count, with initial and rapid thrombocyte decrease. No patients required dis­continued therapy because of marked lymphopenia, neutropenia, or thrombocytopenia. Although several strategies can decrease circulating antibodies, studies have reported that patients with increased pre­transplant PRA levels > 10% tend to have earlier and more severe rejections with significantly lower posttransplant survival.26 We obtained similar results concerning PRA levels in our study.

A simultaneous reduction was observed regar­ding CD3-positive (total T lymphocytes), CD3/CD4-positive (T helper cells), and CD3/CD8-positive (cytotoxic T cells) levels, although occurring faster and lasting longer in group 1 patients.

Although higher dosing of ATG might cause CMV infections, a lower incidence of CMV infection was noted by Zuckermann and associates with high-dose ATG use.15 In our study, we observed no differences in incidence of clinical infections between the 2 groups, and there was no patient loss due to any infectious complications.

Conclusions

Induction treatment has been part of the immuno­suppressive protocol for more than 40 years, but when and how it should be used in heart transplant recipients are not clear. Although a growing pool of data point to its efficacy and tolerability, its use as an induction regimen in heart transplant recipients remains unproven.

The potential of rabbit ATG to inhibit progression to cardiac allograft vasculopathy is of substantial importance, and future research is awaited with interest. Modern therapies in which rabbit ATG dosing is adjusted with lymphocyte count may allow reduced dosing, allowing potential safety and cost benefits without compromising efficacy.

Despite our small sample size, favorable clinical outcomes were observed in terms of less acute rejection episodes and better graft function, at least during the early posttransplant period. Patients with intraoperative ATG treatment needed less inotropic therapy and had less posttransplant complications. Our results support the feasibility of ATG as an early therapy as intraoperative infusion in orthotopic heart transplant. Intraoperative continuous infusion of ATG treatment may have a preventive effect for acute cellular rejection in cardiac transplant patients. Further controlled trials are required to identify the most suitable dosage and to optimize the starting time of this induction protocol. Our study, even with its limited patient number, allowed preliminary insight of the therapy modality and duration that could be used for further larger patient group studies.


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DOI : 10.6002/ect.2017.0230


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From the 1Cardiovascular Surgery Department, Kartal Koşuyolu Yuksek Ihtisas Research and Training Hospital, Istanbul; the 2Medical Genetics and Immunology Department, Marmara University Medical Faculty, Istanbul; the 3Cardiovascular Surgery Department, Okan University Medical Faculty, Istanbul; and the 4Cardiovascular Surgery Department, Yeni Yuzyil University Medical Faculty, Istanbul, Turkey
Acknowledgements: The authors have no sources of funding for this study and have no conflicts of interest to declare.
Corresponding author: Murat Bulent Rabus, Denizer Caddesi, No. 2 Cevizli, Istanbul, Turkey
Phone: +90 216 5001500
E-mail: muratrabus@yahoo.com