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© 2003 European Association of Cardio-Thoracic Surgery
Does contegra xenograft implantation evoke cellular immunity in children? eromskib
a Department of Paediatric Cardiac Surgery, Karol Marcinkowski University of Medical Sciences, 27/33 Szpitalna strasse Poznan 60-572, Poland
* Corresponding author. Tel.: +48-61-8491-277; fax: +48-61-8475-228/8669-130 Received December 23, 2002; received in revised form March 6, 2003; accepted March 7, 2003
The aim of the study was to search for changes in subpopulations of peripheral blood lymphocytes and in their activation as the manifestation of cellular immunity against xenograft in recipients of bovine valved conduit used for right ventricle outflow tract reconstruction. Between 24-06-1999 and 19-10-2000 35 children were operated in ECC, 19 had a xenograft implanted, the rest entered the control group. Immunophenotype of lymphoid cells and T cells activation was evaluated with use of flow cytometer: preoperatively and 3, 6 and 12 months after the operation in both groups. There were no differences in numbers of CD3+, CD4+, CD8+ and natural killer cells between groups. A significant rise of B-cells percentages (from 15.5% to 23%) between 3rd and 6th month was noted. The T-lymphocyte activation study revealed higher numbers of CD69+ (0.17 vs. 0.09 G/l) and CD71+ (0.23 vs. 0.11 G/l) cells one year after the implantation in xenograft recipients. Difference between groups in number of CD69+ and CD71+ cells in 12th month may suggest mild activation (<10%) of these subgroups in xenograft recipients. This data may hint the presence of cellular reactivity. Changes in numbers of B-cells may evidence humoral immunity activation. Influence of these phenomena on graft survival remains to be established.
Key Words: Right ventricle outflow tract; Children; Xenograft; Immune response; Flow cytometry; T-cell activation
Majority of congenital heart defects in children can be corrected in anatomic way, including malformations concerning right ventricle outflow tract obstruction (RVOTO). Reconstruction of the RVOT continuity is also necessary when the native pulmonary valve is transferred to aortic position during Ross operation. RVOT reconstruction requires adequate prosthetic material to replace defective structures. The most frequently used substitutes of pulmonary valve and trunk are homografts. Xenografts are the second choice in RVOT reconstruction but are far easily available. Progressive degradation of structure and function of the implant make difficult the use of biological materials in treatment of congenital heart defects with RVOTO. The pathological mechanism of homo- and xenogeneic implant destruction is not completely elucidated. One of possible causes can be an immunological response. It was observed in case of homografts where the presence of anti-HLA antibodies and specific cellular immunity was found [1] as well as in xenograft recipients [2]. Presence of inflammatory cells on leaflet surfaces and infiltration by giant cells and macrophages in xenogeneic valves was evidenced earlier [3]. The use of xenogeneic valved conduit (Contegra) is one of the potential solutions in RVOT surgery [4]. The early results are satisfactory [5]. However possible immunological reaction leading to late graft dysfunction remains to be clarified. This reaction could be even more accentuated in patients exposed to the second implant due to previous immunization. This study was conducted to search for the changes in subpopulations of peripheral blood lymphocytes and in their activation as the manifestation of cellular immunity against xenograft in recipients of bovine valved conduit used for RVOT reconstruction in children with complex congenital heart defects.
From June 1999 to January 2002 a total of 35 patients underwent correction of congenital heart defect with use of cardiopulmonary bypass (CPB), 19 of them underwent Contegra xenograft implantation during RVOT reconstruction (AI+AS-8; l-TGA+PS-3, ToF-3, dTGA+VSD+PS-2, VSD+PA-2, DORV+PS-1) – Table 1. The control group consisted of 16 patients operated due to various heart defects (ASDII-8, VSD -3, ASDI-1, TI III°-1, MI IV°-1, Ao-LV tunnel-1, SV-1) – Table 1.
No patient had a biological graft implanted before. None of them had either autoimmunologic or allergic disease that could activate immunologic system (interview, office records). Moreover during the follow-up symptoms of above mentioned diseases and other disorders influencing immunity were not detected. No immunomodulating drugs were administered to patients. One-year follow-up was obtained from all patients by direct contact in the department. The Ethics Committee on Human Research approved the study protocol. Informed consent was obtained from patients’ parents. 2.1. Surgical technique and postoperative treatment A surgical treatment was performed in both groups in general anaesthesia through a midline sternotomy. Both venae cavae were cannulated through the right atrium auricle (despite patient who underwent bi-directional cavo-pulmonary shunt). Normothermic or moderate hypothermic CPB was instituted. CPB apparatus was previously primed with blood containing fluids or not (in patients over 20 kg of weight and the hematocrit over 40%). Aortic cross clamping and multiple doses of St. Thomas crystalloid cardioplegia was employed in all patients (initial dose: 20 ml/kg, consecutively a half of initial dose every 30 min). Xenogeneic implant was employed in RVOT reconstruction that was a common part of all procedures in patients from xenograft group. The distal anastomosis (pulmonary artery to outflow part of the xenograft) was performed with monofilament suture at the end of the heart arrest, the proximal one (inflow part of the graft – right ventricle) was carried out on beating heart.After the surgical procedure all patients were transferred to intensive care unit. Stored blood was administered if necessary during the postoperative course. Preparations of blood contained enriching solution (Baxter: dextrose, NaCl, mannitol and adenine), were previously leukocyte and thrombocyte depleted and were transfused according to ABO and Rh groups. In the case of children less than 1 year old blood was irradiated. No leukocyte filters were used during the CPB and blood transfusions. 2.2. Flow cytometry Peripheral venous blood was the source of immunocompetent cells analysed in this study. Blood samples were drawn from two groups of patients before the operation and 3, 6 and 12 months after the operation. Part of the sample was used for white blood cell count. The rest was stored in 4 °C (maximum 20 h) until the cytometric assays carried out on Becton–Dickinson (BD) FACS Scan flow cytometer.Following reagents were used: pH 7,4 phosphate-buffered saline solution, FACS Lysing Solution (BD), fluorochrome (PE – phycoerythrin, FITC – fluorescein) conjugated monoclonal antibodies: anti-CD45–FITC, anti-CD14–PE, anti-HLA-DR-PE, anti-CD4–FITC, anti-CD8–PE, anti-CD19–PE, anti-CD16+anti-CD56-PE from Simultest IMK kit (BD), anti-CD69–PE (BD), anti-CD71–FITC (BD), anti-CD25–PE (DAKO) and anti-CD3-FITC (DAKO), IgG1-FITC and IgG2a-PE mouse antibodies for unspecific antibody binding control. Immunophenotype of following lymphoid cells was evaluated: T-cells – CD3+ and their subsets: CD4+ (helper) and CD8+ (suppressor/cytotoxic), B-cells – CD19+ and natural killers (NK) – CD3-CD16+CD56+. Activation of T cells was evaluated by counting the number of T-cells presenting activation markers: major histocompatibility complex class II antigen – HLA-DR+, interleukin-2 receptor – CD25+, glicoprotein 32/28 – early activation marker – CD69+ and transferrin receptor – CD71+. Counts of lymphoid cell subsets were expressed in percentages of all lymphocytes as well as in absolute numbers by multiplying total white blood cell count by percentages (expressed in decimal fraction) of lymphocytes and adequate subpopulation. CD4+ to CD8+ cell count ratio was calculated too. Chosen immunophenotype parameters were standardised by referencing to the age group (neonates 0–30 days, infants 31 days–1 year, children: 1–3, 4–7, 8–12, 13–17 years old) norm for the Polish population provided by Zeman and co-workers [6]. Each single cytometric measurement in given patient was expressed as a percent of mean norm for age group to which the patient belonged. Obtained values were used for further statistical analyses. 2.3. Statistical analysis The normality of variable distribution was assessed by Shapiro–Wilk test. Accordingly mean and standard deviation or median and ranges described variables. On figures medians and quartiles depicted all variables. Between-group comparisons were performed by Student's T-tests or Mann–Whitney U-tests according do variable distribution. Student's T-test for dependent variables or Wilcoxon test were employed in intra-group comparisons. P-values smaller than 0.05 were considered statistically significant. P-values from range 0.05–0.1 were considered as a trend toward statistical significance.
3.1. Operative variables and transfused blood Patient from xenograft group had significantly longer CPB time and aortic cross-clamp time – Table 1. There were no differences between groups in quantity of transfused blood – Table 1. 3.2. Immunophenotype of blood lymphocytes Median white blood cell counts in both groups did not exceed 9 G/l during the follow-up. Median lymphocyte counts and percentages of lymphocytes (referenced do all leukocytes) did not differ significantly during the whole follow-up.No significant differences between groups were observed in percentages (as well as in absolute counts – not shown) of T-cells during the observation. No significant intra- and between-group differences were detected in percentages and absolute numbers of CD4+ and CD8+ cells during the follow-up. The CD4+ to CD8+ ratios did not differ significantly in both groups. However this parameter was always greater than one in xenograft group.
The significant increase of B-cell percentages was observed only in patients with xenograft between 3 and 6 months after the operation. However the between-group difference had only a trend toward statistical significance (
There were no significant differences between both groups in numbers of NK cells during the study (not shown). 3.3. T-cell activation No statistically significant between and intra-group differences were found in percentages and absolute numbers of T-cells expressing HLA-DR and CD25 antigens during the follow-up. The significant difference between groups were found in CD69+ T-cells absolute count 12 month after the operation – Fig. 2. The significant decrease of this parameter was observed in control group between 3rd and 12th month postoperatively. However the parameter expressed as a percentage did no differ significantly during the whole study. The significant differences between groups were found in CD71+ T-cells percentages and absolute counts 12 month after the operation – Fig. 3. Median percentage of CD71+ cells in xenograft group was reaching 10%.
The use of xenogeneic valved conduits seems to overcome certain difficulties connected with other implants employed in RVOT surgery [4]. However the reaction of the organisms to the implant remains still unknown. Availability and good preliminary results of Contegra reconstructive material suggested a need for prospective study on immune response in recipients that may influence the function of the implant. Studies on immune response against xenorgaft were undertaken in various experimental works [7,14], however there is a scarce number of papers describing clinical studies [8] especially in children with congenital heart defects [2]. The control group was created according to assumptions stated above. Comparing to the xenograft group all preoperative variables were balanced. Though significant differences in operative parameters were noticed. This fact reflects the complexity of surgery in xenograft group and the absence of patients operated for RVOTO without biological materials in control group. Nevertheless creation of control group from population of children with congenital heart defects was indispensable to eliminate possible inter-group differences caused by long-term effects of CPB and stored blood transfusions. The choice of flow cytometry as study method was connected with availability of biological material and little degree of invasiveness of its collection what is of great importance in children. However peripheral blood cells amount only a small percent of all lymphoid cells of immunologic system. As well some immunologic processes may occur in lymphoid organs only with little or even without reflection in immunophenotype changes in peripheral blood [6]. Standardisation of the chosen cytometric parameters was employed in the study to minimise possible bias introduced by age factor in patients from heterogeneous populations. Differences of cytometric parameters of immunophenotype in various age groups are well known [9] – a value that lies within a norm in one age group can exceed it in another. Hence, referencing the data to mean age norm for Polish children population could have rendered the results of analysis more accurate. CPB can be a factor influencing the immune system. Changes of immunophenotype parameters are transient and usually observed during CPB and several days after [10]. Return to the baseline requires most frequently one week, thus current study does not seem to be biased by short-term effect of CPB and its different duration. Long-term CPB effect remains unknown. Blood transfusion is an allogeneic tissue transplantation. Its influence on immunological system is not ultimately explained – according to the literature there are possible immunostimulating and immunosuppressive effects [11]. None of them was clearly visible in our study. Administering only leukocyte depleted and irradiated blood preparations could have probably diminished them. There are several reasons of potential immunogenicity and antigenicity of Contegra xenograft: (1) big surface of contact with host tissues (in comparison to other biological prosthetic valves); (2) presence of cellular remnants (whole endothelial layer) within the implant; and (3) nonhomogenous graft fixation by glutaraldehyde what was demonstrated by studies with fluorescent probes [12]. Presence of circulating preformed xenoantibodies, unspecific complement activation even by fixed tissues [13] and absence of molecular biocompatibility of the endothelium and extracellular matrix can further enhance immune response to xenorgaft. White blood cell counts during the follow-up does not reflect any acute inflammatory reactions and remain within a norm. Absence of differences in absolute numbers and percentages of T-cells and CDR+ cells, balanced CD4+/CD8+ ratios may evidence lack of any stronger immunologic stimuli. A trend towards statistical significance of inter-group difference in percentages of B-cells (CD19+) in 6th month after the operation and significant increase of this parameter in xenograft group (up to 150% of mean age norm) may evidence humoral immunity activation. Facts indicating humoral immune response were described previously [2]. Increased immunoglobulin levels were detected in children with xenograft [2] as well as presence of plasma cells in explanted xenoprosthetic valves. Experimental studies evidence creation of antibodies against fixed xenogeneic material [7,14]. The increase of activated T-cells may suggest the ongoing immunological response. Analysis of T-cell expressing CD25 and HLA-DR markers did not reveal any activation. However there was a significant difference in absolute number of T-cells expressing CD69 antigen and in quantity and percentages of CD71+ T-cells between groups 12 month postoperatively. This may suggest activation of these subgroups of lymphocytes in xenograft recipients. Percentage of CD71+ cells (<10%) hints rather mild activation, present in some patients. This data may hint for some cellular reactivity. Presence of specific cellular response against allogeneic valves was proved in human [1] – sensitised CD4+ and CD8+ T-cells were eluted from explanted homografts. Similarly T-cells were also isolated from xenogeneic valve prostheses [2]. Experimental studies showed presence of inflammatory infiltration into the xenograft as well as T-cells activation by fixed tissues of xenogeneic valve [7]. Presented publications show mainly local reaction to the xenograft. As mentioned above, such a weak local event may not always be manifested in changes of immunophenotype of the peripheral blood. Non-specific local inflammatory reaction may be suggested. The onset of immune response against xenograft and its maximum is an important issue. Certain immunologic reaction was observed in patient with xenograft a year after the implantation. Experimental studies indicate maximum immunologic response about 3 months after the implantation and further extinction of immunity [15]. This discrepancy can be explained by inter-species differences or failure to detect immunologic reactions due to low frequency of measurements. Studies on homograft revealed intensification of immunological response from 1st do 3rd month after implantation [15].
ICVTS on-line discussion Author: Antonio Corno, CHUV, Cardiac Surgery, 46 rue du Bugnon, Lausanne CH 1011, Switzerland Date: 27-May-2003 12:17 Message: A timely and provocative paper. In fact there is an increasing number of clinical reports on the early and medium term results with the bovine jugular vein (Contegra/Medtronic) used as xenograft biological valved conduit from the right ventricle to the pulmonary artery. Even if we contributed to the publication of the first series of implantations within the multicenter clinical trial (Corno et al., Bovine jugular vein as right ventricle-to-pulmonary artery valved conduit. J Heart Valve Dis 2002;11:242-7, PMID 12000167), we acknowledge the lack in the literature of serious clinical studies on immunitary reaction of this type of biological conduit. The Authors provided in an accurate way the evidence of the immunitary reaction subsequent to the clinical implantation, but they didn't extend their report to the potential clinical inferences of the results of their observations, particularly to the suggestion of pre-operative screenings to perform in order to discriminate between good and bad candidates for the implantation of this type of conduit. Another consideration is regarding the known tendance of homografts (still considered the gold standard) to develop immunitary reactions. Since most of the patients requiring implantation of a valved conduit in infancy will require a conduit replacement before adolescence, do the centers using only homografts have to consider alternating a homograft with a Contegra/Medtronic conduit for valved conduit replacement? How the immunitary reaction developed by this type of xenograft can be compared to the same type of reaction observed after homograft use? Author: Manoj Purohit, SPR, Cardiac Surgery, Alderhey Hospital, Eaton Road, Liverpool, L12 2AP, UK Date: 01-Jun-2003 14:42 Message: All the xenograft conduits are getting their foothold because of the non-availability of allografts especially in small sizes, which are the current favourites. Immunological reactions to conduits and its role in conduit failure have remained a burning research issue. Modifications on this basis have been done and even immuno suppression has been tried even for allografts, without any conclusive evidence of improvement in conduit survival. It would be useful to know how these research findings can be correlated with the outcome of the patients. I know the present study is for 12 months only, which is a short period in the natural history of conduit failure but it would be more prudent if findings in such studies could point towards a clinical outcome, for which most of the studies both on allografts and xenografts have failed. With the present day knowledge of immunology and advancement in transplant sciences, it can be extrapolated that there will be immunological reaction but what it means and how it manifests is more important. Is there a real difference in the immunological reaction in early conduit failure patients and those with conduit lasting for longer times? The reasons they have stated for high chances of immunogenicity and antigenicity of Contegra are going to be same for any xenograft conduit fixed with gluteraldehyde and it cannot be compared with biological valves without conduits. The paper has failed to give any direction to further research on this topic, does this study need to be done for a prolonged period so the clinical outcome can also be tested? What about explanted conduits, how can they be tested and help in this research? It would have been interesting as well to keep another control group of allograft conduits.
We express our appreciation to Ms Magdalena Trybus from Department of Clinical Immunology for her invaluable help in performing FACS studies. We also thank Mr RC Quijano and ‘Our Heart Foundation’ for support in conducting this study. doi:10.1016/S1569-9293(03)00058-6
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Abstract
1. Introduction
) – 
median-xenograft group. 
control group. *percent of CD19+ increased significantly (
) between 3rd and 6th month after the operation in xenograft group, while CD19+ percentage in control group remained constant (^). **difference between groups in 6th month had only a trend towards statistic significance (
) difference between groups in 12th month. ^ significant decrease of CD69+ in control group between 3rd and 12th month.
) difference between groups in 12th month.
