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Eur J Cardiothorac Surg 1999;15:672-679
© 1999 Elsevier Science NL

The influence of antibody and complement removal with a Ig-Therasorb column in a xenogeneic working heart model¹

^a Department of Cardiac Surgery, Klinikum Grosshadern, Ludwig–Maximilians-University Munich, Marchioninistr. 15, D-81377 Munich, Germany
^b Institute for Surgical Research, Klinikum Grosshadern, Ludwig–Maximilians-University Munich, Munich, Germany

Received 20 September 1998; received in revised form 23 December 1998; accepted 10 February 1999.

Corresponding author. Tel.: +49-89-7095-4404; fax: +49-89-7095-8897.

	Abstract

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Objective: Organ transplantation is limited by the number of brain-dead human donors. Xenotransplantation could be an alternative to guarantee a constant supply of organs. A major problem of xenotransplantation are xenogeneic natural antibodies (XNAb) directed against species-specific antigens of a discordant donor species (e.g. pig). They trigger the hyperacute xenograft rejection (HXR). Re-usable immunoapheresis (IA)-columns Ig-Therasorb® (Therasorb, Baxter) were used to adsorb these XNAb. The effect of immunoapheresis of the perfusing human blood was investigated in ex vivo working pig hearts. Methods: Hearts of 12 landrace pigs (body weight 14–31 kg) were explanted after inducing cardiac arrest with 4°C Celsior solution. Human blood (500 ml, heparinized) was obtained from healthy volunteers. In group 1 (G1, n=6), blood as perfusate remained untreated. In group 2 (G2, n=6), native blood was separated by plasmapheresis into cellular components and plasma. The latter passed through the Ig-Therasorb column for removal of immunoglobulins (so-called immunoadsorption or immunoapheresis). After back-table preparation the hearts were mounted to the working heart model. After 20 min of reperfusion in Langendorff mode, the working heart mode was established. Blood samples were taken isochronously for measurement of: CK(-MB), LDH, ASAT, troponin, immunoglobulins, complement activity, anti-pig antibodies and others. After cessation of the heart, atrial and ventricular tissue samples were taken for histological examinations (light/electron microscopy and immunohistochemistry). Results: Two cycles of immunoapheresis reduced the levels of IgG by 84%, IgM by 83.3% and IgA by 76%. In G2, the antibody immunoadsorption of blood prolonged the duration of the working heart mode significantly to 335±37.5 min. In contrast, hearts of group 1 (control) failed after 125±31.3 min. Heart rate was significantly different between both groups (G1, 77.3±6.1 beats/min; G2, 86.5±5.5 beats/min). In G2 cardiac output was 118% and mean coronary flow was 154.6% higher than in G1. CK, LDH and ASAT showed no differences in the two groups. Heart weight increased significantly more in group 1 than in G2. Histological examination indicated specific signs of HXR in G1 after 1.5 h, whereas in G2 only slight unspecific damages were found after 6 h. Conclusion: Antibody removal by means of immunoapheresis results in a significantly improved xenogeneic cardiac function. Immunoapheresis may, therefore, become an important adjunct in future pig-to-man clinical xenotransplantation.

Key Words: Working heart perfusion • Xenotransplantation • Hyperacute rejection • Immunoadsorption • Ig Therasorb® column

	Introduction

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The permanently increasing shortage of human organs for transplantation raises the question of whether cardiac xenotransplantation could be an alternative to allotransplantation. The most stringent hurdle to the transplantation of organs in a discordant species combinations (e.g. pig to primate) is the hyperacute xenograft rejection (HXR).

Antigen–antibody interaction with the epitopes on the endothelium of the xenograft particularly activates the complement cascade by both, the classical and the alternative pathway [1]. This leads to a loss of the normal anticoagulant character of the vascular cell surface [2]. Similar to clinical organ transplantation of ABO-incompatible donors and recipients, the presumptive etiology of HXR is the binding of preexisting xenoreactive natural antibodies (XNAb) to glycoproteins and glycolipids of the vascular endothelium. These XNAb develop in the early postnatal period in response to carbohydrate antigens expressed by intestinal bacteria. XNAb are mainly of the IgM and to a lesser extent of IgG type [3]. Histological examinations of such rejected organs show microvascular thrombosis, interstitial edema, hemorrhage, inflammation and patchy necrosis [3] with deposition of immunoglobulins, complement and fibrin within the graft microvasculature [4].

Consequently, the most effective procedure to enhance xenograft survival could be a perioperative antibody depletion. The great variety of therapeutic strategies for removal of XNAb and complement depletion [5] includes plasma exchange, plasmapheresis, xenogeneic organ perfusion, unspecific antibody absorbents, the use of haptens like Gal-1-3Gal-fragments and penicillamine [6]. Plasma exchange and organ perfusion result in a loss of coagulatory and plasma proteins and, therefore, are clinically unattractive.

Alternatively, trials with immunoadsorption using affinity columns of immobilized staphylococcal proteins A and G have been highly effective in the treatment of autoimmune diseases, renal transplant patients with anti-HLA antibodies [7] and in a pig-to-dog renal transplant model [8].

In initial animal experiments in 1981, using antibody-based immunoadsorption as a very specific depletion technique sheep antibodies against LDL-cholesterol in pig plasma were used [9]. The first successful clinical trial was performed in 1983 with LDL-Therasorb column [10]. Columns of polyclonal antibodies directed against human immunoglobulins (Ig) were extremely effective for removing human IgG and IgM XNAb from plasma without a significant impact on coagulatory and plasmatic proteins [11]. In the following experiments we tested immunoapheresis (IA) performed by a re-usable antihuman Ig-Therasorb column (Therasorb, Baxter) in our ex vivo pig heart perfusion system.

The working heart model allows to perform external cardiac work by pumping blood against an afterload, providing quantitative data about the developing HXR by direct measurement of cardiac output, mimicking the in vivo situation of pig-to-man xenotransplantation [12].

	Materials and methods

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Animals
The hearts of 12 male Landrace pigs with an average body weight of 22.5 kg (14–31 kg) were explanted. Two series of experiments were performed: in group 1 (G1), the hearts of six pigs were perfused with xenogeneic untreated human blood and served as a control. In group 2 (G2), the hearts from six Landrace pigs were perfused with human blood which had circulated twice through an immunoapheresis column.

Donor animals were anesthetized with azaperon, ketamine hydrochloride and xylacine. Tracheotomy was performed and the animals were ventilated mechanically. Anesthesia was maintained with N₂O/O₂ (2 l/4 l per min), intravenous pancuronium and fentanyl citrate. After a median sternotomy and intravenous injection of heparin (400 IU/kg) the cardioplegic arrest was induced with 200 ml iced (4°C) Celsior-solution (Imtix) and external topical cooling. The heart–lung block was excised, both venae cavae and hili were ligated and both lungs were removed. Cannulae for the xenoperfusion circuit were inserted into the aortic root and left atrium. A cannula in the pulmonary artery enabled direct measurement of the coronary sinus flow. Perfusion was started after 50±9 min of cold ischemic time.

`Working heart' model
The ex vivo perfusion apparatus ( Fig. 1 ) described earlier [13] was modified by adding electrocardiografic (ECG) monitoring and pacer stimulation with epicardial electrodes placed on both ventricles. For perfusion 500 ml of fresh heparinized human blood (100 IU/ml) was taken from healthy volunteers. After reperfusion for 27.5±12.8 min in Langendorff mode the working heart situation was established. From the main reservoir blood was transported by a roller pump (BP 742, Fresenius, Bad Homburg, Germany) through a hollow fibre pediatric oxygenator with integrated heat exchanger (Dideco module 1500, Dideco GmbH, Puchheim, Germany). Blood temperature (37°C) and blood gases were kept within physiological range controlled on-line with a pH-meter (WTW, Wuppertal, Germany). Glucose (4 mg/h), insulin (15 IU/h) and calcium gluconate (50 mg/h) were continuously substituted. According to the experimental protocol for evaluation of parameters influencing the working heart apparatus, two control experiments (perfusion of the system with pig blood only) and an autologous control group (perfusion of the pig heart with pig blood) were performed.

View larger version (21K): [in this window] [in a new window]   Fig. 1. A schematic design of the working heart model. Way 1: Langendorff-mode (thick black line). Blood is transported by a roller pump via a oxygenator/heater to the afterload reservoir (75 cm). After passive coronary perfusion the right coronary sinus effluent could be measured directly via cannula in pulmonary artery. Way 2: Working heart mode (thick pale line). Blood from the reservoir is pumped to the preload column (15 cm). The left ventricle has to pump the blood to the afterload reservoir (75 cm). From there the overflow was registered directly as cardiac output.

Hemodynamic parameters
In the working heart mode the heart ejected into the arterial chamber which was positioned 750 mm above the heart. A direct measurement of cardiac output (CO) was possible by collecting the overflow from the arterial reservoir and coronary sinus effluent via the pulmonary artery. Data of the arterial afterload, blood pressure and preload pressure were monitored with a transducer (Gould P 23 ID, Gould, Cardiovascular Products Division, Oxnard, Canada) and visualized on a monitor (Sirecust 308d, Siemens, Erlangen, Germany).

Stroke work index (SWI), coronary resistance (CR), specific coronary flow (SCF) and from venous and arterial blood gas the arteriovenous oxygen difference (AVDO₂) was determined (Table 1).

As markers for myocardial damage creatine kinase (CK and CK-MB), lactate dehydrogenase (LDH) and ASAT (aspartate aminotransferase), as well as myoglobin and osmolarity were determined by standard methods.

Histology
Frozen tissue sections of 4–6 µm were stained using hematoxylin and eosin and examined under a light microscope. For transmission electron microscopy tissue sections from both the right and left ventricle and atria were taken at the end of perfusion, embedded in tissue tek (Miles, USA), snap-frozen in liquid nitrogen, and stored at -70°C until use. Other tissue samples were fixed in glutaraldehyde 6.25% and stored until further saccharose (0.2 mol/l) processing. Thin sections (0.5 µm) prepared with epon resin were first coloured with toluidine-methylene blue in order to gain an overview by light microscopy. Ultrathin tissue sections of interest (100 nm) were laid on copper grids and stained with uranylacetate and lead. The examination with transmission electron microscopy (Philips 300) was performed at two magnifications (x10 000 and x16 000). For immunohistochemical analysis cryostat-prepared tissue specimens were stained with FITC-conjugated goat antibodies specific for C3, C4, C5b-9. Tissue deposits of IgA, IgG and IgM were stained according to the avidin-biotin method. Monoclonal antibodies were obtained from Dako (Hamburg, Germany) and Immunotech Diagnostics (Marseille, France).

Statistical analysis
The control group G1 (without treatment, n=6) was compared statistically with group G2 (with immunoadsorption, n=6). The results are given as the mean±SEM. The Wilcoxon test for paired samples was used to compare data from both groups at corresponding experimental times. A P-value of less than 0.05 was considered a statistically significant difference.

	Results

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Cardiac performance
The working heart-modus was significantly prolonged in G2 (335.0±37.5 min, P<0.002) after IA as compared with G1 (125.0±31.3 min). In G1, heart weight increase, expressed in %/h, was significantly higher than in G2 (G1: 0.4±0.08 vs. G2: 0.09±0.023%/h, P<0.05). Heart rate in G2 (86.5±5.5 beats/min) was different to G1 (77.3±6.1 beats/min, P<0.05). Increasingly elevated ST-segments, supraventricular/ventricular extra beats and final ventricular fibrillation were typical ECG-changes in group 1 only. Bradycardia and continuously increasing blood potassium levels were specific for group 2.

The cardiac output in G2 was significantly higher as G1 (G2: 252.6±29.5 vs. G1: 115.8±38.1 ml/min, P=0.018, Table 1). This was due to a rapid hemodynamic deterioration in G1 which started after 120–150 min. The stroke work index (SWI) including heart rate and weight was significantly higher in G2 after 150 and 180 min. After 120 min of perfusion the cardiac output, the coronary flow and specific mean coronary flow (SCF; G2: 0.94±0.12 vs. G1: 0.37±0.14 ml/min per g, P<0.01) were significantly higher in G2 than in G1. After IA the coronary resistance in G1 exceeded that of G2 by almost 2.5 times (P<0.01). Arteriovenous oxygen consumption (AVDO₂) tended to be lower in G2.

Serology
The most important parameter for the effectiveness of IA were the levels of plasma immunoglobulins IgG, IgM and IgA ( Fig. 2 ). The major part of XNAb of the IgM class decreased from 1.2±0.7 to 0.2±0.07 g/l after two cycles of column adsorption (83.3%). Without IA the levels remained constant until 120 min. Afterwards a sudden rise of more than 50% was observed.

View larger version (22K): [in this window] [in a new window]   Fig. 2. In G1 Ig levels were significantly higher than in G2. IgM demonstrated after 3 h a mild increase in G1. Decrease of plasma levels of IgG and IgM after 2 cycles of immunoadsorption (Ig-Therasorb column) (16 and 17.7% of the initial value, G2).

In addition reduction of complement factors C3 and C4 of more than 50% in G2 as compared to G1 could be demonstrated ( Fig. 3 ). Both C3 and C4 of the G1 decreased after 60 min by about 50% to a constant level. Serum complement activity was reduced after 2 cycles of IA in G2 from normal ranges of 1075±17.6% (CH100) and 80±2.1% (AP100) to 100±70.7% (CH100) and 15±10.6% (AP100, P<0.05). After 30 min of perfusion no CH100 and AP100 activity was detected in both groups.

View larger version (21K): [in this window] [in a new window]   Fig. 3. Reduction of complement components C3 and C4 after immunoapheresis. In G1 plasma complement levels decreased after 60 to 180 min of xenogeneic perfusion, which is compatible with HXR. In G2 initial removal of C3 and C4 by immunoadsorption was more than 50%.

View larger version (20K): [in this window] [in a new window]   Fig. 4. Anti pig antibody levels measured by a hemagglutination assay. Immunoadsorption (G2) was highly efficient in removing these species-specific antibodies. In G1 (no adsorption) complete anti-pig antibody elimination was achieved by the pig heart within 60 min.

Macroscopically, hearts of G1 showed massive hemorrhagic damage after 2 h compared with hearts of G2, but no distinct and characteristic changes. Histology showed in G2 open vessels in a nearly inconspicuous myocardium (H/E staining, Fig. 5 a). In contrast specimens of G1 showed interstitial edema, microvascular thrombosis as well as endothelial cell detachment and hemorrhage, which is consistent with signs of HXR. Immunohistochemistry demonstrated depositions of C3, C4 and IgM in G1 but not in G2 ( Fig. 5b).

View larger version (155K): [in this window] [in a new window]   Fig. 5. (a) Histology (light microscopy with hematoxylin/eosin staining) demonstrates massive damage of the myocardium with vascular thrombosis in G1. Group 2 reveals nearly inconspicuous tissue structure with open vessels. (b) Immunohistochemistry showed marked deposition of IgM in G1 as compared to G2.

The myocardium of G2 (EM) showed mild interstitial edema. Cardiomyocytes suffered from a slight detachment of the sarcolemmal membrane with mostly intact architecture of myofibrils after a perfusion time of 335 min. In G1 the myocardium showed typical signs of HXR, such as massive cell necrosis of myocytes, degenerative swelling of mitochondria with rupture of the cristae and vacuolization of mitochondria and hypercontractions of contractile elements after 125 min.

	Discussion

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There is growing evidences that XNAb's of IgM type binding to pig Gal-1-3Gal carbohydrate epitopes [14] and activating of endothelial cells represents a central mechanism of HXR in discordant species combinations [15]. In particular the activation of the complement cascade caused by IgM via the classical pathway leads to a critical loss of vascular integrity [16]. Whether this happens as a result of antibody binding or simply via the alternative pathway is still a matter of debate [17]. Recent results from baboon experiments demonstrated, that XNAb are a central event in HXR and are the trigger of acute vascular and also of chronic cellular rejection [18].

Depletion of antibodies in other xenotransplant models by plasmapheresis or organ perfusion delayed HXR [5]. Disadvantages of these techniques were bleeding complications due to the loss of clotting factors and other plasma proteins. The initial experiments [7] during the 1960s and 1970s obtained excellent results. Pig-to-primate kidney xenotransplantation in combination with splenectomy and plasmapheresis allowed a survival of 22 days [19].

In the first generation of immunoadsorption technique, specific for IgG, IgM and IgA, employed tryptophan and phenylalanine as adsorbents and was only semiselective. The columns could not be regenerated. The second generation was a reusable column which adsorbed all immunoglobulins except the subclass IgG3. The third generation is now a reusable and highly specific column. Immobilized sheep antibodies are directed against one or several human immunoglobulins. Todays Ig-Therasorb column contains polyclonal anti-human-Ig as the immunosorbent. This column is re-usable and represents a safe and effective means of antibody removal with minimal effects upon the coagulation system. It's advantage is a limited impact on plasma levels of Factor V, Factor VIII, transferrin and fibrinogen [20] and a small loading capacity (300 ml). This would allow a safe clinical application over several hours.

Presuming, that XNAb are the most important trigger for HXR, we hypothesized, that the subtotal removal of XNAb by using of Ig-Therasorb column would mitigate and delay HXR in cardiac xenotransplantation. In a baboon model after xenotransplantation rising titres of both anti pig IgG and IgM were observed. Another study using a specific anti-human Ig column with polyclonal anti-human IgM antibodies (µ-chain-specific) conjugated to sepharose showed insufficient results [20]. Thus, we assume that only the removal of all IgM, IgG and IgA is qualified for our purpose. In contrast to recent studies, also the investigation of IgA levels as the activator of the complement system via the alternative pathway was necessary [21]. The release of C3a/C5a anaphylatoxins of the complement cascade causes a leukocyte adhesion (C3bi) and aggregation of platelets to the endothelial cell membrane. It is also described, that C5a induces a loss of thrombomodulin, superoxide dismutase and a loss of cell surface heparan sulfate proteoglycan with thrombolytic effect via antithrombin III. The protein complex C5b7 generates gap formation at endothelial cell junctions [22].

The immunological data of consumption of complement components ( Fig. 3) with massive deposition of IgM, C5b-9, C3 and C4 in the tissue and massive typical tissue damage specific for HXR in light and electron microscopy correlate well with deterioration of hemodynamic parameters like cardiac output, stroke work index and coronary flow with increasing coronary resistance after 90–120 min during HXR which is found only in G1. IA reduced complement components C3 and C4 by more than 50%. They are possibly fixed to the antibodies which were adsorbed to the column or by direct consumption in the extracorporeal immunoadsorption system. This is important, because in recent studies it was postulated that complement depletion additional to antibody removal would be beneficial for prolonging xenograft survival [23]. Since IA also decreased complement activity (CH100 to 10% and AP100 to 15%), XNAb and complement depletion must be made responsible for the advantageous effect. According to Leventhal's observation [20] low antibody titres avoid complement activation.

After two cycles of IA anti pig antibodies of IgM, IgG and IgA decreased to very low levels. Even lower levels of anti-pig antibodies were found after hemoperfusion of the hearts with native blood within the first 30 min. This was compatible with endothelial and interstitial Ig deposits seen in immunohistochemistry. After 90 min of heart perfusion anti-pig antibodies had completely disappeared. IgM shows a moderate rebound after 3–4 h of xenogeneic perfusion. The big SEM of the initial values is caused by a single blood donor with very low anti-pig antibody levels. We assume, that the measurement of anti-pig antibody is the most sensitive parameter for HXR in xenotransplantation of primates and patients.

Exposure of endothelial cell monolayers to XNAb results in gaps between the cells. XNAb also attack the underlying matrix and the cardiomyocytes leading to additional complement deposits on the surface of cardiomyocytes (and endothelial cells) [24].

Group 2 with IA, experiences a 2.7-fold prolonged perfusion period in the working heart mode without histological signs of HXR and with less than 25% of weight increase compared with G1 as a sign of minute myocardial damage.

The autologous and void perfusion experiments demonstrated that the working heart mode is limited to 6–7 h. Endothelial cell damage after ischemia and reperfusion injury, hemolysis and inflammation is the response to extracorporeal circulation. The substantial increase of cellular enzymes and electrolytes, especially potassium, were due to enhanced release. It was possibly caused by an accumulation of metabolites due to missing renal and hepatic clearance function.

Generally, the ex vivo working heart model is highly sensitive to even minimal myocardial damage. Especially our modified version, with epicardial ECG monitoring, demonstrated ischemic ST-segment elevation in G1 in the early HXR. As the heart performs external work by pumping blood against an afterload, the model gives plenty of quantitative data indicating the pace of HXR by direct measurement.

Previous ex vivo heart models of retrograde Langendorff perfusion do not equally correspond to the physiological in vivo situation, since the left ventricle is empty and significant myocardial damage may remain undetected.

In conclusion all data confirm, that column IA could delay HXR by antibody and complement removal. To achieve the most efficient elimination of IgG, IgA and IgM and complement in primate experiments and later clinical application, an exact measurement of the immunoglobulins, complement levels and especially anti-pig antibodies by an assay are a prerequisite to control IA with regard to frequency and number of cycles when using Ig-Therasorb columns. In combination with organs from hDAF transgenic pigs and new immunosuppressive strategies the reusable immunoadsorption with an automated Ig Therasorb column regeneration process could eliminate XNAb in HXR and eventually also in antibody-mediated mechanisms of acute vascular and chronic cellular rejection of xenografts. It is certainly an effective and clinically applicable technique for antibody and complement removal also in a future clinical setting.

	Footnotes

 
Presented at the 12th Annual Meeting of the European Association for Cardio-thoracic Surgery, Brussels, Belgium, September 20–23, 1998.

	Appendix A. Conference discussion

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Mr D. Wheatley (Glasgow, UK): How long can you keep a heart going where there is no immune problem? Your average was 5.5 h. That seems pretty good. Is that near to what you can achieve under ideal conditions with no immune issues?

Dr Brenner: Normally, in the working-heart mode run time was 2 h without immunoadsorption, 5.5 h with immunoadsorption, the longest was about 8 h. It's a very long period. We also terminated perfusion in further studies of a colleague after 3 h. But our aim was now to see how long it could be performed at all. Thus we had these very good results. We had also another two groups as control. One was the autologous perfusion of pig hearts perfused with pig blood, and they also had a run time of 6–7 h.

Mr Wheatley: So you've achieved what you would get with an autologous perfusion?

Dr Brenner: If in another control group only blood circulated in the system for 6 h, it was shown that, for example, potassium increased and this led to the end of perfusion.

Dr G. Steinhoff (Hannover, Germany): DAF-transgenic pigs are able to degrade most of the effects of antibodies binding to the pig organs. Could you speculate about the applicability of these adsorption columns? Apparently they may not be necessary to overcome hyperacute rejection, but could they be used in other clinical situations?

Dr Brenner: We are doing experiments in the next months with hDAF-transgenic pigs from Cambridge. And in our Institute of Surgical Research we also performed experiments with liver perfusion, one group with hDAF-transgenic livers and another group only with immunoadsorption. And interestingly, there were similar results.

There was the problem in the Cambridge group, that in baboon xenotransplantation after 4 or 5 days anti-pig antibodies were increasing. So we are now interested in a combination of both. We are performing baboon experiments with orthotopic/heterotopic transplantation of hDAF-transgenic organs in combination with immunoadsorption because immunoadsorption is a reusable technique and it is practicable under control of anti-pig antibodies, which were measured every day.

	References

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