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J Thorac Cardiovasc Surg 1999;118:805-814
© 1999 Mosby, Inc.

CARDIOTHORACIC TRANSPLANTATION

CHARACTERIZATION OF A PIG-TO-GOAT ORTHOTOPIC LUNG XENOTRANSPLANTATIONMODEL TO STUDY BEYOND HYPERACUTE REJECTION

From the ^aDepartment of Thoracic and Vascular Surgery, andHeart-Lung Transplantation and Experimental Surgical Laboratory, HôpitalMarie-Lannelongue, Le Plessis Robinson, Paris-Sud University.^bINSERMU 504 Villejuif; and the ^cLaboratory of Experimental Surgery, FondationTransplantation, Strasbourg, France.

Address for reprints: Paolo Macchiarini, MD, PhD, Department of Thoracicand Vascular Surgery, Heidehaus Hospital (Hannover Medical School), Am Leineufer,70, 30419 Hannover, Germany (E-mail: pmacchiarini{at}compuserve.com).

	Abstract

Top Abstract Introduction Material and methods Results Discussion Appendix: Discussion References

 
Background: A pig-to-goat orthotopiclung xenograft model was developed to test whether depletion of goat xenoreactiveantibodies against pig red blood cells would prolong pig lung xenograft survival.
Methods: Adult goats with anti-pig xenoreactive antibodiesunderwent left pneumonectomy followed by orthotopic transplantation of pigleft lung (group 1) or immunodepletion of their xenoreactive antibodies byextracorporeal right pig lung perfusion before transplantation without (group2) or with (group 3) complete clampage of the right pulmonary artery. In group4, goat left lungs were orthotopically transplanted into pigs and served asnegative controls (pig serum does not have anti-goat xenoreactive antibodies).Each study group included 5 animals. Immunosuppression in surviving recipientsincluded cyclosporine and azathioprine.
Results: Group 1 recipients died 7 ± 3 hours after xenograft reimplantationof severe pulmonary hypertension and dysfunction and vasogenic shock, withlittle evidence of histologic xenograft injury. Group 2 xenografts had a stablecirculatory and respiratory function on reperfusion and survived 9 ±4 days. Group 3 animals also tolerated complete occlusion of the right pulmonaryartery, and xenografts assured the total respiratory support for 4 ±1 days. After immunodepletion, goat serum showed no detectable titers of xenoreactiveantibodies, which began to reappear by postoperative day 2, where xenograftsshowed histologic stigmata of acute (humoral and cellular-mediated) rejectionthat evolved to a complete xenograft necrose at death. Group 4 xenograftsshowed scattered features of acute rejection 5 ± 1 days after the operation.
Conclusions: Pig left lung xenografts can provide prolongedand complete respiratory support after depletion of goat xenoreactive antibodies,but they ultimately necrose once recipient xenoreactive antibodies returnto pretransplantation values.

	Introduction

Top Abstract Introduction Material and methods Results Discussion Appendix: Discussion References

 
Lung transplantation across widely disparate species (eg, pig-to-human)is restrained by the hyperacute rejection. This reaction, primarily initiatedby preformed anti--galactosyl (-Gal) xenoantibodies that arepresent in human serum and directed against -Gal epitopes expressedon the vascular porcine endothelium, ¹ activates complement and causes irreversible xenograft damage withinminutes to hours on revascularization. ^2-4 Although removal or inhibitionof these anti-Gal xenoantibodies may prolong xenograft survival, ^5,6 this down-regulation is only transitory, and the events occurringbeyond the hyperacute rejection are unknown.

Goats and pigs belong to the order of ungulates and, like all the otherlower mammals, do not have anti–-Gal natural antibodies in theirserum ^7,8; therefore a moderate type of hyperacute rejection could be expected.However, recent evidence exists that other unrecognized non-Gal–relatedhumoral factors may trigger hyperacute rejection. ⁶ Moreover, goats are far more sensitive to xenogeneicwhole blood than other species ⁹ and represent a well-established large animal model in cardiothoracicsurgery. ^10-13 Therefore we first investigated the existenceof natural non-Gal xenoantibodies in goats and pigs and, on the basisof these results, tested whether their depletion would prolong an orthotopicxenograft lung in a discordant model.

	Material and methods

Top Abstract Introduction Material and methods Results Discussion Appendix: Discussion References

 
Animals and study design.
Large White (La Roche Cormier, Vendome, France) outbred pigs weighing20 to 30 kg served as lung donors. Similar weight adult goats (Saalen, INRA,Jouy en Josas, France) served as recipients. All animals received care incompliance with the "Principles of Laboratory Animal Care" formulatedby the National Society for Medical Research and the "Guide for theCare and Use of Laboratory Animals" prepared by the Institute of LaboratoryAnimal Resources, National Research Council, and published by the NationalAcademy Press, revised 1996.

The first step of this study was to demonstrate whether the goat/pigcombination would be a model of discordant xenotransplantation. This was madeby performing blood measurements of natural antibody titers and ex vivo studiesof the isolated lungs in both directions. Because these studies provided evidencethat adult goats have anti-pig xenoantibodies and not vice versa, we nextrandomly studied 4 types of orthotopic lung xenotransplantation. Adult goatsunderwent left pneumonectomy followed by orthotopic transplantation of pigleft lung without (group 1) or with (groups 2 and 3) immunodepletion of theirxenoantibodies by extracorporeal right pig lung perfusion before transplantation;the allocation of the animals was randomly performed. Group 3 goats also hadcomplete clampage of the right pulmonary artery (RPA). In group 4, goat leftlungs were orthotopically transplanted into pigs and served as negative controls.

Ex vivo lung xenoperfusion model.
Pigs were premedicated with intramuscular ketamine hydrochloride (25mg/kg) and atropine sulfate (1 mg/kg) and anaesthetized with intravenous sodiumpentobarbital (25 mg/kg). Pigs or goat left lungs were then harvested andex vivo perfused and ventilated according our previously developed model. ³ The arteriovenous oxygen difference(AVO₂; milliliters of oxygen per 100 mL blood) was calculated accordingto the formula
AVO₂ = ([1.34 x Hb] x [S_art]) – ([1.34 x Hb] x [S_ven])
where Sis the arterial (S_art) or venous (S_ven) oxygen saturationand Hb is hemoglobin concentration (grams per deciliter). Blood flow (flowprobe, Statham SP2202; Biomedical Division, Oxnard, Calif) and pulmonary arterypressure (millimeters of mercury per milliliter per minute; Kipp and ZonenBD112, Amsterdam, The Netherlands) measurements allowed calculation of thepulmonary vascular resistance (PVR) as pulmonary artery pressure (millimetersof mercury) and blood flow (milliliters per minute).

Orthotopic left lung transplantation.
Pig left lungs acting as xenografts to be implanted orthotopicallywere harvested as for the ex vivo model. The right lung and middle lobe andthe heart of the same pig donor absorbed goat xenoantibodies after the followingareas were stapled: (1) origin of the pulmonary trunk and left pulmonary artery,(2) right and left pulmonary veins beyond their left atrium takeoff, (3) venaecavae, and (4) left main bronchus. The ascending aorta was sutured with acontinuous 6-0 Prolene suture (Ethicon, Inc, Somerville, NJ) just before theorigin of the 2 coronary arteries to avoid myocardial perfusion. These xenograftswere then placed in 2 sterile plastic bags containing cold (4°C) Euro-Collinssolution.

Goats were premedicated with acepromazine (0.05-0.1 mg/kg) and atropinesulfate (0.2 mg/kg) and anesthetized with intravenous sodium pentobarbital(1 mg/kg). Adequacy of ventilation and oxygenation was assessed by arterialblood gas analysis and pulse oximeter (Finger Pulse Sensor; Epic Medical,Plano, Tex). They were subsequently selectively intubated through an endotrachealdouble-lumen tube and ventilated at 100% oxygen by a mechanical ventilator(Siemens, Elema, Sweden; tidal volume of 15 mL/kg at 17-20 breaths/min with5 cm H₂O positive end-expiratory pressure). Light anesthesia wasmaintained with 1% to 2% fluothane (Halotane; Zeneca Pharma, Cergy, France).A left posterolateral thoracotomy in the 4th intercostal space was the usualapproach to enter the left pleural cavity, and 1 mg pancuronium bromide wasinjected intra-arterially as needed for complete muscle relaxation.

The left internal thoracic artery was dissected, and a polyvinyl catheterwas inserted to record blood pressure and heart rate with a pressure transducer(Kipp and Zonen) and to serve as an arterial sampling line. After the divisionand ligation of the pulmonary inferior ligament and a left lower vein thatdrained the right middle lobe, the pericardium was opened; the aortopulmonaryligament was ligated, and the pulmonary trunk and RPA were dissected and encircled.We next placed polyvinyl catheters and flow probes into the left atrium, rightappendage, and in and around the thoracic aorta, pulmonary trunk, and originof the RPA to measure systemic and pulmonary pressures and cardiac output(Statham SP2202; Biomedical Division).

In goats of group 1, pig left-lung xenografts were immediately orthotopicallyimplanted with the use of the standard implantation technique. In goats ofgroups 2 and 3, goat cardiac output was first passed for at least 15 minutesinto pig right lung-middle lobe and heart block to immunoabsorb the goat xenoantibodies(Fig 1). At completion, pig leftlungs were implanted. Goats in group 3 also underwent ligation of the RPA.The pleural spaces were then closed with 2 chest tubes, and the chest wallwas closed with interrupted polyglactin 1 sutures.

View larger version (44K): [in this window] [in a new window]   Fig. 1. An aortic root cannula (DLP, Inc, Grand Rapids, Mich) was inserted into the descending aortaand connected to a tubing system and a cannula (William Harvey, CRBard, Inc, Tewsburg, Mass) to allow the anterograde flow of goatcardiac output through pig right lung, middle lobe, and heart. A venting multihooledcatheter drained the immunoabsorbed blood into the distal left azygos vein.During this immunoabsorption time, the right lung and middle lobe were ventilatedthrough a mechanical ventilator (MV) to avoidgas embolism. To measure transpulmonary pressure and/or stabilize the recipientduring this procedure, a pressure transducer was connected to the tubing system,and intermittent tubing clamps and peripheral vasoconstrictive and inotropicagents were used.

Medical support and immunosuppression.
Before xenograft reperfusion, recipients received 1 g of indomethacinto block thromboxane production. Animals received intravenous antibiotics(cephalothin, 500 mg/daily), oral acetylsalicylic acid (100 mg/daily) andprednisolone (40 mg, days 1 and 3 after the operation), and intramuscularcyclosporine (INN: ciclosporin) (5-10 mg/kg/day) to maintain plasma levelsof 250 to 350 ng/mL and oral azathioprine (2.5 mg/kg daily). Animals wereplaced in cages and fed standard laboratory food and water ad libitum.

Fiberoptic tracheal and chest radiographic examinations were performeddaily. Open lung biopsies were performed 2 and 4 days after the operation.Animals were killed at the clinical or histologic occurrence of xenograftnecrosis or at the first evidence of discomfort.

Dilution effect.
Plasma dilution was assessed by the measurement of total plasma proteinlevels, as described previously. ¹⁴ No dilution of plasma was evidenced after pig lung perfusion, whereasa dilution of 1.6- ± 0.15-fold was observed in plasma after pig lungtransplantation (as compared with plasma before transplantation).

Measurement of serum xenoantibodies and their elution from lung tissue.
Serum samples were obtained from donors and recipients before and dailyafter xenotransplantation to measure xenoantibody titers by hemagglutination.Pig red cells were washed 3 times with 20 volumes of phosphate-buffered salinesolution (PBS). Twofold serially diluted 50 µL aliquots of goat serum andlung eluates were tested against equal volumes of 2% pig red cells in PBS,and results were expressed as the reciprocal of the highest dilution at whichagglutination was observed. After perfusion with goat whole blood, the rightimmunoabsorbent lungs were washed with PBS, then the fixed antibodies wereeluted with NH₄OH 1% (pH 11). Aliquots of 50 mL of eluate werecollected and dialyzed against PBS and stored frozen at –20°C untilused.

Preparation of endothelial cell extracts.
Porcine endothelial cells were explanted from the aorta of large Whitepigs and cultured until confluence in Roswell Park Memorial Institute mediumthat contained 20% fetal calf serum and 1 mmol/L sodium pyruvate, as describedpreviously. ¹⁴ Endothelialcell monolayers were washed, scraped from the flasks, and pelleted by centrifugationat 900g for 5 minutes. The cells were resuspendedand lysed in 1 volume of 50 mmol/L Tris-hydroxymethyl-amino methane (Tris;pH 7.2) containing 2% t-octylphenoxypolyoxyethanol (Triton X-100), 5 mmol/Lethylenediaminetetra-acetic acid and Pefablock, 1 mmol/L benzamidine, 15 µmol/Lpepstatin and 10 µmol/L leupeptin for 30 minutes on ice. After centrifugationat 10,000g for 30 minutes, the supernatantwas collected and stored at –70°C until used.

Sodium dodecylsulfate-polyacrylamide gel electrophoresis and Westernblot analysis.
To characterize the specificity of anti-pig antibodies that were removedafter pig lung transplantation or pig lung perfusion, the reactivity of nativeand xenoantibody-depleted goat serum samples was analyzed on porcine endothelialcell extracts.

Samples of pig endothelial-cell extracts were electrophoresed on a 7.5%gel under reducing conditions. Proteins resolved in this way were transferredto 0.45 µm polyvinylidene difluoride membranes. ¹⁴ After saturation with 3% low-fat dry milk in Tris-bufferedsaline solution (TBS) overnight at 25°C, the membranes were washed 3 timeswith 0.3% Tween-20 in TBS and stored in this buffer until used. Goat sera(diluted 1:20 in TBS) or lung eluates (diluted 1:20, 1:10, and 1:5 in TBS)were incubated overnight at 25°C. After being washed with 0.3% Tween-20in TBS, the antibody binding was detected by incubating blots with alkalinephosphatase–conjugated affinity purified rabbit anti-goat IgG (H+L;The Jackson Laboratories, Bar Harbor, Maine) diluted 1:1000 in 0.5% bovineserum albumin in TBS for 2 hours at 25°C. The expression of -Galresidues was analyzed with biotinylated isolectin B4 from Griffonia simplicifolia (GSI-B4) as described previously. ¹⁵ The immunoblots were revealedwith nitroblue tetrazolium (Biorad, Hercules, Calif).

Histologic information.
Xenografted and recipient native lungs were macroscopically assessed.At least 4 samples of representative areas were taken, and fragments werefixed in 10% formaldehyde and routinely processed into paraffin wax. Sectionswere cut at 5 µm and stained with hematoxylin and eosin.

Statistical analysis.
In the ex vivo model, xenograft failure was defined as such when theex vivo lung perfusion was associated with no gas exchange, severe pulmonaryhypertension, and gross evidence of pulmonary hemorrhage and edema. In theorthotopic model, experiments were stopped at the onset of recipient discomfortor xenografts dysfunction. Data are expressed as mean ± SD of the numberof observations and analyzed by 1-way analysis of variance with Fisher’sprotected least significance difference (StatView 4.02; Abacus Concepts, Inc,Berkeley, Calif). The a priori level of significance was set at a P value of less than .05.

	Results

Top Abstract Introduction Material and methods Results Discussion Appendix: Discussion References

 
Ex vivo lung studies.
Goat sera contains xenoantibodies to pig red cells, but pig serum demonstratedno detectable titers of xenoantibodies against goat red blood cells. Unsurprisingly,goat lungs perfused with pig blood displayed almost normal xenograft functionover the entire 5-hour study period, although a marked pulmonary hypertensionand fall in AVO₂ occurred when pig lungs were perfused with goatblood (Fig 2), but both ex vivo models were working after the 5-hour studyperiod. Goat anti-pig xenoantibodiesfall from 6.4 ± 1.6 before the ex vivo perfusion to 0.8 ± 1.0on completion of the study period. Goat lungs perfused with pig blood werenormal, although pig lungs perfused with goat blood were congested and edematouswithout pulmonary microvessel thrombosis (Fig 3).

View larger version (19K): [in this window] [in a new window]   Fig. 2. Hemodynamics (A) and oxygen extraction (B) atbaseline (time 0) and at specific time intervals after ex vivo lung xenograftsperfusion with either goat or pig blood. Data are expressed as mean ±SD (error bars) of the experiments. The differencewas statistically significant (P < .001).During the time interval between –30 and 0 minutes, the xenografts wereperfused with autogenic blood to stabilize the perfusion system; before reperfusion,they were washed with 1.5 L saline solution to avoid hemolyzation of the residualautogenic blood.

View larger version (113K): [in this window] [in a new window]   Fig. 3. Histologic features of pig lungsperfused with goat blood, displaying alveolar edema but no microvessel thrombosis.Biopsy specimens were taken at the experiment’s completion. (Originalmagnification, x180.)

View larger version (108K): [in this window] [in a new window]   Fig. 4. Histologic features of pig leftlungs orthotopically implanted into the goats show a normal structure, exceptthe presence of rare peribronchial inflammatory cells. Biopsy specimens weretaken at the experiment’s completion. (Original magnification, x50.)

View larger version (31K): [in this window] [in a new window]   Fig. 5. Natural anti-pig xenoantibodiesdetected by standard hemagglutination in goat sera. The rise of xenoantibodieswas earlier for group 3 than for group 2 goats. The xenoantibodies were expressedas the reciprocal of the highest dilution at which agglutination was observed.

View larger version (104K): [in this window] [in a new window]   Fig. 6. A, Biopsy specimen taken at day2 shows signs of mild vascular rejection, as demonstrated by the presenceof lymphoid infiltration within the wall of a pulmonary venule (group 2; originalmagnification, x50) or (B) severe rejectionas demonstrated by an aspect of follicular lymphocytic bronchiolitis (group3; original magnification, x12.5).

View larger version (140K): [in this window] [in a new window]   Fig. 7. The same pig xenotransplantedlung (as illustrated on Fig 6) shows a complete ischemicnecrosis without inflammatory cells (postoperative day 6). (Original magnification,x25.)

View larger version (74K): [in this window] [in a new window]   Fig. 8. Western blot analysis of thereactivity of goat anti-pig natural antibodies. Porcine aortic endothelialcells membrane extracts were separated by sodium dodecyl sulfate-polyacrylamidegel electrophoresis, blotted onto polyvinylidene difluoride, and stained asfollows: lane 1, goat serum, before in vivo pig lung transplantation; lane2, goat serum, after in vivo pig lung transplantation; lane 3, goat serum,before ex vivo pig lung perfusion; lane 4, goat serum, after ex vivo pig lungperfusion; lanes 5 and 6, eluates from 2 independent lung perfusion experiments;lane 7, G simplicifolia isolectin B4. Antibodybinding was revealed with alkaline phosphatase-conjugated rabbit anti-goatIgG (H+L) antibodies.

	Discussion

Top Abstract Introduction Material and methods Results Discussion Appendix: Discussion References

 
The goat and pig combination represent, despite their phylogenetic vicinity,a promising -Gal antibody-free model, which permits the study of discordantorthotopic lung xenotransplantation beyond the occurrence of hyperacute rejection.Although its clinical relevance may remain unclear, we have confirmed in vivoour previous ex vivo hypotheses that a variety of non-Gal antibodiesmay trigger hyperacute rejection as well. ⁶ Moreover, by depleting these non-Gal antibodies with a cross-perfusionof pig immunoadsorbent organ, hyperacute rejection may be prevented and completexenograft function and survival can be prolonged temporarily. We also providedevidence that xenograft failure depends on the reappearance of anti-pig xenoantibodiesin recipient goats and confirmed that conventional immunosuppression doesnot alter this outcome at all. ¹

In the ex vivo perfusion model, pig lung xenografts were working aftera 5-hour perfusion with goat blood, despite severe pulmonary hypertensionand oxygen dysfunction. By contrast, the same situation in the untreated orthotopicmodel was characterized by the occurrence of refractory severe pulmonary hypertensionand dysfunction and vasogenic shock, leading to death in 7 ± 3 hoursafter xenograft reperfusion. One may argue that this difference may be relatedto the intrapulmonary or systemic synthesis of thromboxane in goats. ⁹ However, the thromboxane activitywas always neutralized by intravenous administration of indomethacin beforexenograft reperfusion. Yet, the only design difference between goats experiencing(group 1) or not experiencing (groups 2 and 3) circulatory and pulmonary failurewas the cross-perfusion of pig organ. Curiously, this clinical scenario wasassociated with little histologic xenograft injury except alveolar edema,and we may assume that this and the absence of pulmonary microvessel thrombosismay be related to the limited goat blood flow through the pig xenograft becauseof the severe pulmonary hypertension.

By cross-perfusing goat blood through pig immunoabsorbent organs, whichretained the anti-pig antibodies, we were able to prevent hyperacute rejection;and our immunologic evaluation clearly demonstrated that this phenomenon wasrelated to the disappearance of anti-pig xenoantibodies from the circulationof the recipients. Using Western blot analysis of the eluted antibodies, wedemonstrated that goat antibodies recognize numerous protein bands on porcineendothelial cells. This result is in line with the reactivity observed inother discordant species ^15-17 and with our previous ex vivo observation thatmultiple human anti-pig xenoantibodies were absorbed on perfusion of pig lungswith human blood. ⁶ Unsurprisingly,the pattern of reactivity did not parallel the presence of -Gal–bearingproteins, as assessed with the G simplicifoliaisolectin B4. Whether goat anti-pig natural antibodies belong to an homogeneousrepertoire with limited specificity against a common epitope shared by targetproteins (as -Gal epitope for the pig-to-primate combination) is notknown. The complement or complement cascade may have also played an unspecifictriggering role, as in other discordant models, ¹⁸ even in the presence of very low xenoantibody titers.However, because there are no specific reagents to test goat complement, wecould not test this hypothesis.

Yet, even when hyperacute rejection was prevented and xenografts assuredcomplete respiratory support, they ultimately were rejected and became necrosed.The first step of this rejection process was the appearance of an acute vascularrejection similar to that observed in lung allotransplantation where perivascularor peribronchial lymphoid infiltration are found. Of note, the acute vascularrejection included stigmates of both humoral-mediated and cellular-mediatedxenograft rejection. The humoral-mediated rejection included endothelial hyperplasiaand interstitial congestions, and this corresponded well to the working hypothesisthat acute vascular rejection arises under conditions in which hyperacuterejection is avoided and is governed by the physiologic state of the vascularendothelial cell. ¹⁹ Thecellular-mediated rejection included perivascular and/or peribronchial lymphoidcell infiltration, and this type of rejection was only observed 2 days aftertransplantation. In other terms, the only event-free xenograft survival timewas when xenoantibodies were absent because the appearance of the initialxenorejection injuries occurred when goats again displayed anti-pig xenoantibodiesin their sera. It might be possible that these xenoantibodies may have stimulatednatural killer cells or a T-cell–mediated process to induce furtherendothelial cell dysfunction and procoagulant activity.

All these rejection injuries appeared earlier, and the cellular-mediatedrejections were more vigorous in goats belonging to group 3 than to group2, and this fits well with the fact that the entire cardiac output was flowingthrough the pig xenograft. Indirectly, these observations involve that T-cell–mediatedxenograft rejection is often more severe than T-cell–mediated allograftrejection ²⁰ and that, invivo, pig xenografts are almost certain to provoke vigorous direct or indirectT-cell xenoresponses within the first week after transplantation. Ultimately,xenografts become completely destroyed by an ischemic necrosis, and this mainlyoccurs when the titers of anti-pig xenoantibody return to pretransplantationvalues. Of note, the ischemic necrosis typically spared recipients’bronchial, arterial, and left atrium stumps, mirroring to some extent theobservations reported in the precyclosporin lung allotransplantation era anddemonstrating that conventional immunosuppressive agents may be less effectiveat prolonging xenograft survival than at prolonging allograft survival.

We observed that goat serum contains natural antibodies that bind topig antigens whereas the opposite does not hold, as was already observed inother species combinations. ¹⁴ Moreover, the presence of natural antibodies was correlated with theseverity and tempo of graft rejection, because hyperacute rejection was seenin pig-to-goat xenografts (group 1) but not in goat-to-pig xenografts (group4). These results are in accordance with previous work that reported the absenceof hyperacute rejection in pig-to-newborn goat cardiac xenografts becauseof very low levels of natural antibodies in newborn. ¹³ They also allow to define of the pig-to-goat combinationas a discordant xenograft model, whereas the goat-to-pig model would be concordant,according to a classification based on the immunologic response. ²¹

In summary, we have developed an orthotopic -Gal antibody-freelung xenotransplantation model that reports a refractory vasogenic shock andpulmonary hypertension and dysfunction after 7 ± 3 hours on pig xenograftreperfusion. This form of hyperacute rejection is consistently prevented bythe immunodepletion of goat anti-pig xenoantibodies before xenograft implantation.Unfortunately, this prevention is only temporary because both humoral- andcellular-mediated rejection occur and evolve to xenograft necrosis once goatsresynthesize anti-pig xenoantibodies. This pig-to-goat combination could bea valuable working discordant model for the evaluation of strategies aimedat preventing antibody-mediated rejection mechanisms.

	Appendix: Discussion

Top Abstract Introduction Material and methods Results Discussion Appendix: Discussion References

 
Dr Mark K. Ferguson (Chicago, Ill). You have shown that you get very complete immunodepletion with the absorption model that you have used. Is there some commercial or mechanical way that you can achieve that same result?

Dr Macchiarini. This way could be achieved in the swine-to-human combination with -Gal antibody columns. However, in this combination we do not need anti-Gal columns. Commercially, yes. You need to develop a system like a membrane oxygenator incorporating carbohydrates to absorb target antibodies.

Dr David M. Kulick (Minneapolis, Minn). In the vascular rejection of your xenografts did you look at immunohistochemistry or fluorescence-activated cell sorter analysis to identify the types of lymphocytes or mononuclear cells that were infiltrating around the vascular structures in the lungs that were transplanted?

Dr Macchiarini. No. We did only simple histologic examination that showed leukocyte infiltration.

Dr Kulik. In the pig-to-primate combination for heart transplantations, we have seen a marked increase in CD68-positive cells, and I was curious if you had seen that in the lungs as well.

Dr Macchiarini. No.

	Acknowledgments

 
We thank Chantal Verriest, Rémi Burel, Hegésippe Langouste,Bruno Baudet, and Aline Perrin for technical assistance and Denis Petraz forphotographic contributions.

	Footnotes

 
Read at the Seventy-ninth Annual Meeting of The American Association for Thoracic Surgery, New Orleans, La, April 18-21, 1999.

Supported by the Xenotransplantation project within the European Communityprogram DGXII Biotechnology (shared cost BI04-CT97-2242 and concerted action3026PL950004) and Fondation Transplantation.

	References

Top Abstract Introduction Material and methods Results Discussion Appendix: Discussion References

 

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