Xenoreactive Natural Antibodies Research Articles

Altered Blood Levels of Anti-Gal Antibodies in Alzheimer's Disease: A New Clue to Pathogenesis?

Alzheimer’s disease is a neurodegenerative disorder whose pathological mechanisms, despite recent advances, are not fully understood. However, the deposition of beta amyloid -peptide and neuroinflammation, which is probably aggravated by dysbiotic microbiota, seem to play a key role. Anti-Gal are the most abundant xenoreactive natural antibodies. They are supposed to stem from immunization against the gut microbiota and have been implicated in the pathogenesis of several diseases, including multiple sclerosis. These antibodies target the alpha-Gal epitope, expressed on the terminal sugar units of glycoprotein or glycolipid of all mammals except apes, Old World monkeys and humans. The alpha-Gal is constitutively expressed in several bacteria constituting the brain microbiota, and alpha-Gal-like epitopes have been detected in gray matter, amyloid plaque, neurofibrillary tangles and corpora amylacea of the human brain, suggesting a potential link between anti-Gal and Alzheimer’s disease etiopathogenesis. For the first time, our study searched for possible alterations of anti-Gal immunoglobulin levels in Alzheimer’s disease patients. IgG and IgM blood levels were significantly lower, and IgA significantly higher in patients than in healthy subjects. These results suggest that such immunoglobulins might be implicated in Alzheimer’s disease pathogenesis and open new scenarios in the research for new biomarkers and therapeutic strategies.

Investigation of the influence of xenoreactive antibodies on activation of complement and coagulation in an ex vivo perfusion animal study using porcine kidneys.

During pig-to-primate xenotransplantation or perfusion of porcine organs with human blood, a xenogeneic coagulopathy with consecutive development of thrombotic microangiopathy (TMA) can be observed. The aim of this study was to elucidate the influence of the reduction of xenoreactive natural antibodies on the coagulopathy using an exvivo perfusion system. Thirteen perfusion experiments using landrace wild-type porcine kidneys were performed in three different experimental groups: autologous, xenogeneic, and immunoadsorption. During and after perfusion, blood and tissue samples were collected to assess markers of coagulation, complement, inflammation, and endothelial activation. Immunoadsorption prior to perfusion did not prolong perfusion time (174min ±28) compared to xenogeneic (182min ±22) experiments, whereas autologous perfusion was possible for maximum of 240min in all experiments. Activation of coagulation was similar comparing perfusions after immunoadsorption (D-Dimer 24186μg/l ±5813; TAT 566μg/l ±34) to xenogeneic (D-Dimer 22175μg/l ±7826, TAT 600μg/l ±0) experiments. But antibody-mediated complement activation was reduced in the immunoadsorption group. TNF-alpha and markers of endothelial cell activation were lower in the immunoadsorption group compared to the xenogeneic experiments. In this exvivo perfusion model, we observed that marked removal of xenogeneic antibodies can reduce complement activation via the classical pathway as well as endothelial cell activation and inflammation. Immunoadsorption cannot prevent the activation of the terminal complement cascade and coagulation.

The production of multi-transgenic pigs: update and perspectives for xenotransplantation.

The domestic pig shares many genetic, anatomical and physiological similarities to humans and is thus considered to be a suitable organ donor for xenotransplantation. However, prior to clinical application of porcine xenografts, three major hurdles have to be overcome: (1) various immunological rejection responses, (2) physiological incompatibilities between the porcine organ and the human recipient and (3) the risk of transmitting zoonotic pathogens from pig to humans. With the introduction of genetically engineered pigs expressing high levels of human complement regulatory proteins or lacking expression of α-Gal epitopes, the HAR can be consistently overcome. However, none of the transgenic porcine organs available to date was fully protected against the binding of anti-non-Gal xenoreactive natural antibodies. The present view is that long-term survival of xenografts after transplantation into primates requires additional modifications of the porcine genome and a specifically tailored immunosuppression regimen compliant with current clinical standards. This requires the production and characterization of multi-transgenic pigs to control HAR, AVR and DXR. The recent emergence of new sophisticated molecular tools such as Zinc-Finger nucleases, Transcription-activator like endonucleases, and the CRISPR/Cas9 system has significantly increased efficiency and precision of the production of genetically modified pigs for xenotransplantation. Several candidate genes, incl. hTM, hHO-1, hA20, CTLA4Ig, have been explored in their ability to improve long-term survival of porcine xenografts after transplantation into non-human primates. This review provides an update on the current status in the production of multi-transgenic pigs for xenotransplantation which could bring porcine xenografts closer to clinical application.

Inhibition of complement component C5 prevents clotting in an ex vivo model of xenogeneic activation of coagulation.

Xenogeneic thrombotic microangiopathy (TMA) and acute vascular rejection (AVR) prevent long-term survival of porcine xenografts after transplantation into non-human primates. Preformed xenoreactive natural antibodies (XNA) cause endothelial damage and activate the complement system. Mechanisms of xenogeneic coagulation and platelet activation are only poorly characterized. A microfluidic flow chamber was used to study platelet activation and thrombus formation of human platelet-rich plasma (PRP) upon perfusion over wild-type (WT) or α-1,3- galactosyltransferase knockout (GTKO) and human CD46 (hCD46) transgenic porcine aortic endothelial cells (PAEC). Activation of plasma coagulation (thrombin-anti-thrombin complex; TAT) and complement (C3a, C5a) was studied in human platelet-free plasma (PFP) after co-incubation with PAEC. The activation of PAEC (E-Selectin, tissue factor, ICAM-1, ICAM-2, VCAM-1) was studied after incubation with human serum. Eculizumab (200 μg/ml) was used to inhibit terminal complement activation in all experiments. WT-PAEC perfused with human PRP showed thrombus formation at different shear rates (3 dyn/cm(2) : 23 ± 10%; 10 dyn/cm(2) : 17 ± 10% of flow chamber viewing field). GTKO/hCD46 PAEC exhibited reduced, but not fully prevented thrombus formation (3 dyn/cm(2) : 12 ± 12%). Porcine PRP caused little or no thrombus formation (3.0 ± 4% and 0.5 ± 0.9%, respectively). Flow cytometry of human platelets after perfusion over WT-PAEC revealed an increase in platelet CD62P expression (29.5 ± 3%), compared to non-perfused PRP (7 ± 2%) or PRP running through empty flow chambers (12.7 ± 0.3%). Incubation of human PFP with PAEC resulted in an increase of TAT that correlated with C5a activation. Specific inhibition of complement by eculizumab prevented thrombus formation (WT-PAEC: 1.6 ± 2% at 3 dyn/cm(2) and 0.24 ± 0.33% at 10 dyn/cm(2) , GTKO/hCD46 PAEC: 0.2 ± 0.3% at 3 dyn/cm(2) ) as well as activation of coagulation and platelets. Induction of endothelial E-Selectin and VCAM-1 in WT-PAEC upon incubation with human serum was significantly reduced by eculizumab. Eculizumab did not reduce thrombin generation capacity of human PRP or normal platelet aggregation. Thrombus formation in this ex vivo model of xenogeneic TMA was closely linked with complement activation. Specific inhibition of complement C5 by eculizumab prevented endothelial cell activation, but also coagulation and platelet activation without compromising thrombin generation capacity of human blood or normal platelet function.

Anti-Gal IgM, IgA and IgG natural antibodies in childhood

The target for the most abundant xenoreactive natural antibodies in humans is the α-Gal epitope. Anti-Gal could provide natural immune defense against pathogens that express the α-Gal epitope. Anti-Gal natural antibodies are usually studied in adult individuals. Data demonstrating the incidence and concentration of anti-Gal natural antibodies in childhood are in short supply and incomplete. In the present study we prospectively quantified anti-Gal IgM, IgA and IgG levels in different age groups of children from delivery to 24 months of age and compared these levels to the level of these antibodies in their respective mothers. Measurement of anti-Gal antibodies may broaden the spectrum of specific antibodies that are available for determination of specific antibody responses in physiological and pathological conditions in children.Plasma was collected from umbilical cord blood of full term newborn, from blood of infants at age 6, 12 and 24 months and from their respective mothers at time of delivery. Quantitative determination of anti-Gal antibodies IgM, IgA and IgG were made with the enzyme immunoassays Human Anti-Alpha Galactosyl IgM ELISA, IgG ELISA and IgA ELISA. Hemagglutination activity was titrated against rabbit erythrocytes.The kinetic processes for the formation of natural antibodies in the first two years of life, in general, compared with the kinetics for the formation of total immunoglobulins IgM, IgA and IgG. There were no detectable anti-Gal IgM and IgA in the cord blood, whereas anti-Gal IgG were found at similar levels in both neonate cord blood and peripheral blood of their respective mothers. When comparing the percentage of natural antibodies in the plasma of children, the level of natural antibodies in children at the age of two years was approximately 37% for IgM, 25% for IgG and 15% for IgA. The titration of antibodies required for agglutination of rabbit red blood cells over the 24 month period followed the same trend observed for the formation of natural antibodies.Our study demonstrates the kinetics of formation of anti-Gal IgM, IgA and IgG natural antibodies in the first two years of life. The relative lack of these antibodies in this period should be taken into account when assessing for humoral immunodeficiencies, particularly with regards to the potential for children to mount an anti-carbohydrate response.

Gene transfer of the adaptor Lnk (SH2B3) prevents porcine endothelial cell activation and apoptosis: implication for xenograft’s cytoprotection

Targeting protective gene expression to porcine endothelium by genetic modification of the donor could improve xenograft survival by controlling cell activation and death. We previously found that, in endothelial cells (EC), the molecular adaptor Lnk (SH2B3) is a negative regulator of cytokine signaling. We also have shown that Lnk is upregulated in pig EC (PAEC) in response to tumor necrosis factor-α (TNF) and xenoreactive natural antibodies (XNA) binding. The present study investigated whether ectopic expression of human Lnk using gene transfer may be efficient to control signaling pathways associated with inflammation and apoptosis in porcine aortic endothelial cells (PAEC). Endothelial cells cultures were established from WT and Gal(-/-) pigs and transduced with a recombinant adenovirus encoding human Lnk. Phenotype and functions of transduced PAEC expressing Lnk were analyzed by flow cytometry, western blot and XNA and complement-dependent assays. The regulatory functions of Lnk toward inflammation were assessed in TNF-activated EC, and the protective functions were tested toward TNF-induced apoptosis and anoïkis. Apoptosis assays included DNA content analysis and caspase-3/7 activity. First, we found that as a result of adenoviral transduction, human Lnk was efficiently and similarly expressed in EC from WT or Gal(-/-) pigs. Lnk expression or EC transduction caused no significant change in the binding of XNA (IgG and IgM) to PAEC and has no effect on complement activation and C5b-9 formation. We demonstrated that expression of human Lnk efficiently inhibits TNF signaling in PAEC and decreases VCAM-1 induction by 46.3 ± 1.2% compared to controls (n = 6, **P < 0.01). Furthermore, expression of Lnk was associated with a significant decrease in the percentage of caspase-3/7-dependent apoptosis caused by TNF in the presence of actinomycin D and also reduces cell death by anoïkis by 25.0 ± 1.9% compared to controls (n = 5, **P < 0.01). Together, these findings indicate that the signaling adaptor Lnk is effective to reduce PAEC activation and apoptosis. Thus, Lnk is a potential candidate for the modulation of signaling pathways to protect vascular EC from inflammation in xenotransplantation.

Histopathology of xenografts in pig to non‐human primate discordant xenotransplantation

Xenotransplantation could provide a solution to the critical shortage of organs for transplantation in humans. Swine have been proposed as a suitable donor species. Swine organs, however, when transplanted to primates, are rapidly rejected by hyperacute rejection (HAR) and acute humoral xenograft rejection (AHXR). Both HAR and AHXR are triggered by xenoreactive natural antibodies directed against a specific epitope (galactose alpha1-3 galactose: Gal) on porcine vascular endothelium. In attempt to prevent HAR and AHXR, alpha1,3-galactosyltransferase gene knockout (GalT-KO) pigs have been produced. GalT-KO pig organs do not express the Gal epitope (antigen), and it therefore can eliminate the anti-Gal antibody--Gal antigen immunoreaction in xenotransplantation. We reported our initial study of kidney transplantation from GalT-KO miniature swine to baboons with either immunosuppression protocol or with a tolerance inducing protocol. Here, we discussed the pathology of xenografts in GalT-KO pig to non-human primate kidney transplantation.

Research of xenograft hyperacute rejection by perfusing pig kidneys with human blood in vitro

Objective Ex vivo studies by perfusing pig kidneys with differents blood to evaluate human antiporcine xenograft hyperacute rejection. Methods Pig kidneys were perfused for 30 minutes with pig blood (group 1; n =3),human blood (group 2; n =5),complement heat innactivated human blood (group 3; n =5), and xenoreactive natural antibodies (XNA) -depleted human blood (group 4; n=5). Renal blood flow rate and vascular resistance were caculated very 5 minutes. Histologic and immunohistologic examinations were carried out after perfusion. Results Pig kidneys perfused with human blood, group 2, showed HAR, higher vascular resistance and lower blood flow rate. Kidneys in Group 1,3,4 had no histologic evidence of HAR. Blood flow rate and vasular resistance kept steady. Conclusion The depletion of complement or XNA in human blood protected pig kidneys form HAR. Key words: Xenotransplant; Rejection; Kidney

Intrasplenic Preconditioning: A Model for the Study of Xenostimuli Accommodation

Discordant xenotransplantation, the grafting of organs from one phylogenic species to another, results in hyper-acute rejection (HAR). HAR is associated with the deposition of recipient preformed xenoreactive natural antibodies and complement on the endothelium of the donor organ, leading to activation and apoptosis of the endothelium, an event associated with xenograft rejection. Endothelial resistance to HAR, termed "accommodation," an active protection of graft endothelium, may be achieved by previous stimulation of endothelial cells by discordant xenoantibodies. Forty-eight male Wistar rats were used to evaluate HAR induction in an isolated, dually perfused in-situ rat liver transfused with human blood. This ex-vivo model served to mimic rat-to-human liver xenotransplantation. Preconditioning of the liver endothelium was induced by rat intrasplenic injection of human blood (n=8) or effluent of previously xenotransfused rat liver (n=8), i.e., high versus low xenoantibody solution, each undertaken 1d before liver xenotransfusion. Two other groups were not preconditioned. Preconditioned and non-preconditioned rats were perfused directly with human blood, and eight rats were used as controls (non-preconditioned Krebs-perfused). Eight rats were perfused directly with human blood, and eight rats were used as controls. The effluent that exited these first-line livers was used to perfuse the second-line livers. Portal and hepatic artery perfusion pressures, resistances, rates of oxygen extraction, lactic acid and pH, and wet-to-dry weight ratio values were significantly increased in livers xenotransfused with blood indicating HAR, compared with unchanged values in livers perfused with Krebs solution. Portal pressure and resistance were best protected from HAR by the blood preconditioning in the blood perfused group, while the hepatic artery perfusion system was better protected by the perfusate precondition-blood perfused group. The physiologic effects of HAR were attenuated in most second-line livers. Attenuation of HAR in rats' livers is achieved by preconditioning with xenoantibodies and/or by "filtering out" xenoantibodies present in the circulation, and is suggestive of accommodation. This novel method may be useful in future studies aimed at refining methods for accommodating xenotransplantation.

Expression of Tissue Factor and Initiation of Clotting by Human Platelets and Monocytes After Incubation With Porcine Endothelial Cells

Intravascular thrombosis remains a major barrier to successful pig-to-primate xenotransplantation. However, the precise factors initiating thrombosis are unknown. In this study, we investigated the contribution of recipient platelets and monocytes. Primary pig aortic endothelial cells (PAECs) were incubated with combinations of fresh or heat-inactivated human plasma, platelets, or monocytes, after which they were separated and analyzed individually by flow cytometry for tissue factor (TF) expression and for their ability to clot recalcified normal or factor-VII-deficient plasma. Procoagulant porcine TF was induced in PAECs only by fresh human plasma, and not by heat-inactivated plasma, platelets, or monocytes. In contrast, procoagulant human TF was induced on platelets and monocytes after incubation with PAEC, irrespective of whether the plasma was present or not. In addition, human platelets caused the shedding of procoagulant TF-expressing aggregates from PAEC. This work defines a cell-based in vitro assay system to address complex interactions among PAECs, human platelets, and monocytes. The induction of procoagulant TF on PAECs by fresh human plasma was most likely dependent on xenoreactive natural antibody and complement present in fresh human plasma. In contrast, the shedding of procoagulant platelet-PAEC aggregates, induced by human platelets, and the induction of procoagulant TF on human platelets and monocytes by PAEC, occurred independently of these factors. These results suggest that different mechanisms may contribute to the initiation of thrombosis after xenotransplantation, some of which may not be influenced by the further manipulation of the immune response against pig xenografts.

Resistance to anti-xenogeneic response by combining α-Gal silencing with HO-1 upregulation

Background A major barrier to clinical xenotransplantation is preformed xenoreactive natural antibodies (XNA) found in higher primates which react to Galα(1,3)Gal (α-Gal) epitopes found on lower species. Accommodation of organs to xenogeneic recipients involves upregulation of cytoprotective genes and resistance to complement dependent cytotoxicity (CDC). Methods To develop methods of increasing these organ-protective effects, we established an in vitro CDC model utilizing human serum as the source of XNA and porcine endothelial cells (pEC) as targets. Results Using this system we demonstrated that downregulation of α-Gal epitopes by siRNA silencing of α1,3-galactosyltransferase (α-GT) led to marginal protection from CDC while α-Gal silencing combined with Griffonia simplicifolia isolectin B4 (GS-IB4), a lectin that specifically binds to α-Gal epitopes, led to complete protection. Interestingly, α-Gal silencing and GS-IB4 mediated effects were not associated with inhibition of XNA binding to cells, but with significant decreased E-selectin expression and cytoprotective gene HO-1 upregulation. PI3K inhibitor LY294002 could block the elevation of HO-1 protein expression and reverse the protective effect of α-Gal silencing and GS-IB4 against CDC. Conclusion These data support the use of combination approaches targeting independent accommodation mechanisms to synergistically enhance donor organ survival in a xenogeneic setting.

Normal human serum contains high levels of anti‐Galα1‐4GlcNAc antibodies

Natural xenoreactive antibodies (Abs) directed against the Bdi-epitope (Gal alpha 1-3Gal beta) on the cells of non-primate mammals take part in hyperacute rejection of xenotransplanted organs. We found that some Abs, which were one-step affinity purified on Bdi-Sepharose, cross-reacted with the disaccharide Gal alpha 1-4GlcNAc beta. The epitope Gal alpha 1-4GlcNAc has not been identified on mammals or bacterial polysaccharides yet. To isolate the antibodies of the corresponding specificity the disaccharide was immobilized on Sepharose and antibodies were affinity purified from pooled serum of blood group O individuals. These one-step purified Abs cross-reacted with Bdi, but after a prior absorption step on Bdi-Sepharose no cross-reactivity with Bdi was observed any longer. Surprisingly, the quantity of anti-Gal alpha 1-4GlcNAc isolated from the same serum pool, 4-7 microg/ml, was equal to that of anti-Bdi or more. Independently of ABO blood groups all the tested healthy donors had anti-Gal alpha 1-4GlcNAc Abs at a similar level. Monospecific anti-Gal alpha 1-4GlcNAc Abs were not cytotoxic towards porcine cells. 1. The actual concentration of monospecific, xenoreactive Gal alpha 1-3Gal beta Abs in blood may be considerably lower than the value referred to in the literature for 'anti-alpha Gal' or 'anti-Galili' antibodies. 2. Anti-Gal alpha 1-4GlcNAc Abs seem not to be important for xenotransplantation.

Specific Non-Responsiveness to Pig in Baboons Receiving BMT from GalT-KO Pigs.

Background: The induction of stable hematopoietic cell chimerism through bone marrow transplantation (BMT) has been demonstrated to induce donor-specific tolerance in rodent, porcine, nonhuman primate, and human clinical allogenic models, and has also been successful in concordant rodent and nonhuman primate xenogeneic models, as well as in the pig-to-NOD/SCID humanized mouse xenogenic model. However, stable chimerism and tolerance has been difficult to achieve in the discordant pig-to-baboon xenotransplantation model, possibly due in part to the presence in baboons of pre-formed natural xeno-reactive antibodies to a1,3-galactose (Gal) determinants expressed in pigs, but not in Old World primates and humans. The recent availability of miniature swine homozygous for a disruption in the gene encoding a1,3-galactosyltransferase (GalT-KO pigs) has now made it possible to study pig-to-baboon xenografts in the absence of effects of anti-Gal antibodies. We have investigated the GalT-KO pig-to-baboon model further by modifying the conditioning and immunosuppression regimen to facilitate engraftment and tolerance through bone marrow transplantation.Methods: BM was harvested from GalT-KO swine (n=3). Baboons (n=3) were pre-treated with whole body (3 Gy) and thymic (7 Gy) irradiation, Sangstat rabbit anti-thymocyte globulin (ATG), LoCD2b (rat IgG2b anti-primate CD2) and splenectomy, and received FK506 immunosuppressive and supportive therapy for 28 days. The baboons were monitored for the presence of pig cells by flow cytometry, for porcine progenitor cells in the bone marrow by porcine cytochrome b specific PCR of colony-forming units (CFUs), and for cellular reactivity to pig cells by MLR and CML. Antibody formation to LoCD2b and ATG was tested by enzyme-linked immunosorbent assay (ELISA), and antibody reactivity to GalT-KO pig cells was tested by flow cytometry and antibody mediated cytotoxicity assay.Results: A mean of 1.4 × 109 BM cells/kg was infused into each baboon. Although pig cells were undetectable in the peripheral blood of the baboons by flow cytometry, porcine progenitor cell engraftment as well as chimerism in the bone marrow and thymus was detected by PCR in the first baboon on day 28. ELISA results indicated the presence of antibodies to rat (LoCD2b) and rabbit (ATG) immunoglobulin within two weeks; however, no antibodies to pig cells could be detected by flow cytometry or cytotoxicity assay. The second baboon had undetectable serum antibodies to pig cells for 60 days despite the presence of induced antibodies to rat LoCD2b and rabbit ATG. Porcine progenitor cell engraftment was confirmed by PCR of CFUs on day 60 and MLR showed no response to pig although the animal regained alloresponses by this time. The third baboon, in contrast, had detectable induced serum antibodies to pig cells as well as rat and rabbit immunoglobulin by day 14 following BMT.Conclusions: Engraftment has been achieved following GalT-KO pig-to-baboon BMT with evidence of specific humoral and cellular non-responsiveness to pig cells (2/3 baboons), suggesting the possibility that this protocol may facilitate xenograft tolerance.

Human neutrophil gene expression profiling following xenogeneic encounter with porcine aortic endothelial cells: the occult role of neutrophils in xenograft rejection revealed

The role of innate immune cells in the recognition and activation of xenogeneic endothelium has always been considered secondary to the initial insult of xenoreactive natural antibodies (XNA) and complement. It was argued, however, that innate immune cells are capable of recognizing and activating xenogeneic endothelium in the absence XNA and complement. Here, we show that porcine aortic endothelial cells (PAECs) activate human neutrophils directly. This contact-dependent activation causes a transient calcium rise leading to increased reactive oxygen metabolite (ROM) production. Neutrophil gene-expression profiling using an adenylate uridylate-rich element-based microarray revealed a dramatic change in the neutrophil gene profiles upon exposure to PAECs. The PAEC-dependent neutrophil transcriptional activity was further confirmed by real-time polymerase chain reaction, which revealed a rapid increase in the mRNA message of a number of inflammatory cytokines. The activation of human neutrophils by PAECs was independent of galactose alpha1,3-galactose (Galalpha1,3-gal) structures, as inclusion of saturating concentrations of anti-Galalpha1,3-gal l antibodies had no significant effect. Furthermore, this activation was inhibited in the presence of the calcium chelator 1,2-bis(O-aminophenyl-ethane-ethane)-N,N,N',N'-tetraacetic acid-acetoxymethyl ester and the ROM inhibitor diphelylene iodonium. Our data illustrate the direct activation of innate immune cells by PAECs in the absence of XNA and complement and suggest alternative recognition sites between PAECs and human innate immune cells.

Hyperacute Lung Rejection in the Pig-to-Human Model 4: Evidence for Complement and Antibody Independent Mechanisms

We assessed whether the combination of complement regulation and depletion of xenoreactive antibodies improves the outcome of pulmonary xenografts compared with either strategy alone. Lungs from pigs heterozygous (hDAF(+/-)) or homozygous (hDAF(+/+)) for the human decay accelerating factor transgene (hDAF) or their nontransgenic litter mates (hDAF(-/-)) were perfused with heparinized whole human blood. In additional groups, xenoreactive natural antibodies (XNA) were depleted by pig lung perfusion (hDAF(-/-)/AbAbs, hDAF(+/-)/AbAbs) before the experiment. This combined approach was augmented by adding soluble complement receptor 1 (sCR1) to the perfusate in one further group (hDAF(+/-)/AbAbs/sCR1). HDAF(-/-) lungs perfused with unmodified human blood were rejected after 32.5 min (interquartile range, IQR 5 to 210). HDAF(+/-) lungs survived for 90 min (IQR 10 to 161, P = 0.54). Both groups showed a rapid rise in pulmonary vascular resistance (PVR), which is a characteristic feature of hyperacute rejection (HAR). This phenomenon was blunted in the hDAF(+/+) group, although survival (48 min, IQR 14 to 111) was not further prolonged. Antibody depletion (AbAbs) led to a significant increase in survival time (hDAF(-/-)/AbAbs: 315 min, IQR 230 to 427; hDAF(+/-)/AbAbs: 375 min, IQR 154 to 575), reduced PVR and less complement production. Addition of sCR1 reduced complement elaboration but did not further improve survival (200 min, IQR 128 to 580) and surprisingly tended to increase PVR. Depletion of xenoreactive antibodies is more effective than membrane-bound complement regulation to blunt hyperacute rejection of pulmonary xenografts, but even the combined approach including soluble-phase complement inhibition is not sufficient to reliably prevent organ failure within hours. It therefore seems likely that other factors independent of antibody and complement contribute to HAR in this model.

208 CYTOLYTIC ANALYSIS AND NUCLEAR TRANSFER OF hCD46-TRANSGENIC PORCINE EMBRYONIC GERM CELLS TO DEVELOP AND IN VITRO MODEL OF XENOTRANSPLANTATION

Pigs are considered the most likely source of organs for xenotransplantation due to their anatomical and physiological similarities to humans. Production of transgenic pigs including addition of human complement-regulatory protein genes and deletion of alpha-1,3-galactosyl transferase gene may overcome hyperacute rejection (HAR), the first and currently the most critical immunological hurdle in the development of xenogeneic organs for human transplantation. However, even after resolving HAR in pig-to-human xenotransplantation, a series of other transgenic pigs may be required to alleviate subsequent acute and chronic rejection and incompatibility of porcine proteins to human counterparts. The production of transgenic pigs is not only labor-intensive, time-consuming, and costly, but also the usefulness of such pigs in transplantation to humans is unpredictable. For these reasons, development of a reliable in vitro procedure to pre-evaluate effectiveness of the transgenic approach would be beneficial. This study was preformed to establish an in vitro model of xenotransplantation using porcine embryonic germ (EG) cells, undifferentiated stem cells derived from culture of primordial germ cells. Porcine EG cells were maintained in feeder-free state in DMEM containing 15% (v/v) fetal bovine serum and 1000 units/mL leukemia inhibitory factor. Human complement down-regulator hCD46 (also known as MCP, membrane cofactor protein) gene under the regulation of cytomegalovirus promoter was introduced into porcine EG cells. Transfected cells were selected by antibiotic treatment and confirmed by PCR. To test the resistance of hCD46-transgenic EG cells to human xenoreactive natural antibody and complement, EG cells were cultured for 1.5 days in DMEM containing 15% (v/v) normal human serum. The treatment with human serum did not affect the survival of hCD46-transgenic EG cells, whereas with the same treatment approximately one half of non-transfected EG cells failed to survive (P &lt; 0.01). Transgenic EG cells presumably capable of overcoming HAR were used as nuclear donors for subsequent transfer of nuclei into enucleated oocytes. Among 110 reconstituted oocytes, 19 (17.3%) developed to the blastocyst stage. Analysis of individual nuclear transfer embryos by PCR indicated that 89.5% (17/19) of embryos contained transgene hCD46. The PCR-negative embryos might be due to an incomplete antibiotic selection of cells after transfection. Overall, the results of the present study demonstrate that the cell culture-based model of xenotransplantation may validate the usefulness of particular transgenic pigs prior to actual production. Further experiments on differentiation of transgenic EG cells into various cell types, cytolytic analysis of such cells to assess efficiency of xenotransplantation, and subsequent production and transfer of transgenic clone embryos to recipients may provide a useful new procedure to accelerate xenotransplantation research.

A novel approach to xenotransplantation combining surface engineering and genetic modification of isolated adult porcine islets

Effective cytoprotection to xenoislets would circumvent the major tissue limitation for pancreatic islet transplantation (PIT). Cell-surface engineering with poly[ethylene glycol] (PEG) derivatives can successfully prevent antibody binding to the surface antigens. Gene transfer of the antiapoptotic Bcl-2 gene has been shown to decrease cytotoxicity mediated by xenoreactive natural antibodies and complement. In this study, we assessed survival and function of surface-engineered porcine islets genetically modified to overexpress Bcl-2. Incorporation of PEG derivatives into the islet surface and adenovirus-mediated gene transfer of Bcl-2 (AdBcl-2) was accomplished within 24 hours post-isolation. Cytotoxicity induced by human xenoreactive natural antibodies was evaluated by islet intracellular lactate dehydrogenase release and microscopic analysis using membrane-integrity staining. Islet functionality was assessed by static incubation and after intraportal infusion (5000 IEQ) into diabetic NOD-SCID mice reconstituted with human lymphocytes (5 x 10 8 /intraperitoneally/15 days before PIT). No significant change in islet viability, morphology, and functionality was demonstrated after the incorporation of PEG-mono-succimidyl-succinate (MSPEG), or PEG-di-succimidyl-succinate "end"-capped with albumin (DSPEG) with or without gene transfer of Bcl-2. Islets treated with MSPEG presented a significant reduction in lactate dehydrogenase release compared with controls (41.2 +/- 3 vs 72.1 +/- 7, respectively, P <.05). Further protection was accomplished by DSPEG or AdBcl-2. The maximal cytoprotection was achieved by DSPEG +AdBcl-2 (15.5 +/- 4.9%, P <.001). Nonfasting glucose >200 mg/dL was found in 100% of the animals given control islets (n = 6) within 48 hours post-transplant. In contrast, euglycemia was achieved in 100% of the animals given islets modified with DSPEG + AdBcl-2 during the observation time. Surface-engineering with functionalized PEG derivatives in combination with genetic modification with Bcl-2 significantly reduced islet loss after PIT. Application of this novel technology may improve results in xenoislet transplantation.

Acute vascular rejection of xenografts: roles of natural and elicited xenoreactive antibodies in activation of vascular endothelial cells and induction of procoagulant activity.

Hyperacute rejection of vascularized discordant xenografts can now be effectively managed. However, acute vascular rejection (AVR) then ensues, resulting in graft destruction, coagulopathy, or both within weeks. The aim of this study was to determine associations between humoral responses to the xenograft and the induction of AVR, coagulopathy, or both. In vitro, heat-inactivated, naive or sensitized baboon sera containing xenoreactive natural or elicited antibodies were used to activate porcine aortic endothelial cells (PAEC) in vitro. Tissue factor expression on PAEC was determined as an index of heightened procoagulant activity. In vivo, porcine renal xenografts were transplanted into immunosuppressed baboons, and at the time of rejection or the development of a consumptive coagulopathy, biopsy specimens were obtained for studies of xenoreactive antibody binding and tissue factor expression. In vitro, incubation of PAEC with naive baboon sera containing natural anti-Galalpha1,3Gal (Gal) antibodies resulted in minimal tissue factor induction; the addition of complement boosted procoagulant responses. Elicited xenoreactive antibodies, and to non-Gal epitopes alone, induced high amounts of procoagulant activity on PAEC; the addition of complement resulted in overt cytotoxicity. In vivo, AVR was associated with xenoreactive antibody deposition in the graft. When vascular endothelial binding of xenoreactive antibody was combined with the expression of tissue factor, consumptive coagulopathy developed irrespective of histopathologic features of AVR. Our in vitro results indicate that elicited antibodies, potentially to non-Gal epitopes, induce endothelial cell activation and tissue factor expression; in vivo, a consumptive coagulopathy occurred when there was xenoreactive antibody deposition and increase of tissue factor.

Natural antibodies prevent in vivo transmission of porcine islet-derived endogenous retrovirus to human cells.

The discovery of porcine endogenous retroviruses (PERV) has raised concerns regarding the safety of porcine xenotransplantation. However, transmission of PERV had not been observed in humans exposed to porcine tissue. We examined whether PERV derived from porcine pancreatic islet cells could infect human cells in vivo and the role of natural antibodies in inhibiting PERV infection. In vivo infective potential of PERV was studied in SCID mice reconstituted with human peripheral blood leucocytes. Porcine islets were transplanted under the kidney capsule. PERV infection was determined by analyzing PERV gene expression in graft infiltrating lymphocytes (GIL) harvested 21 days posttransplantation. Mice were administered normal human serum prior to and 2 days posttransplantation to study their role in protection of human cells against PERV infection. PERV genes were expressed in all porcine tissues examined, including purified porcine islets. PERV expression was detected in GILs from three of five human-SCID mice. Administration of human serum blocked PERV infection in GILs in five of five human-SCID mice. These results indicate that PERV from porcine islets can infect human cells in vivo. Normal human serum blocks transmission of retrovirus in vivo, suggesting that natural xenoreactive antibodies can prevent PERV infection.

Rechazo celular agudo en modelo ex vivo de xenotrasplante renal cerdo-hombre

Cellular Acute Rejection Pig To Human In An Ex Vivo Renal Transplant Model ObjetiveEx vivo perfusion model of pig-to-human kidney xenotransplantation to evaluate human antiporcine xenograft rejection once hyperacute rejection (HAR) is avoided. methodsPig kidneys were perfused for 3 hours with pig blood (group 1; n=5), human blood (group 2; n=5), complement heat innactivated human blood (group 3; n=5), platelet-depleted human blood (group 4; n=5); and xenoreactive natural antibodies (XNA)-depleted human blood (group 5; n=5). Tissue samples were studied with immunoperoxidase techniques. ResultadosPig kidneys perfused with human blood, group 2, showed HAR with interstitial haemorrhage, vascular thrombi and glomerular injury. Pig kidneys perfused with manipulated human blood (groups 3 to 5) had no histologic evidence of HAR, but showed signs of acute cellular rejection with different degrees of interstitial infiltrate of mononuclear cells, specially in the XNA-depleted group. ConclusionesThe model may be valuable in the isolated evaluation of the elements involved in the pig-tohuman xenorejection. The depletion of complement, platelets and XNA protected porcine kidneys form HAR in our study and allowed the development of an acute cellular rejection, a very unusual fact in this short time, 3 hours.