Anti-pig IgM Research Articles

Development of immunochromatographic strip assay to detect recent infection of Japanese encephalitis virus in swine population

Japanese Encephalitis (JE) is a mosquito borne re-emerging viral zoonotic disease. Sero-conversion in swine occurs 2–3 weeks before human infection, thus swine act as a suitable sentinel for predicting JE outbreaks in humans. The present study was undertaken with the objective of developing immunochromatographic strip (ICS) assay to detect recent infection of Japanese Encephalitis virus (JEV) in swine population. The two formats of ICS assay were standardized. In the first format, gold nanoparticles (GNP) were conjugated with goat anti-pig IgM (50 μg/ml) followed by spotting of recombinant NS1 protein (1 mg/ml) of JEV on NCM as test line and protein G (1 mg/ml) as control line. In the format-II, GNP were conjugated with rNS1 protein (50 μg/ml) followed by spotting of Goat anti-pig IgM (1 mg/ml) as test line and IgG against rNS1 (1 mg/ml) as control line. To decrease the non- specific binding, blocking of serum and nitrocellulose membrane (NCM) was done using 5% SMP in PBS-T and 1% BSA, respectively. Best reaction conditions for the assay were observed when 10 μl of GNP conjugate and 50 μl of 1:10 SMP blocked sera was reacted on BSA blocked NCM followed by reaction time of 15 mins. Samples showing both test and control line were considered positive whereas samples showing only control line were considered negative. A total of 318 field swine sera samples were screened using indirect IgM ELISA and developed ICS assay. Relative diagnostic sensitivity and specificity of format-I was 81.25% and 93.0% whereas of format-II was 87.50% and 62.93%, respectively. Out of 318 samples tested, 32 were positive through IgM ELISA with sero-positivity of 10.06% while sero-positivity with format-I of ICS was 8.1%. Owing to optimal sensitivity and higher specificity of format-I, it was validated in three different labs and the kappa agreement ranged from 0.80 to 1, which signifies excellent repeatability of the developed assay to test field swine sera samples for detecting recent JEV infection.

In vitro and in vivo immune assessments of genetically-engineered pig skin grafts in New World (squirrel) monkeys.

Half a million patients in the USA alone require treatment for burns annually. Following an extensive burn, it may not be possible to provide sufficient autografts in a single setting. Genetic manipulations (GM) of pigs offer the possibility of reducing primate humoral and cellular rejection of pig skin xenografts and thus extending graft survival. We compared the survival of skin grafts from pigs with 9-GM with that of autografts and allografts in squirrel monkeys. Monitoring for rejection was by (1) macroscopic examination, (2) histopathological examination of skin biopsies, and (3) measurement of anti-monkey and anti-pig IgM and IgG antibodies. Autografts (n = 5) survived throughout the 28 days of follow-up without histopathological features of rejection. Median survival of allografts (n = 6) was 14 days and of pig xenografts (n = 12) 21 days. Allotransplantation was associated with an increase in anti-monkey IgM, but the anticipated subsequent rise in IgG had not yet occurred at the time of euthanasia. Pig grafts were associated with increases in anti-pig IgM and IgG. In all cases, histopathologic features of rejection were similar. 9-GM pig skin xenografts survive at least as long as monkey skin allografts (and trended to survive longer), suggesting that they are a realistic clinical option for the temporary treatment of burns. Although monkeys with pig skin grafts developed anti-pig IgM and IgG antibodies, these did not cross-react with monkey antigens, indicating that a primary 9-GM pig skin graft would not be detrimental to a subsequent monkey skin allograft.

Antibody reactivity with new antigens revealed in multi-transgenic triple knockout pigs may cause early loss of pig kidneys in baboons.

Recent advances in gene editing technology have enabled the production of multi-knockout (KO) and transgenic pigs in order to overcome immunologic barriers in xenotransplantation (XTx). However, the genetic manipulations required to produce these changes may have the unintended consequence of producing or revealing neoantigens reactive with natural antibodies present in baboons. In this study, we examined whether the neoantigens that develop in multi-transgenic (mTg) GalT, Cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH), β-1,4-N-acetyl-galactosaminyl transferase 2 (B4) KO pigs can cause rejection of xenografts in baboons. Five baboons that had <35% cytotoxicity against GalT-KO peripheral blood mononuclear cells (PBMCs) in a pre-screening assay received pig kidneys and vascularized thymic grafts (VT+K) from multi-transgenic hCD47, human thrombomodulin (hTBM), human endothelial protein C receptor (EPCR) with/without hCD46 and hCD55 with GalT-KO/NeuGC-KO/B4-KO (mTg Tri-KO) swine. In order to further examine the effects of anti-donor non-Gal natural antibody (nAb), anti-pig preformed IgM and IgG nAb binding against the GalT-KO PBMCs was compared with the donor-type PBMCs using donor pretransplant sera as well as 5 additional naïve baboon sera by flow cytometric analysis. Five baboons that received VT+K grafts had stable renal function in the first 11days (serum creatinine<1.5mg/dL). Two of the five baboons had higher binding of preformed IgG to mTg Tri-KO PBMCs than to GalT-KO PBMCs (mTg Tri-KO>GalT-KO), and they rejected their grafts at POD 20. In contrast, the other three baboons demonstrated either mTg Tri-KO=GalT-KO or mTg Tri-KO<GalT-KO, and they maintained renal function 43, 52, and 154days without rejection. Among 10 baboon sera, two had less antibody binding against PBMCs that were syngeneic to the mTg Tri-KO than against GalT-KO PBMCs (mTg Tri-KO<GalT-KO); three had similar binding to mTg Tri-KO and GalT-KO PBMCs (mTg Tri-KO=GalT-KO); and five had higher binding to m Tg Tri-KO than to GalT-KO PBMCs (mTg Tri-KO>GalT-KO). These data suggest that neoantigens associated with mTg Tri-KO promote acute xenograft rejection in a pig-to-baboon VT+K XTx model. The screening assays may be useful to select "safe" recipients to receive mTg Tri-KO kidneys.

Xenoantigenicity of porcine decellularized valves

BackgroundThe xenoantigenicity of porcine bioprosthetic valves is implicated as an etiology leading to calcification and subsequent valve failure. Decellularization of porcine valves theoretically could erase the antigenicity of the tissue leading to more durable prosthetic valves, but the effectiveness of decellularization protocols in regard to completely removing antigens has yet to be verified. Our hypothesis was that decellularization would remove the more abundant α-gal antigens but not remove all the non α-gal antigens, which could mount a response.MethodsPorcine aortic valves were decellularized with 1% sodium dodecyl sulfate for 4 days. Decellularized cusps were evaluated for α-gal epitopes by ELISA. To test for non α-gal antigens, valves were implanted into sheep. Serum was obtained from the sheep preoperatively and 1 week, 1 month, and 2 months postoperatively. This serum was utilized for anti-porcine antibody staining and for quantification of anti-pig IgM and IgG antibodies and complement.ResultsDecellularized porcine cusps had 2.8 ± 2.0% relative α-gal epitope as compared to fresh porcine aortic valve cusps and was not statistically significantly different (p = 0.4) from the human aortic valve cusp which had a 2.0 ± 0.4% relative concentration. Anti-pig IgM and IgG increased postoperatively from baseline levels. Preoperatively anti-pig IgM was 27.7 ± 1.7 μg/mL and it increased to 71.9 ± 12.1 μg/mL average of all time points postoperatively (p = 0.04). Preoperatively anti-pig IgG in sheep serum was 44.9 ± 1.5 μg/mL and it increased to 72.6 ± 6.0 μg/mL average of all time points postoperatively (p = 0.01). There was a statistically significant difference (p = 0.00007) in the serum C1q concentration before valve implantation (2.5 ± 0.2 IU/mL) and at averaged time points after valve implantation (5.3 ± 0.3 IU/mL).ConclusionsDecellularization with 1% sodium dodecyl sulfate does not fully eliminate non α-gal antigens; however, significant reduction in α-gal presence on decellularized cusps was observed. Clinical implications of the non α-gal antigenic response are yet to be determined. As such, evaluation of any novel decellularized xenografts must include rigorous antigen testing prior to human trials.

Transplantation of hepatocytes from genetically engineered pigs into baboons.

Some patients with acute or acute-on-chronic hepatic failure die before a suitable human liver allograft becomes available. Encouraging results have been achieved in such patients by the transplantation of human hepatocyte progenitor cells from fetal liver tissue. The aim of the study was to explore survival of hepatocytes from genetically engineered pigs after direct injection into the spleen and other selected sites in immunosuppressed baboons to monitor the immune response and the metabolic function and survival of the transplanted hepatocytes. Baboons (n=3) were recipients of GTKO/hCD46 pig hepatocytes. All three baboons received anti-thymocyte globulin (ATG) induction and tapering methylprednisolone. Baboon 1 received maintenance immunosuppressive therapy with tacrolimus and rapamycin. Baboons 2 and 3 received an anti-CD40mAb/rapamycin-based regimen that prevents sensitization to pig solid organ grafts. The baboons were euthanized 4 or 5weeks after hepatocyte transplantation. The baboon immune response was monitored by the measurement of anti-non-Gal IgM and IgG antibodies (by flow cytometry) and CFSE-mixed lymphocyte reaction. Monitoring for hepatocyte survival and function was by (i) real-time PCR detection of porcine DNA, (ii) real-time PCR for porcine gene expression, and (iii) pig serum albumin levels (by ELISA). The sites of hepatocyte injection were examined microscopically. Detection of porcine DNA and porcine gene expression was minimal at all sites of hepatocyte injection. Serum levels of porcine albumen were very low-500-1000-fold lower than in baboons with orthotopic pig liver grafts, and approximately 5000-fold lower than in healthy pigs. No hepatocytes or infiltrating immune cells were seen at any of the injection sites. Two baboons (Baboons 1 and 3) demonstrated a significant increase in anti-pig IgM and an even greater increase in IgG, indicating sensitization to pig antigens. As a result of this disappointing experience, the following points need to be considered. (i) Were the isolated pig hepatocytes functionally viable? (ii) Are pig hepatocytes more immunogenic than pig hearts, kidneys, artery patch grafts, or islets? (iii) Does injection of pig cells (antigens) into the spleen and/or lymph nodes stimulate a greater immune response than when pig tissues are grafted at other sites? (iv) Did the presence of the recipient's intact liver prevent survival and proliferation of pig hepatocytes? (v) Is pig CD47-primate SIRP-α compatibility essential? In conclusion, the transplantation of genetically engineered pig hepatocytes into multiple sites in immunosuppressed baboons was associated with very early graft failure. Considerable further study is required before clinical trials should be undertaken.

Xenotransplanted Pig Sertoli Cells Inhibit Both the Alternative and Classical Pathways of Complement-Mediated Cell Lysis While Pig Islets Are Killed.

Xenotransplantation has vast clinical potential but is limited by the potent immune responses generated against xenogeneic tissue. Immune-privileged Sertoli cells (SCs) survive xenotransplantation long term (≥90 days) without immunosuppression, making SCs an ideal model to identify xenograft survival mechanisms. Xenograft rejection includes the binding of natural and induced antibodies and the activation of the complement cascade. Using an in vitro cytotoxicity assay, wherein cells were cultured with human serum and complement, we demonstrated that neonatal pig SCs (NPSCs) are resistant to complement-mediated cell lysis and express complement inhibitory factors, membrane cofactor protein (MCP; CD46), and decay- accelerating factor (DAF; CD55) at significantly higher levels than neonatal pig islets (NPIs), which served as non-immune-privileged controls. After xenotransplantation into naive Lewis rats, NPSCs survived throughout the study, while NPIs were rejected within 9 days. Serum antibodies, and antibody and complement deposition within the grafts were analyzed. Compared to preformed circulating anti-pig IgM antibodies, no significant increase in IgM production against NPSCs or NPIs was observed, while IgM deposition was detected from day 6 onward in both sets of grafts. A late serum IgG response was detected in NPSC (days 13 and 20) and NPI (day 20) recipients. Consistently, IgG deposition was first detected at days 9 and 13 in NPSC and NPI grafts, respectively. Interestingly, C3 was deposited at days 1 and 3 in NPI grafts and only at day 1 in NPSC grafts, while membrane attack complex (MAC) deposition was only detected in NPI grafts (at days 1-4). Collectively, these data suggest NPSCs actively inhibit both the alternative and classical pathways of complement-mediated cell lysis, while the alternative pathway plays a role in rejecting NPIs. Ultimately, inhibiting the alternative pathway along with transplanting xenogeneic tissue from transgenic pigs (expressing human complement inhibitory factors) could prolong the survival of xenogeneic cells without immunosuppression.

Studies of Pig Complement: Measurement of Pig CH50, ACH50, and Components

BackgroundOn the basis of a comparison of the hemolytic complement titer in pigs with that in humans, the complement system of pigs was investigated. The response of innate immunity, such as the natural antibodies, against humans was also examined. MethodsHemolytic complement activity of pig serum was measured with the use of a microtitration technique. CH50 was determined according to the method of Mayer. ACH50 was assayed according to the methods of Platts-Milles and Ishizaka. Hemolytic activities of C1, C4, C2, C3, C5, C8, and C9 were estimated through the use of intermediate cells and reagents, as described previously. In addition, the pig natural anti-human antibody was studied with the use of human peripheral blood mononuclear cells (PBMCs). Human PBMCs were stained with 5% pig serum, followed by staining with fluorescein isothiocyanate–labeled goat anti-pig IgG and IgM. The resulting stained cells were quantified by use of a FACScalibur system. The alternative pathway of pig complement was also measured with the use of human erythrocytes and normal pooled pig serum with or without Mg++EGTA. ResultsBoth the CH50 and ACH50 titers were lower than those of humans. Concerning the components, except for C3, each component, that is, C1, C4, C2, C5, C8, and C9, was also lower than that of humans, based on measured values for human complement components. Pig serum clearly contains natural antibodies, IgG and IgM, to human PBMCs. The alternative pathway of pig complement reacted with human erythrocytes. ConclusionsAs a whole, pig innate immunity, the complement system and natural antibody, recognizes the surfaces of human cells.

Pig Artery Patch Transplantation (Tx) as a Surrogate For Heart Tx in Baboons.

Background: Pig heart Tx in baboons is time-consuming, expensive, and associated with complications. Graft failure can result from the development of (i) elicited anti-pig antibodies or (ii) thrombotic microangiopathy in the graft. We have developed a simple substitute model of pig artery patch Tx and have compared the results with those following heart Tx. Methods: Baboons received artery patch Tx into the wall of the abdominal aorta) (Gp 1; n=28) or heterotopic heart Tx (Gp 2; n=17) from GTKO pigs (+/- transgenes aimed at complement or coagulation regulation [n=22] +/- T cell regulation [a human mutant MHC class II transactivator, CIITA, n=9]). Nine recipients (patch 4, heart 5) received no immunosuppressive therapy (IS). The remainder (patch 24, heart 12) received IS based on blockade of either the CD40:CD154 pathway (anti-CD154 or anti-CD40 mAbs) or the CD28:B7 pathway (CTLA4-Ig - abatacept or belatacept) or of both pathways (anti-CD40mAb+belatacept). Monitoring was by MLR, anti-pig IgM and IgG, histopathology, and various coagulation (platelet count, fibrinogen, D-dimer) and inflammatory (C-reactive protein, CRP) parameters. Results: In both models, (i) with no IS, grafts induced a significant T cell response; (ii) anti-CD154mAb prevented a T cell response, whereas CTLA4-Ig did not; and (iii) anti-CD40mAb+belatacept prevented T cell proliferative and elicited antibody responses and cellular infiltration of the graft. Expression of CIITA in a patch graft reduced the elicited antibody response. Despite heparin therapy, thrombocytopenia, fall in fibrinogen, and increases in D-dimer and CRP were documented in baboons with heart grafts that did not express a human coagulation-regulatory transgene, but only increases in D-dimer and CRP were seen after patch Tx. Treatment with anti-CD40mAb+belatacept did not prevent the rise of D-dimer or CRP in either model. An IL-6R antagonist prevented a rise in CRP after patch Tx. Conclusions: Outcomes of patch and heart Tx are similar. Expression of CIITA reduces the elicited antibody response to a patch graft. To replace anti-CD154mAb, blockade of both the CD28 and CD154 pathways is essential. Patch Tx provides some (but not all) information on the coagulation and inflammatory responses to a heart graft. IL-6R blockade may reduce the inflammatory response. DISCLOSURE:Ayares, D.: Employee, Revivicor.

Are there advantages in the use of specific pathogen‐free baboons in pig organ xenotransplantation models?

Baboons have natural antibodies against pig antigens. We have investigated whether there are differences in anti-non-Gal pig antibody levels between baboons maintained under specific pathogen-free (SPF) conditions and those housed under conventional conditions (non-SPF) that might be associated with improved outcome after pig-to-baboon organ transplantation. Baboons (n=40) were housed indoors (SPF n=8) or in indoor/outdoor pens (non-SPF n=32) in colonies of similar size and structure. Non-SPF colonies harbor a number of pathogens common to non-human primate species, whereas many of these pathogens have been eliminated from the SPF colony. Complete blood cell counts (CBC), blood chemistry, and anti-non-Gal IgM and IgG levels were monitored. There were no significant differences in CBC or blood chemistry between SPF and non-SPF baboons. Anti-non-Gal IgM levels were significantly lower in the SPF baboons than in the non-SPF baboons (MFI 7.1 vs. 8.8, P<0.05). One SPF and two non-SPF baboons had an MFI >20; if these three baboons are omitted, the mean MFIs were 4.8 (SPF) vs. 7.5 (non-SPF) (P<0.05). Anti-non-Gal IgG was minimal in both groups (MFI 1.0 vs. 1.0). As their levels of anti-non-Gal IgM are lower, baboons maintained under SPF conditions may be beneficial for xenotransplantation studies as the initial binding of anti-pig IgM to an α1,3-galactosyltransferase gene-knockout pig organ may be less, thus resulting in less complement and/or endothelial cell activation. However, even under identical SPF conditions, an occasional baboon will express a high level of anti-non-Gal IgM, the reason for which remains uncertain.

Boosted Rat Natural Xenoantibodies Cross-React with Enterococcus faecalis by Targeting Melibiose and L-Rhamnose

Natural antibodies include a subset described as xenoantibodies considered to be directed at microorganisms and also cross-react with antigens of unrelated species. In this study, we generated T-cell-independent (TI) and T-cell-dependent (TD) xenoantibodies in Lewis rats with hamster and pig blood injections. TI anti-hamster and anti-pig IgM and IgG xenoantibodies cross-reacted with Enterococcus faecalis but not with Escherichia coli isolated from the blood of Lewis rats after cecal ligation and puncture (CLP). TI anti-pig IgM xenoantibodies also showed some reactivity with two human blood isolates of E. faecalis. In contrast, TD xenoantibodies did not show any reactivity with rat or human bacteria. TI and TD anti-hamster and anti-pig IgM and IgG xenoantibodies showed cross-reactivity with lymphocytes and endothelial cells from species distinct to that used for immunization. Glycan array analysis and inhibition assays identified antibodies against melibiose and <smlcap>L</smlcap>-rhamnose as mediators of anti-hamster and anti-porcine xenoantibody cross-reactivity with E. faecalis. A rise in TI anti-hamster and anti-pig xenoantibodies was accompanied by decreased survival of Lewis rats in a low-severity sepsis model of CLP. Therefore, TI xenoantibodies in the rat include anti-carbohydrate antibodies reactive to bacteria of endogenous flora. Enhancement of these antibodies may result in more severe infectious diseases caused by these microorganisms.

Minimal effect of bortezomib in reducing anti‐pig antibodies in human leukocyte antigen‐sensitized patients: a pilot study

Bortezomib, a proteasome inhibitor used to treat multiple myeloma, has been administered (± plasma exchange ± intravenous immunoglobulin [IVIg]) in attempts to reduce antibodies against human leukocyte antigens (HLA) in sensitized patients undergoing organ transplantation. To our knowledge, bortezomib has not been investigated for its effect on natural anti-pig antibodies. If bortezomib could reduce the production of anti-pig antibodies, this would likely be beneficial to the outcome of pig organ grafts in primates. Nine patients received bortezomib either to reduce anti-HLA antibody levels before organ allotransplantation or to treat antibody-mediated rejection. Patients at the Mayo Clinic (Group 1; n=4) received bortezomib alone, whereas at the UPMC (Group 2; n=5), this was combined with plasmaphereses ± IVIg in some cases. Anti-pig IgM and IgG levels against wild-type (WT) and α1,3-galactosyltransferase gene knockout (GTKO) pig aortic endothelial cells (flow cytometry-relative mean fluorescence intensity) and anti-Gal IgM and IgG (ELISA-OD480nm ) were measured pre- and post-bortezomib therapy. Mean anti-pig IgM levels were 11.2 (WT) and 1.9 (GTKO) pre-bortezomib treatment and 9.4 (WT: P=0.02) and 1.7 (GTKO: P=0.33) post-bortezomib treatment, respectively. Mean anti-pig IgG levels were 4.3 (WT) and 1.5 (GTKO) pre-bortezomib treatment and 3.6 (WT: P=0.21) and 1.4 (GTKO: P=0.20) post-bortezomib treatment, respectively. Mean anti-Gal IgM and IgG levels were 0.7 and 1.1, respectively, pre-treatment, and 0.6 (P=0.03) and 1.1 (NS), respectively, post-treatment. When the data were analyzed in Groups 1 and 2 separately, there were no significant differences between the pre- and post-bortezomib levels of anti-pig, anti-non-Gal, or anti-Gal IgM or IgG. From this limited study, we conclude that bortezomib might reduce anti-Gal IgM levels in primates, but, in this respect alone, is unlikely to have any significant effect on the outcome of GTKO pig organ transplantation.

Development and partial validation of a recombinant E2-based indirect ELISA for detection of specific IgM antibody responses against classical swine fever virus

Detecting classical swine fever virus specific antibody responses is critical for prevention and control of CSF. In this study, a ΔE2-based indirect ELISA was developed to detect specific IgM antibodies against CSFV. The optimized conditions that were determined experimentally are: a ΔE2 antigen concentration of 0.5μg/ml, a serum sample dilution of 1/100 incubated at 37°C for 1.5h, and a HRP conjugated rabbit anti-pig IgM dilution of 1/50,000 incubated at 37°C for 1h. Three hundred clinical sera were tested with ΔE2-IgM-ELISA and IDEXX ELISA and the positive rates were 77.3% (232/300) and 71.7% (215/300), respectively. Concordance rate between them was 80.3% (241/300). The 59 inconsistent sera were tested further: among the 21 IDEXX ELISA +/ΔE2-IgM-ELISA − and 38 IDEXX ELISA +/ΔE2-IgM-ELISA − samples, 17 and 24 were determined positive by virus neutralization test; 15 and 25 were tested positive by ΔE2-IgG-ELISA, respectively. In addition, the E2-specific IgM antibody response in 15 vaccinated piglets could be detected 2 weeks post-vaccination and earlier than specific IgG antibody. It increased regularly and reached high levels by 6 weeks post-vaccination. The ΔE2-IgM-ELISA could be used for clinical detection and for exploring the kinetics of IgM antibody response.

Costimulation blockade in pig artery patch xenotransplantation - a simple model to monitor the adaptive immune response in nonhuman primates.

CD154 blockade-based immunosuppression successfully prevents both humoral and cellular adaptive immune responses in baboons receiving α1,3-galactosyltransferase gene-knockout (GTKO) pig organs. Using a GTKO pig artery transplantation model in baboons, we evaluated the efficacy of CD28/B7 costimulatory pathway blockade in comparison with CD154 blockade. Baboons received artery patch grafts from GTKO pigs, with no (Group1), anti-CD154mAb-based (Group2), or CTLA4-Ig-based (Group3) immunosuppressive therapy. Anti-pig IgM and IgG antibody and cellular responses were monitored. Xenografts were immunohistologically evaluated for antibody and complement deposition, and cellular infiltration. Group1 baboons developed increased IgM and IgG antibody and cellular responses against GTKO antigens. In Group2, anti-CD154mAb alone prevented the development of both IgM and IgG antibody and cellular responses,but not cellular infiltration of the graft. In the single baboon that received anti-thymocyte globulin (ATG) + mycophenolate mofetil (MMF) + anti-CD154mAb, cellular infiltration of the graft was not seen. In Group3, CTLA4-Ig with ATG + MMF inhibited the cellular proliferative response to pig antigens but did not prevent the IgG response or cellular infiltration. (i) Artery patch transplantation is a simple model to monitor the adaptive immune response to xenografts; (ii) anti-CD154mAb prevents sensitization but not cellular infiltration (but, without anticoagulation, may result in early thrombosis of a pig xenograft); (iii) although in only one baboon, the addition of ATG and MMF prevents cellular infiltration and (iv) replacement of anti-CD154mAb by CTLA4-Ig (at the doses used), even in combination with ATG and MMF, prevents the cellular proliferative response to GTKO pig antigens but is insufficient to prevent the development of anti-pig antibodies.

Soluble Galα(1,3)Gal conjugate combined with hDAF preserves morphology and improves function of cardiac xenografts

Cytotoxic anti-Galalpha(1,3)Gal antibodies play a key role in the rejection of pig organs transplanted into primates. Regimens reducing anti-Galalpha(1,3)Gal antibodies were associated with severe side effects unable to prevent antibody rebound until soluble synthetic oligosaccharides with terminal Galalpha(1,3)Gal inhibiting antigen binding became available. We displayed kinetics of anti-pig and anti-Galalpha(1,3)Gal IgM and IgG antibody levels using GAS914, a Galalpha(1,3)Gal trisaccharide conjugated to poly-l-lysine, and investigated corresponding changes of parameters of heart function. Using a working heart model, hDAF pig hearts were perfused with human blood containing GAS914 (group 1). As controls hDAF pig hearts (group 2) and landrace pig hearts (group 3) were perfused with human blood only. Levels of anti-Galalpha(1,3)Gal (IgM, IgG) and anti-pig antibodies were assessed to prove the effectiveness of GAS914. As parameters of heart function, cardiac output (CO), stroke work index (SWI), coronary blood flow (CBF) and coronary resistance were measured. Creatine phosphokinases, lactate dehydrogenase and aspartate aminotransferase were evaluated as markers of myocardial damage. Histological and immunohistochemical investigations were performed at the end of perfusion. In group 1 an immediate and extensive reduction in both IgM and IgG anti-Galalpha(1,3)Gal was found. Anti-pig antibodies were eliminated accordingly. Antibody binding to GAS914 was complete before the start of organ perfusion. Corresponding to rapid antibody elimination in group 1 GAS914 not only was able to significantly prolong the beating time of the heart in hDAF pigs, but also to clearly improve functional parameters. When switching to the working heart mode hDAF pig hearts perfused with human blood containing GAS914 (group 1) revealed a CO starting at a significantly higher level than hDAF (group 2) and non-transgenic pig hearts (group 3) perfused with human blood only. Similarly, in group 1 SWI was significantly increased at the beginning of perfusion compared to that of group 2 and group 3. The increase in CBF during perfusion and the corresponding fall of coronary resistance occurred without significant differences between the groups revealing the independence of hDAF and GAS914. Due to an immediate and profound reduction in Galalpha(1,3)Gal-specific antibodies, soluble Galalpha(1,3)Gal conjugates not only prolong survival, but also improve the hemodynamic performance of the heart in DAF pigs.

THREE MONTH IMMUNE PROTECTION OF MICROENCAPSULATED PORCINE ISLETS TRANSPLANTED IN PRIMATES WITHOUT IMMUNOSUPPRESSION: PRELIMINARY RESULTS

P1072 Aims: Survival of xenogeneic pig pancreatic islets into primates has been casuistically reported. Although the major hurdle for pig islets graft remains the control of primary non-function, acute vascular and cellular rejection, there is no uniform regimen which is able to overcome all immunological problems. The aim of this study was to assess the ability of alginate micro-encapsulation to protect islets in the gold standard discordant pig to primate model. Methods: Pancreases were obtained from adult (>2 years) Landrace pigs. All pig islets isolations were performed with Liberase PI following a static digestion method. Pig islets encapsulation with alginate solution (1% w/v), was carried out by vibrating nozzle and cross-linked with calcium ions (100mM). The mean size of capsules was 600-800μm and contained an average of three pig islets. After microencapsulation, pig islets (15,000 islets equivalent/kg) were transplanted under the capsule of both kidneys of non-diabetic Cynomolgus (n=2) and streptozotocin-induced diabetic primates (n=2). Porcine C-peptide and anti-pig IgM and IgG antibodies were assessed in serum prior and up to 90 days after transplantation by radioimmunoassay and flow cytometry, respectively. As controls, two animals received either non-encapsulated pig islets or empty capsules. Primate kidneys were removed after 30, 60 and 90 days post-transplantation, fixed in Bouin solution and immunostained for insulin. Viability of islets was assessed by Trypan blue and dithyzone staining. Results: The first non-diabetic animal underwent a total pancreatectomy 30 days after graft and serum porcine C-peptide was detected at 0.121ng/ml thereby demonstrating that islets could still produce C-peptide even without immunosuppression. This animal unfortunately died three days after surgery for unknown reasons. The two kidneys of the second primate were removed 60 and 90 days after graft. The alginate capsules were intact and a majority of islets were positive for dithyzone coloration and insulin immunostaining as well as negative by trypan blue. To induce diabetes in the third animal, high dose of STZ (130mg/kg) was used. After encapsulated pig islet graft, the non-fasting blood glucose decreased from 600 to 214 mg/dl day 2 after transplantation and the porcine C-peptide level went from 0 to 3.6 ng/ml. Unfortunately, this animal died on post-graft day 3 due toxic STZ side-effects (liver steatosis and acute tubular necrosis (creatinine 9mg/dl)). In the fourth primate, diabetes was induced with a lower STZ dose (55mg/kg). The kidneys were removed 30 and 60 days after graft. The alginate capsules were intact and insulin and dithyzone positive cells could be evidenced. One additional animal received empty capsules and the kidney removed 146 days after transplantation demonstrated the integrity of the capsules. The animal which received non-encapsulated pig islets demonstrated 10 days after the graft a complete destruction of the cells, a cellular infiltration and major fibrosis. In all these animals, but one which received empty capsules, there was a strong anti-porcine IgM and IgG antibody response thereby demonstrating the xenogeneic sensitization. Conclusions: A calcium-alginate microencapsulation is able to protect, for at least 90 days, pig islets again a strong discordant humoral immune response without any immunosuppression. Functional beta-cells were present after one month in two animals since swine C-peptide could be detected. This method must be confirmed in additional diabetic primates.

Removal of bowel aerobic gram-negative bacteria is more effective than immunosuppression with cyclophosphamide and steroids to decrease natural alpha-galactosyl IgG antibodies.

Natural alpha-Galactosyl (Gal) antibodies play an important role in the rejection of pig xenografts by humans and Old World monkeys. In this study we investigate the efficacy of two different strategies to reduce the serum level of natural anti-Gal antibodies. On the one hand, removal of aerobic gram-negative bacteria from the intestinal flora, because anti-Gal antibodies appear to be produced as a result of the continuous sensitization by these microorganisms. On the other hand, we studied the effect on these antibodies of an immunosuppressive regimen of cyclophosphamide and steroids. Ten baboons were treated for three months with norfloxacin (Nor Group; n=6) or cyclophosphamide and steroids (CyP Group; n=4). A further four baboons did not receive any treatment (Control Group). Aerobic gram-negative bacteria became negative in stools of the Nor Group after two weeks of treatment, and remained undetectable until week 7. Thereafter, a gradual increase on the fecal concentration of aerobic gram-negative bacteria was observed despite the norfloxacin treatment. The mean anti-Gal IgG in the Nor Group gradually declined from week 4 to 9 to a mean of 62.7 +/- 18% of the baseline level, and during this period were significantly lower than in the CyP (P<0.02) and the Control (P<0.05) groups. No differences were observed between the three groups during the 16 weeks of follow-up in serum levels of anti-Gal IgM, hemolytic anti-pig antibodies, total IgG, IgM and IgA. In conclusion, removal of normal aerobic gram-negative bacteria from the intestinal flora is more effective than immunosuppression with CyP and steroids in reducing the level of natural anti-Gal antibodies, although there is no discernible effect on IgM antibodies.

Specific depletion of preformed IgM natural antibodies by administration of anti-mu monoclonal antibody suppresses hyperacute rejection of pig to baboon renal xenografts.

The elimination of circulating anti-porcine preformed antibodies is crucial for avoiding hyperacute vascular rejection (HAVR) of primarily vascularized xenograft in discordant pig to baboon model. Previously described methods used for eliminating natural antibodies, however, constantly removed both anti-porcine IgM and IgG antibodies, as well as often complement proteins. To study specifically the role of preformed anti-porcine IgM antibodies, a specific anti-IgM monoclonal antibody (mAb) has been designed and evaluated in vivo. Iterative injections of anti-IgM mAb (LO-BM2) at high dose (20 mg/kg) depleted to undetectable level the circulating IgM and therefore anti-porcine IgM antibodies but did not change the concentration of anti-pig IgG antibodies. The serum concentration of IgM and IgG antibodies was assessed by ELISA and the level of anti-pig natural IgM and IgG antibodies by flow cytometry (FC). Anti-rat sensitization was assessed by specific ELISA as well as the serum concentration of LO-BM2. Iterative injections of LO-BM2 allowed to specifically eliminate the anti-porcine IgM antibodies to undetectable levels at ELISA. Despite a normal serum level of anti-porcine IgG and complement proteins, HAVR was avoided. Without immunosuppression, the specific elimination of preformed anti-porcine IgM prolonged the survival of a renal xenograft in baboon up to 6 days, whereas without IgM antibody elimination, the renal xenografts were hyperacutely rejected within hours. The lost of activity of LO-BM2 after 10 days was concomitant to an IgM and IgG antibody rebound, which caused an acute vascular rejection of the xenograft. Specific elimination of natural anti-porcine IgM antibodies allows to avoid HAVR of a pig to baboon renal xenograft, whereas anti-porcine IgG antibodies and complement proteins were present in the serum. This result confirms previous in vitro reports and demonstrates for the first time in vivo that preformed IgM antibodies alone are responsible for HAVR, while preformed anti-porcine IgG antibodies are unable alone to cause HAVR. Anti-IgM therapy appears as an important tool to transiently but completely eliminates xeno-IgM antibodies in vivo.

Comparative study of antibody removal before pig-to-baboon and human ABO-incompatible renal transplantation

THE MECHANISM of rejection following pig-to-baboon xenotransplantation (XenoTx) has been compared with that following ABO-incompatible allotransplantation (ABO-Tx), because both involve natural antibody (Ab) against carbohydrate epitopes. Recently, ABO-Tx has shown excellent outcomes following pretreatment with double filtration plasmapheresis (DFPP). We compared changes in anti-donor Ab and histopathology of the renal graft between ABO-Tx and XenoTx. Before renal transplantation (Tx), DFPP was performed on days 26, 24, 22, and 21 in ABO-Tx (n 5 25) and on days 22 and 0 (immediately before reperfusion) in XenoTx (n 5 4). Cyclosporine (or tacrolimus), steroid, and cyclophosphamide were administered to both patients and baboons. Baboons were divided into two groups according to the extent of Ab removal: XenoTx Group I, Ab removal to the same extent as in ABO-Tx (n 5 2); XenoTx Group II, nearly total removal of Ab (n 5 2). Anti-A/B and anti-pig IgM (IgG) Abs were measured by flow cytometry. Twenty-five patients underwent ABO-Tx. All grafts remain functioning, except in two cases where the grafts were lost from recurrence of the original disease after 6 months and from chronic rejection after 5 years, although 3 had reversible humoral rejection. In pig-to-baboon renal transplantation without DFPP pretreatment, hyperacute rejection (HAR) was observed 60 and 90 minutes after transplantation. In XenoTx Group I, no HAR was observed. Grafts were rejected on days 6 and 7, and showed coagulative necrosis caused by vascular rejection; biopsies on days 1, 4, and 5 showed only minimal changes. In XenoTx Group II, the baboons could not tolerate the extra DFPP and died 4 hours and 2 days after transplantation. Immunohistochemical study of the grafts showed IgM, IgG, and C3 binding to endothelial cells within 1 hour post-Tx in XenoTx Group I, and IgM and C3 binding as early as 1 hour post-Tx in XenoTx Group II, while no Ab binding was detected at any time (1 hour to 2 years) after ABO-Tx. The same extent of Ab removal in XenoTx Group I as in ABO-Tx inhibited HAR but could not prevent delayed vascular rejection. Even when anti-pig Ab was depleted by 98% (XenoTx Group II), the small amount of remaining Ab still had the ability of binding to the pig graft within 1 hour. For successful xenografting, we suggest that the concentration of xenoantigen epitopes expressed on pig grafts may need to be reduced by genetic engineering to the level of the ABO antigens on human kidneys.

Comparative study of theefficacy of removal of anti-ABO and anti-gal antibodies by double filtration plasmapheresis.

Successful clinical ABO-incompatible renal transplantation has been achieved by the removal of anti-A or anti-B antibodies using double filtration plasmapheresis (DFPP). We have compared changes in the levels of anti-donor antibodies and the histopathology of the renal grafts following human ABO-incompatible allotransplantation and pig-to-baboon xenotransplantation using pretransplant DFPP. DFPP was performed on days 6, -4, -2 and -1 before the ABO-incompatible transplants (n=25) and on days -2 and 0 (immediately before reperfusion) in the xenotransplants (n=4). In two baboons (XenoTx Group I) the extent of antibody removal was comparable to that in the ABO-incompatible patients, and an even greater level of removal was achieved in another two baboons (XenoTx Group II). Anti-A and anti-B and anti-pig IgM and IgG antibodies were measured by flow cytometry. All clinical ABO-incompatible renal grafts are functioning, except two which were lost from recurrence of the original disease or from chronic rejection. Three other grafts underwent humoral rejection episodes, which were successfully treated. DFPP reduced the mean anti-A/B IgM and IgG antibody levels to 8% and 13% of pretreatment levels, respectively. After kidney transplantation, they were maintained at 37% and 46% of pre-DFPP level. No antibody binding to the transplanted kidney was detected at any time (1 h to 2 yr) after ABO-incompatible allotransplantation. In contrast, in XenoTx Group I, the same extent of antibody removal (90%) prevented hyperacute rejection, but the two grafts were rejected on days 6 and 7, respectively, from acute vascular rejection. In XenoTx Group II, the additional DFPP that was required to deplete the remaining 10% of anti-pig antibody was poorly tolerated and the two baboons died 4 h and 2 days, respectively, after renal transplantation. Although anti-pig IgM antibodies were reduced to 2% of pre-treatment level, IgM and C3 binding were detected in the graft as early as 1 h posttransplantation. These data suggest that the concentration of xeno-antigen epitopes expressed on pig organs may need to be reduced by genetic engineering to the much lower level of blood group A/B antigens on human kidneys if discordant xenotransplantation is to be successful.

Long-term discordant xenogeneic (porcine-to-primate) bone marrow engraftment in a monkey treated with porcine-specific growth factors.

Mixed allogeneic hematopoietic chimerism has previously been reliably achieved and shown to induce tolerance to fully MHC-mismatched allografts in mice and monkeys. However, the establishment of hematopoietic chimerism has been difficult to achieve in the discordant pig-to-primate xenogeneic model. To address this issue, two cynomolgus monkeys were conditioned by whole body irradiation (total dose 300 cGy) 6 and 5 days before the infusion of pig bone marrow (BM). Monkey anti-pig natural antibodies were immunoadsorbed by extracorporeal perfusion of monkey blood through a pig liver, immediately before the intravenous infusion of porcine BM (day 0). Cyclosporine was administered for 4 weeks and 15-deoxyspergualin for 2 weeks. One monkey received recombinant pig cytokines (stem cell factor and interleukin 3) for 2 weeks, whereas the other received only saline as a control. Both monkeys recovered from pancytopenia within 4 weeks of whole body irradiation. Anti-pig IgM and IgG antibodies were successfully depleted by the liver perfusion but returned to pretreatment levels within 12-14 days. Methylcellulose colony assays at days 180 and 300 revealed that about 2% of the myeloid progenitors in the BM of the cytokine-treated recipient were of pig origin, whereas no chimerism was detected in the BM of the untreated control monkey at similar times. The chimeric animal was less responsive by mixed lymphocyte reaction to pig-specific stimulators than the control monkey and significantly hyporesponsive when compared with a monkey that had rejected a porcine kidney transplant. To our knowledge, this is the first report of long-term survival of discordant xenogeneic BM in a primate recipient.