Development of a consensus protocol to quantify primate anti‐non‐<scp>G</scp>al xenoreactive antibodies using pig aortic endothelial cells

Azimzadeh, Agnes M.; Byrne, Guerard W.; Ezzelarab, Mohamed; Welty, Emily; Braileanu, Gheorghe; Cheng, Xiangfei; Robson, Simon C.; McGregor, Christopher G.A.; Cooper, D. K. C.; Pierson, Richard N.

doi:10.1111/xen.12125

Cited by 22 publications

(21 citation statements)

References 59 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Serum incubation for 30min has been the most common time used 3,10–12,14–37 . A uniform method to measure natural and induced antibody responses to nonGal epitopes was recommended in 2014 11 . In this study, differences in serum concentration affected antibody binding to nonGal antigens, which we have confirmed in our present study.…”

Section: Discussionmentioning

confidence: 99%

The impact of serum incubation time on IgM/IgG binding to porcine aortic endothelial cells

Zhang

Gao

Zhao

et al. 2017

Xenotransplantation

Self Cite

View full text Add to dashboard Cite

Background The results of the assay for measuring anti-nonGal antibodies (which affect pig xenograft survival) in recipients is important. Serum incubation time and concentration may be important factors in the extent of antibody binding to the graft. The aim of this in vitro study was to determine the optimal incubation time and serum concentration for measuring anti-nonGal antibody binding to porcine aortic endothelial cells (pAECs). Materials and Methods Pooled human, naive and sensitized baboon sera were incubated with wild-type, α1,3-galactosyltransferase gene-knockout (GTKO), and GTKO/human CD55 pAECs. IgM/IgG binding to pAECs after varying serum incubation times (0.5, 1, 2, 3h) and concentrations (5, 10, 20, 40μL) was determined by flow cytometry. Results An increase in incubation time from 30min to 2h was associated with increases in anti-nonGal IgM/IgG binding to GTKO and GTKO/hCD55 pAECs of pooled human, naive and sensitized baboon sera (P<0.05). Pooled human serum showed a significant increase in anti-nonGal IgM (x1.5), and a minimal increase in anti-nonGal IgG antibody binding. IgM/IgG binding of sensitized baboon serum to GTKO pAECs after 2h incubation was ×1.5 and ×2 greater than after 30min incubation, respectively whereas naïve baboon sera showed minimal (non-significant) increase in anti-nonGal IgM/IgG antibody binding. With 2h incubation, increasing the serum concentration from 5μL to 20μL significantly increased antibody binding to nonGal antigens in pooled human and sensitized baboon serum. With naïve baboon serum, only IgG was significantly increased. Conclusions Increasing the serum incubation time contributed to improve the sensitivity of detecting anti-nonGal antibodies, without affecting cell viability in vitro.

show abstract

Section: Discussionmentioning

confidence: 99%

The impact of serum incubation time on IgM/IgG binding to porcine aortic endothelial cells

Zhang

Gao

Zhao

et al. 2017

Xenotransplantation

Self Cite

View full text Add to dashboard Cite

show abstract

“…(F) Macrophage infiltration and (G) natural killer (NK) cell infiltration were both significantly higher in GTKO NPIs than in AIs (P = .02 and P = .05, respectively) costimulation blockade generated a xenospecificIgM response, which was not prevented by our experimental regimen. Indeed, non-Gal natural antibodies exist,[43][44][45] and our findings may point to the need for immunosuppressive interventions addressing the initial immune activation and additional genetic modifications of porcine islets to minimize natural IgM binding to non-Gal xenoantigens. Screening for these antibodies and selecting…”

mentioning

confidence: 80%

Early barriers to neonatal porcine islet engraftment in a dual transplant model

Samy

Davis

Gao

et al. 2018

American Journal of Transplantation

View full text Add to dashboard Cite

Porcine islet xenografts have the potential to provide an inexhaustible source of islets for β cell replacement. Proof-of-concept has been established in nonhuman primates. However, significant barriers to xenoislet transplantation remain, including the poorly understood instant blood-mediated inflammatory reaction and a thorough understanding of early xeno-specific immune responses. A paucity of data exist comparing xeno-specific immune responses with alloislet (AI) responses in primates. We recently developed a dual islet transplant model, which enables direct histologic comparison of early engraftment immunobiology. In this study, we investigate early immune responses to neonatal porcine islet (NPI) xenografts compared with rhesus islet allografts at 1 hour, 24 hours, and 7 days. Within the first 24 hours after intraportal infusion, we identified greater apoptosis (caspase 3 activity and TUNEL [terminal deoxynucleotidyl transferase dUTP nick end labeling])-positive cells) of NPIs compared with AIs. Macrophage infiltration was significantly greater at 24 hours compared with 1 hour in both NPI (wild-type) and AIs. At 7 days, IgM and macrophages were highly specific for NPIs (α1,3-galactosyltransferase knockout) compared with AIs. These findings demonstrate an augmented macrophage and antibody response toward xenografts compared with allografts. These data may inform future immune or genetic manipulations required to improve xenoislet engraftment.

show abstract

“…Anti‐non‐Gal IgM and IgG levels were monitored pre‐transplant and then at twice‐weekly intervals post‐transplantation by flow cytometry using donor pig aortic endothelial cells, as reported previously . Pooled human sera were used to indicate “control” levels.…”

Section: Methodsmentioning

confidence: 99%

Early graft failure of GalTKO pig organs in baboons is reduced by expression of a human complement pathway‐regulatory protein

Azimzadeh

Kelishadi

Ezzelarab

et al. 2015

Xenotransplantation

Self Cite

View full text Add to dashboard Cite

Background We describe the incidence of early graft failure (EGF, defined as loss of function from any cause within 3 days after transplant) in a large cohort of GalTKO pig organs transplanted into baboons in three centers, and the effect of additional expression of a human complement pathway-regulatory protein, CD46 or CD55 (GalTKO.hCPRP). Methods Baboon recipients of life-supporting GalTKO kidney (n = 7) or heterotopic heart (n = 14) grafts received either no immunosuppression (n = 4), or one of several partial or full immunosuppressive regimens (n = 17). Fourteen additional baboons received a GalTKO.hCPRP kidney (n = 5) or heart (n = 9) and similar treatment regimens. Immunologic, pathologic, and coagulation parameters were measured at frequent intervals. Results EGF of GalTKO organs occurred in 9/21 baboons (43%). hCPRP expression reduced the GalTKO EGF incidence to 7% (1/14; p<0.01 vs GalTKO alone). At 30 min, complement deposits were more intense in organs in which EGF developed (p<0.005). The intensity of peri-transplant platelet activation (as β-thromboglobulin release) correlated with EGF, as did the cumulative coagulation score (p<0.01). Conclusions We conclude that (i) the transgenic expression of a hCPRP on the vascular endothelium of a GalTKO pig reduces the incidence of EGF, and reduces complement deposition, (ii) complement deposition and platelet activation correlate with early GalTKO organ failure, and (iii) the expression of a hCPRP reduces EGF but does not prevent systemic coagulation activation. Additional strategies will be required to control coagulation activation.

show abstract

Development of a consensus protocol to quantify primate anti‐non‐Gal xenoreactive antibodies using pig aortic endothelial cells

Cited by 22 publications

References 59 publications

The impact of serum incubation time on IgM/IgG binding to porcine aortic endothelial cells

The impact of serum incubation time on IgM/IgG binding to porcine aortic endothelial cells

Early barriers to neonatal porcine islet engraftment in a dual transplant model

Early graft failure of GalTKO pig organs in baboons is reduced by expression of a human complement pathway‐regulatory protein

Contact Info

Product

Resources

About