Anti-C1 domain antibodies that accelerate factor VIII clearance contribute to antibody pathogenicity in a murine hemophilia A model.

Essentials Inhibitor formation remains a challenging complication of hemophilia A care. The Bethesda assay is the primary method used for determining bleeding risk and management. Antibodies that block factor VIII binding to von Willebrand factor can increase FVIII clearance. Antibodies that increase clearance contribute to antibody pathogenicity. SUMMARY: Background The development of neutralizing anti-factor VIII (FVIII) antibodies remains a challenging complication of modern hemophilia A care. In vitro assays are the primary method used for quantifying inhibitor titers, predicting bleeding risk, and determining bleeding management. However, other mechanisms of inhibition are not accounted for in these assays, which may result in discrepancies between the inhibitor titer and clinical bleeding symptoms. Objectives To evaluate FVIII clearance in vivo as a potential mechanism for antibody pathogenicity and to determine whether increased FVIII dosing regimens correct the associated bleeding phenotype. Methods FVIII-/- or FVIII-/- /von Willebrand factor (VWF)-/- mice were infused with anti-FVIII mAbs directed against the FVIII C1, C2 or A2 domains, followed by infusion of FVIII. Blood loss via the tail snip bleeding model, FVIII activity and FVIII antigen levels were subsequently measured. Results Pathogenic anti-C1 mAbs that compete with VWF for FVIII binding increased the clearance of FVIII-mAb complexes in FVIII-/- mice but not in FVIII-/- /VWF-/- mice. Additionally, pathogenic anti-C2 mAbs that inhibit FVIII binding to VWF increased FVIII clearance in FVIII-/- mice. Anti-C1, anti-C2 and anti-A2 mAbs that do not inhibit VWF binding did not accelerate FVIII clearance. Infusion of increased doses of FVIII in the presence of anti-C1 mAbs partially corrected blood loss in FVIII-/- mice. Conclusions A subset of antibodies that inhibit VWF binding to FVIII increase the clearance of FVIII-mAb complexes, which contributes to antibody pathogenicity. This may explain differences in the bleeding phenotype observed despite factor replacement in some patients with hemophilia A and low-titer inhibitors.

The hDAF exogene protects swine endothelial and peripheral blood mononuclear cells from xenoreactive antibody mediated cytotoxicity in hDAF transgenic pigs.

Xenoreactive antibody-induced complement activation and cytotoxicity poses a major obstracle to xenograft survival in humans. Previously, we have generated transgenic pigs carrying the hDAF exogene to help overcome this problem. In this study, we examined whether the hDAF exogene in various swine cells shows an equally protective effect for the complement-mediated cytotoxicity induced by human xenoreactive antibodies. Pig peripheral blood mononuclear cells (PBMCs) and aortic endothelial cells (PAECs) were used as targets. Fresh human serum was harvested from a single healthy human donor as the source of human xenoreactive antibodies and complement. The target cells cocultured with medium containing various concentrations of human serum for 24 hours were evaluated using the 3-[4,5-dimethylthiaolyl]-2,5-diphenyl-tetrazolium bromide assay for cellular viability. We observed that xenoreactive antibody plus human complement-mediated cellular cytoxicity dose-dependently correlated with the concentration of human serum in the culture medium. As compared with the PBMCs from the normal pigs, PBMCs from hDAF transgenic pigs showed significantly better survival after treatment with human serum (P < .05). Similarly, the survival of PAECs from the hDAF transgenic pig were also significantly higher than that from normal pigs (P < .05). Our data demonstrated a protective effect from human xenoreactive antibodies and complement-mediated cytoxicity of hDAF exogenes in pig PBMCs and PAECs. These observations support the clinical value of the hDAF transgenic pig as an organ donor in xenotransplantation.

Teratogenic effects of transplacental transfusion of heterologous antisera simulated in an experimental model using in vitro whole rat embryo culture.

The effects of the transplacental transfusion of heterologous rabbit-anti-rat antiserum (RAR antiserum) and subsequent immunological interaction on the development of 9-10 days old rat embryos (stages 8-10 somites) were studied using an in vitro whole rat embryo culture. Transplacental transfusion was simulated by the embryonic intracardiac microinjection of approximately 0.5 microliter RAR antiserum, followed by an incubation period of 24 and 48 hours. All the tested embryos survived the incubation period. Embryos taken from the incubator after 24 hours showed signs of growth retardation and axial non-rotation, a delayed closure of the neural tube and ear vesicle, and a delayed formation of the foregut. They also had a moderate number of areas with local pathogenetic cell degeneration. Embryos taken from the incubator after 48 hours demonstrated signs of growth retardation and incomplete axial rotation. The formation of the foregut and closure of the neural tube was complete, with the exception of one embryo with a persisting open neuroporus posterior. All embryos displayed a considerable number of areas with local pathogenetic cell degeneration. The intracardiac injection technique is an elegant method to test the effects of teratogens administered directly into the embryonic circulation. The results demonstrate that heterologous antisera have teratogenic potential, believed to be due to an immunological reaction, with a particular sensitivity of the neurectoderm in 9-10 day old embryos.

A novel cytomedical vehicle capable of protecting cells against complement.

We have developed "Cytomedicine," which consists of functional cells entrapped in semipermeable polymer, and previously reported that APA microcapsules could protect the entrapped cells from injury by cellular immune system. However, microencapsulated cells were not protected from humoral immune system. Here, we developed a novel APA microcapsule, in which APA microbeads (APA(Ba) microbeads) were modified to contain a barium alginate hydrogel within their centers in an attempt to make it more difficult for antibody and complement to permeate the microcapsules. The permeability of APA(Ba) microbeads was clearly less than that of APA microcapsules, presumably due to the presence of barium alginate hydrogel. Cells encapsulated within APA(Ba) microbeads were protected against treatment with xenogeneic anti-serum. Furthermore, murine pancreatic beta-cells encapsulated in APA(Ba) microbeads remained viable and continued to secrete insulin in response to glucose. Therefore, APA(Ba) microbeads may be a useful carrier for developing anti-complement device for cytomedical therapy.

Venular thrombosis is the key event in the pathogenesis of antibody-mediated cardiac rejection.

A review of the histopathologic features of serial biopsies and excised grafts of 117 experimental and clinical cardiac allografts and xenografts revealed a common sequence in the development of histopathologic changes in grafts showing antibody-mediated (hyperacute and acute vascular) rejection. Based on these observations, we propose the new concept that thrombosis of cardiac veins and venules is the initial key event in antibody-mediated rejection. This is followed by the development of congestion in the subtended venules and capillaries accompanied by interfascicular and, later, intermyocyte edema. Subsequently, focal or diffuse interstitial hemorrhage affecting the subendocardium, extending sometimes to involve the inner half of the ventricular myocardium, is observed. Antibody-mediated rejection therefore appears to be analogous to incomplete venous infarction of the heart. The observed histopathology (in which venular thrombosis plays a key role) favors a thrombogenic basis for the classical features of antibody-mediated rejection, namely edema, vascular thrombi and interstitial hemorrhage. A key role for venular thrombosis would explain the non-uniform distribution of the changes and may suggest new ways of preventing antibody-mediated xenograft rejection.