Haruko Ogawa

Potential value of human thrombomodulin and DAF expression for coagulation control in pig‐to‐human xenotransplantation

Miwa Y, Yamamoto K, Onishi A, Iwamoto M, Yazaki S, Haneda M, Iwasaki K, Liu D, Ogawa H, Nagasaka T, Uchida K, Nakao A, Kadomatsu K, Kobayashi T. Potential value of human thrombomodulin and DAF expression for coagulation control in pig‐to‐human xenotransplantation. Xenotransplantation 2010; 17: 26–37.

Antibody-mediated accommodation of heart grafts expressing an incompatible carbohydrate antigen.

Background. Accommodation in patients transplanted with ABO incompatible allografts describes a state in which antibodies are produced against the incompatible blood group carbohydrate antigen; however, the graft is not rejected. The present study describes an experimental model for antibody-mediated accommodation of organs expressing incompatible carbohydrate antigens. Methods. The model includes &agr;1,3galactosyltransferase knockout mice that lack the &agr;-gal epitope (Gal&agr;1–3Gal&bgr;1–4GlcNAc-R), transplanted heterotopically with wild-type (WT) hearts expressing this epitope. The mice are irradiated and receive memory anti-Gal B cells by adoptive transfer. Immunization of these mice with pig-kidney membranes induces the production of large amounts of anti-Gal, which binds specifically to &agr;-gal epitopes. Results. Under the described accommodation protocol, transplanted mice produce anti-Gal that binds to &agr;-gal epitopes on endothelial cells of the grafted WT heart; however, the WT hearts continued to function for months. Second WT hearts transplanted into accommodating, anti-Gal producing mice, were not rejected. Anti-Gal in accommodating mice was not cytolytic, whereas anti-Gal in rejecting mice readily induced complement-mediated lysis of cells expressing &agr;-gal epitopes. In addition, accommodating mice displayed a preferential increase in the anti-Gal immunoglobulin (Ig)G2b subclass. Conclusions. The immune system may be manipulated to accommodate grafts expressing incompatible carbohydrate antigens by preferential production of noncytolytic anticarbohydrate antibodies.

Elimination of anti-Gal B cells by alpha-Gal ricin1.

BACKGROUND A major barrier in pig to human organ transplantation is the binding of human anti-Gal to alpha-gal epitopes (Gal alpha 1-3Gal beta 1-4GlcNAc-R) on pig cells, resulting in hyperacute and acute vascular rejection of pig xenografts. Moreover, the immune system in xenograft recipients is activated by these epitopes to produce high affinity anti-Gal, which is also detrimental to xenografts. Production of anti-Gal can be prevented by specific elimination of anti-Gal B cells. This was achieved with the toxin ricin A, coupled to human alpha1-acid glycoprotein modified to carry alpha-gal epitopes. This complex, designated alpha-gal ricin, is targeted in vivo to anti-Gal B cells by interaction with the immunoglobulin molecules (i.e., B cell receptors) on these cells. METHODS Carbohydrate chains on alpha 1-acid glycoprotein were converted to carry alpha-gal epitopes by enzymatic treatment with recombinant alpha 1,3 galactosyltransferase (alpha 1,3GT). This molecule and ricin A were biotinylated and coupled by avidin to generate alpha-gal ricin. The efficacy of alpha-gal ricin in eliminating anti-Gal B cells was studied in the experimental model of alpha 1,3GT knockout (KO) mice. These mice produce large amounts of anti-Gal immunoglobulin G when immunized with pig kidney membranes, as measured by ELISA with alpha-gal epitopes linked to bovine serum albumin (BSA). In the absence of anti-Gal B cells, these mice lack the ability to produce anti-Gal. RESULTS Repeated administration of alpha-gal ricin into alpha1,3GT KO mice resulted in elimination of anti-Gal B cells, thereby preventing production of anti-Gal immunoglobulin G after immunization with pig kidney membranes. This prevention of anti-Gal production occurred with doses of alpha-gal ricin that were not toxic to the mice and did not affect production of antibodies with other specificities. CONCLUSIONS Administration of alpha-gal ricin results in specific elimination of anti-Gal B cells in alpha 1,3GT KO mice. The elimination of these B cells may prove to be helpful in attempts to achieve immune tolerance to alpha-gal epitopes in primates.

Mechanism of the antiviral effect of hydroxytyrosol on influenza virus appears to involve morphological change of the virus.

Hydroxytyrosol (HT), a small-molecule phenolic compound, inactivated influenza A viruses including H1N1, H3N2, H5N1, and H9N2 subtypes. HT also inactivated Newcastle disease virus but not bovine rotavirus, and fowl adenovirus, suggesting that the mechanism of the antiviral effect of HT might require the presence of a viral envelope. Pretreatment of MDCK cells with HT did not affect the propagation of H9N2 virus subsequently inoculated onto the cells, implying that HT targets the virus but not the host cell. H9N2 virus inactivated with HT retained unaltered hemagglutinating activity and bound to MDCK cells in a manner similar to untreated virus. Neuraminidase activity in the HT-treated virus also remained unchanged. However, in the cells inoculated with HT-inactivated H9N2 virus, neither viral mRNA nor viral protein was detected. Electron microscopic analysis revealed morphological abnormalities in the HT-treated H9N2 virus. Most structures found in the HT-treated virus were atypical of influenza virions, and localization of hemagglutinin was not necessarily confined on the virion surface. These observations suggest that the structure of H9N2 virus could be disrupted by HT.

Comparative study on signal transduction in endothelial cells after anti-a/b and human leukocyte antigen antibody reaction: implication of accommodation.

Background. Recent development of immunosuppressive therapy has provided a platform for clinical human leukocyte antigen (HLA)- and ABO-incompatible kidney transplantation. However, the prognosis seems to be different between the two. Accommodation, the condition of no injury even in the presence of antidonor antibody, is one of the key factors for successful transplantation with antidonor antibody. The purpose of this study was to compare signal transduction between anti-A/B and anti-HLA antibody reaction and to elucidate the mechanisms underlying accommodation. Methods. Blood type A- or B-transferase gene was transfected into human EA.hy926 endothelial cells. After cell sorting, A- or B-expressing cells at high levels were obtained. The effects of anti-HLA and anti-A/B antibody binding on complement-mediated cytotoxicity and signal transduction were examined. Results. Preincubation with anti-HLA antibodies only at low levels (<10% of saturation level) or anti-A/B antibodies at high levels (even at near saturation levels) for 24 hr resulted in resistance to complement-mediated cytotoxicity. Anti-A/B antibody ligation inactivated ERK1/2 pathway and increased complement regulatory proteins such as CD55 and CD59, whereas anti-HLA ligation activated PI3K/AKT pathway and increased cytoprotective genes such as hemeoxygenase-1 and ferritin H. Conclusion. Complement inhibition by upregulation of CD55 and CD59 through ERK1/2 inactivation might play a substantial role in accommodation after ABO-incompatible transplantation, which could also explain the intriguing finding of C4d deposition in the graft without rejection.

In vivo targeting of vaccinating tumor cells to antigen-presenting cells by a gene therapy method with adenovirus containing the |[alpha]|1,3galactosyltransferase gene

Poor uptake by antigen-presenting cells (APC) is a major reason for low immunogenicity of autologous tumor vaccines. This immunogenicity may be increased by exploiting the natural anti-Gal antibody that is present in humans as ∼1% of circulating IgG. Anti-Gal binds to α-gal epitopes (Galα1-3Galβ1-4GlcNAc-R) on vaccinating tumor cells and opsonizes them for effective uptake by APC. This epitope is synthesized in human tumor cells by transduction with AdαGT- a replication deficient adenovirus containing the α1,3galactosyltransferase (α1,3GT) gene. Protection against tumors by immunization with AdαGT-transduced tumor cells was studied in α1,3GT knockout (KO) mice, challenged with the highly tumorigenic BL6 melanoma cells. These mice lack α-gal epitopes and can produce anti-Gal. Immunization of KO mice with AdαGT-transduced BL6 cells protects many of the mice against challenge with live BL6 cells lacking α-gal epitopes. Immunization with AdαGT transduced autologous tumor cells may serve as adjuvant immunotherapy delivered after completion of standard therapy. This method may complement another gene therapy method in which GM-CSF-secreting vaccinating tumor cells recruit APC to vaccination sites. Anti-Gal-opsonized vaccinating tumor cells will be effectively internalized by GM-CSF recruited APC and transported to draining lymph nodes for processing and presentation of tumor antigens. Alternatively, injection of AdαGT directly into solid tumor masses of cancer patients may result in anti-Gal-mediated destruction of the transduced tumor cells in a manner similar to xenograft rejection. The subsequent uptake of anti-Gal-opsonized tumor membranes by APC results in their effective transportation to lymph nodes where processed tumor antigens may elicit a protective antitumor immune response.

Mouse-heart grafts expressing an incompatible carbohydrate antigen. II. Transition from accommodation to tolerance

Background. Immune response to incompatible ABO antigens on allografts may result in rejection, accommodation, or immune tolerance. Our objective has been to develop a model for studying these three types of immune response to incompatible carbohydrate antigen in &agr;1,3galactosyltransferase knockout (KO) mice. KO mice lack the &agr;-gal epitope and can produce the anti-Gal antibody against it after immunization with pig kidney membranes (PKM) that express this epitope. Methods. KO mice were transplanted with syngeneic wild-type (WT) heart expressing &agr;-gal epitopes. Subsequently, the mice were lethally irradiated and received lymphocytes including memory anti-Gal B cells from PKM immunized KO mice. Immune response to incompatible &agr;-gal epitopes on the graft was determined by transplanted-heart function and by production of anti-Gal after PKM immunizations. Results. Anti-Gal B cells exposed for 1 to 2 weeks to &agr;-gal epitopes of WT hearts differentiate into cells producing noncytolytic accommodating antibodies. Exposure for longer periods (2–4 weeks) induces a transition from accommodation into tolerance, indicated by the inability of mice to produce anti-Gal antibodies despite repeated PKM immunizations. WT hearts in accommodating and in tolerized mice continue to function for months. Conclusions. In the absence of T-cell help, anticarbohydrate B cells exposed to incompatible carbohydrate antigens of transplanted organs differentiate first into cells capable of producing accommodating antibodies, but, after prolonged exposure, these B cells gradually become tolerized. These findings suggest that prolonged T-cell suppression in recipients of ABO-incompatible allografts may result in a similar induction of tolerance to incompatible blood-group antigens.

Inactivation of high and low pathogenic avian influenza virus H5 subtypes by copper ions incorporated in zeolite-textile materials

The effect of cotton textiles containing Cu(2+) held by zeolites (CuZeo-textile) on the inactivation of H5 subtype viruses was examined. Allantoic fluid (AF) containing a virus (AF virus) (0.1 ml) was applied to the textile (3×3-cm), and incubated for a specific period at ambient temperature. After each incubation, 0.9 ml of culture medium was added followed by squeezing to recover the virus into the medium. The recovered virus was titrated using Madin-Darby canine kidney (MDCK) cells or 10-day-old embryonated chicken eggs. The highly pathogenic H5N1 and the low pathogenic H5N3 viruses were inactivated on the CuZeo-textile, even after short incubation. The titer of A/chicken/Yamaguchi/7/04 (H5N1) in MDCK cells and in eggs declined by >5.0 log(10) and 5.0 log(10), respectively, in 30 s. The titer of A/whooper swan/Hokkaido/1/08 (H5N1) in MDCK cells declined by 2.3 and 3.5 in 1 and 5 min, respectively. When A/whistling swan/Shimane/499/83 (H5N3) was treated on the CuZeo-textile for 10 min, the titer declined by >5.0 log(10) in MDCK cells and by >3.5 log(10) in eggs. In contrast, no decrease in the titers was observed on cotton textiles containing zeolites alone (Zeo-textile). Neither cytopathic effects nor NP antigens were detected in MDCK cells inoculated with the H5N1 virus treated on the CuZeo-textile. The viral genes (H5, N1, M, and NP) were amplified from the virus treated on the CuZeo-textile by RT-PCR. The hemagglutinating activity of the CuZeo-textile treated virus was unaffected, indicating that virus-receptor interactions were maintained. Electron microscopic analysis revealed a small number of particles with morphological abnormalities in the H5N3 virus samples recovered immediately from the CuZeo-textile, while no particles were detectable in the 10-min treated sample, suggesting the rapid destruction of virions by the Cu(2+) in the CuZeo-textile. The loss of infectivity of H5 viruses could, therefore, be due to the destruction of virions by Cu(2+). Interestingly, CuCl(2) treatment (500 and 5000 μM) did not have an antiviral effect on the AF viruses (H5N1 and H5N3) even after 48 h of incubation, although the titer of the purified H5N3 virus treated with CuCl(2) declined greatly. The antiviral effect was inhibited by adding the AF to the purified H5N3 virus prior to the CuCl(2) treatment. The known antibacterial/antifungal activities of copper suggest that the CuZeo-textile can be applied at a high level of hygiene in both animals and humans.

Genetic diversity and mutation of avian paramyxovirus serotype 1 (Newcastle disease virus) in wild birds and evidence for intercontinental spread.

Avian paramyxovirus serotype 1 (APMV-1), or Newcastle disease virus, is the causative agent of Newcastle disease, one of the most economically important diseases for poultry production worldwide and a cause of periodic epizootics in wild birds in North America. In this study, we examined the genetic diversity of APMV-1 isolated from migratory birds sampled in Alaska, Japan, and Russia and assessed the evidence for intercontinental virus spread using phylogenetic methods. Additionally, we predicted viral virulence using deduced amino acid residues for the fusion protein cleavage site and estimated mutation rates for the fusion gene of class I and class II migratory bird isolates. All 73 isolates sequenced as part of this study were most closely related to virus genotypes previously reported for wild birds; however, five class II genotype I isolates formed a monophyletic clade exhibiting previously unreported genetic diversity, which met criteria for the designation of a new sub-genotype. Phylogenetic analysis of wild-bird isolates provided evidence for intercontinental virus spread, specifically viral lineages of APMV-1 class II genotype I sub-genotypes Ib and Ic. This result supports migratory bird movement as a possible mechanism for the redistribution of APMV-1. None of the predicted deduced amino acid motifs for the fusion protein cleavage site of APMV-1 strains isolated from migratory birds in Alaska, Japan, and Russia were consistent with those of previously identified virulent viruses. These data therefore provide no support for these strains contributing to the emergence of avian pathogens. The estimated mutation rates for fusion genes of class I and class II wild-bird isolates were faster than those reported previously for non-virulent APMV-1 strains. Collectively, these findings provide new insight into the diversity, spread, and evolution of APMV-1 in wild birds.

Production of cloned pigs expressing human thrombomodulin in endothelial cells.

Yazaki S, Iwamoto M, Onishi A, Miwa Y, Hashimoto M, Oishi T, Suzuki S, Fuchimoto D‐I, Sembon S, Furusawa T, Liu DG, Nagasaka T, Kuzuya T, Ogawa H, Yamamoto K, Iwasaki K, Haneda M, Maruyama S, Kobayashi T. Production of cloned pigs expressing human thrombomodulin in endothelial cells. Xenotransplantation 2012; 19: 82–91.