Production of human CD59-transgenic pigs by embryonic germ cell nuclear transfer

This study was performed to produce transgenic pigs expressing the human complement regulatory protein CD59 (hCD59) using the nuclear transfer (NT) of embryonic germ (EG) cells, which are undifferentiated stem cells derived from primordial germ cells. Because EG cells can be cultured indefinitely in an undifferentiated state, they may provide an inexhaustible source of nuclear donor cells for NT to produce transgenic pigs. A total of 1980 NT embryos derived from hCD59-transgenic EG cells were transferred to ten recipients, resulting in the birth of fifteen piglets from three pregnancies. Among these offspring, ten were alive without overt health problems. Based on PCR analysis, all fifteen piglets were confirmed as hCD59 transgenic. The expression of the hCD59 transgene in the ten living piglets was verified by RT-PCR. Western analysis showed the expression of the hCD59 protein in four of the ten RT-PCR-positive piglets. These results demonstrate that hCD59-transgenic pigs could effectively be produced by EG cell NT and that such transgenic pigs may be used as organ donors in pig-to-human xenotransplantation.     

Porcine endogenous retrovirus transmission characteristics of galactose alpha1-3 galactose-deficient pig cells

Galactose alpha1-3 galactose (Gal) trisaccharides are present on the surface of wild-type pig cells, as well as on viruses particles produced from such cells. The recognition of Gal sugars by natural anti-Gal antibodies (NAb) in human and Old World primate serum can cause the lysis of the particles via complement-dependent mechanisms and has therefore been proposed as an important antiviral mechanism. Recently, pigs have been generated that possess disrupted galactosyl-transferase (GGTA1) genes. The cells of these pigs do not express Gal sugars on their surface, i.e., are Gal null. Concerns have been raised that the risk of virus transmission from such pigs may be increased due to the absence of the Gal sugars. We investigated the sensitivity of porcine endogenous retrovirus (PERV) produced from Gal-null and Gal-positive pig cells to inactivation by purified NAb and human serum. PERV produced in Gal-null pig cells was resistant to inactivation by either NAb or human serum. In contrast, although Gal-positive PERV particles were sensitive to inactivation by NAb and human serum, they required markedly higher concentrations of NAb for inactivation compared to the Gal-positive cells from which they were produced. Complete inactivation of Gal-positive PERV particles was not achievable despite the use of high levels of NAb, indicating that NAb-mediated inactivation of cell-free PERV particles is an inefficient process.     

Human CD59 Incorporation into Porcine Endogenous Retrovirus Particles: Implications for the Use of Transgenic Pigs for Xenotransplantation

Transgenic pigs have been engineered to express human CD59 (hCD59) in order to suppress hyperacute rejection of xenotransplants in human recipients. In this study, porcine endogenous retrovirus (PERV) was produced in a porcine cell line expressing hCD59 in order to examine the effect of this complement control protein on PERV neutralization by human sera. hCD59 was found to be incorporated into PERV particles produced from engineered ST-IOWA cells. PERV incorporation of hCD59 resulted in a dramatic inhibition of complement-mediated virolysis by human serum. However, incorporation of hCD59 had no effect on neutralization of PERV by human serum, as measured in infectivity assays. Our results suggest that the use of organs from hCD59 transgenic pigs will inhibit complement-mediated virolysis, but will not compromise the protective effects of human sera on the neutralization of PERV particles.     

Generation of GTKO <Emphasis Type="Italic">Diannan</Emphasis> Miniature Pig Expressing Human Complementary Regulator Proteins hCD55 and hCD59 via T2A Peptide-Based Bicistronic Vectors and SCNT

Pig-to-human organ transplantation has drawn attention in recent years due to the potential use of pigs as an alternative source of human donor organs. While GGTA1 knockout (GTKO) can protect xenografts from hyperacute rejection, complement-dependent cytotoxicity might still contribute to this type of rejection. To prolong the xenograft survival, we utilized a T2A-mediated pCMV-hCD55-T2A-hCD59-Neo vector and transfected the plasmid into GTKO Diannan miniature pig fetal fibroblasts. After G418 selection combined with single-cell cloning culture, four colonies were obtained, and three of these were successfully transfected with the hCD55 and hCD59. One of the three colonies was selected as donor cells for somatic cell nuclear transfer (SCNT). Then, the reconstructed embryos were transferred into eight recipient gilts, resulting in four pregnancies. Three of the pregnant gilts delivered, yielding six piglets. Only one piglet carried hCD55 and hCD59 genetic modification. The expression levels of the GGTA1, hCD55, and hCD59 in the tissues and fibroblasts of the piglet were determined by q-PCR, fluorescence microscopy, immunohistochemical staining, and western blotting analyses. The results showed the absence of GGTA1 and the coexpression of the hCD55 and hCD59. However, the mRNA expression levels of hCD55 and hCD59 in the GTKO/hCD55/hCD59 pig fibroblasts were lower than that in human 293T cells, which may be caused by low copy number and/or CMV promoter methylation. Furthermore, we performed human complement-mediated cytolysis assays using human serum solutions from 0 to 60%. The result showed that the fibroblasts of this triple-gene modified piglet had greater survival rates than that of wild-type and GTKO controls. Taken together, these results indicate that T2A-mediated polycistronic vector system combined with SCNT can effectively generate multiplex genetically modified pigs, additional hCD55 and hCD59 expression on top of a GTKO genetic background markedly enhance the protective effect towards human serum-mediated cytolysis than those of GTKO alone. Thus, we suggest that GTKO/hCD55/hCD59 triple-gene-modified Diannan miniature pig will be a more eligible donor for xenotransplantation.     

Reduction of the Gal-alpha1,3-Gal epitope of mouse endothelial cells by transfection with the N-acetylglucosaminyltransferase III gene

In order to prevent hyperacute rejection in pig-to-human xenotransplantation, it would be very useful to be able to down-regulate the Gal alpha1-3 Galbeta 1-4 GlcNAc-R (alpha-Gal epitope) in mouse and swine tissues. When the beta-D-mannoside beta-1,4-N-acetylglucosaminyl-transferase III (GnT-III) gene was introduced into mouse aorta endothelial cells (MEC) their susceptibility to complement-mediated cell lysis by normal human serum (NHS) was reduced. Expression of GnT-III also suppressed the antigenicity of MEC to human natural antibodies as shown by binding of Griffonia simplicifolia 1 isolectin (GS1B4 lectin) to the alpha-Gal epitope. Western blot analysis indicated that the reactivity of the glycoproteins of the transfectants to NHS and GSIB4 lectin was reduced to approximately the same extent. Thus GnT-III, a key enzyme involved in the formation of branched N-linked sugars, reduces the expression of xenoantigens, suggesting that this approach may be of value in clinical xenotransplantation.     

Genetic modification of pigs as organ donors for xenotransplantation

Transgenic pigs are promising donor organisms for xenotransplantation as they share many anatomical and physiological characteristics with humans. The most profound barrier to pig‐to‐primate xenotransplantation is the rejection of the grafted organ by a cascade of immune mechanisms commonly referred to as hyperacute rejection (HAR), acute humoral xenograft rejection (AHXR), immune cell‐mediated rejection, and chronic rejection. Various strategies for the genetic modification of pigs facilitate tailoring them to be donors for organ transplantation. Genetically modified pigs lacking alpha‐1,3‐Gal epitopes, the major xenoantigens triggering HAR of pig‐to‐primate xenografts, are considered to be the basis for further genetic modifications that can address other rejection mechanisms and incompatibilities between the porcine and primate blood coagulation systems. These modifications include expression of human complement regulatory proteins, CD39, endothelial protein C receptor, heme oxygenase 1, thrombomodulin, tissue factor pathway inhibitor as well as modulators of the cellular immune system such as human TNF alpha‐related apoptosis inducing ligand, HLA‐E/beta‐2‐microglobulin, and CTLA‐4Ig. In addition, transgenic strategies have been developed to reduce the potential risk of infections by endogenous porcine retroviruses. The protective efficacy of all these strategies is strictly dependent on a sufficiently high expression level of the respective factors with the required spatial distribution. This review provides an overview of the transgenic approaches that have been used to generate donor pigs for xenotransplantation, as well as their biological effects in in vitro tests and in preclinical transplantation studies. A future challenge will be to combine the most important and efficient genetic modifications in multi‐transgenic pigs for clinical xenotransplantation. Mol. Reprod. Dev. 77: 209–221, 2010. © 2009 Wiley‐Liss, Inc.     

Down-regulation of the alpha-Gal epitope expression in N-glycans of swine endothelial cells by transfection with the N-acetylglucosaminyltransferase III gene. Modulation of the biosynthesis of terminal structures by a bisecting GlcNAc.

The down-regulation of the alpha-Gal epitope (Galalpha1,3Galbeta-R) in swine tissues would be highly desirable, in terms of preventing hyperacute rejection in pig-to-human xenotransplantation. In an earlier study, we reported that the introduction of the beta1,4-N-acetylglucosaminyltransferase (GnT) III gene into swine endothelial cells resulted in a substantial reduction in the expression of the alpha-Gal epitope. In this study, we report on the mechanism for this down-regulation of the alpha-Gal epitope by means of structural and kinetic analyses. The structural analyses revealed that the amount of N-linked oligosaccharides bearing the alpha-Gal epitopes in the GnT-III-transfected cells was less than 10% that in parental cells, due to the alteration of the terminal structures as well as a decrease in branch formation. In addition, it appeared that the addition of a bisecting GlcNAc, which is catalyzed by GnT-III, leads to a more efficient sialylation rather than alpha-galactosylation. In vitro kinetic analyses showed that the bisecting GlcNAc has an inhibitory effect on alpha-galactosylation, but does not significantly affect the sialylation. These results suggest that the bisecting GlcNAc in the core is capable of modifying the biosynthesis of the terminal structures via its differential effects on the capping glycosyltransferase reactions. The findings may contribute to the development of a novel strategy to eliminate carbohydrate xenoantigens.     

Production of heterozygous alpha 1,3-galactosyltransferase (<Emphasis Type="Italic">GGTA1</Emphasis>) knock-out transgenic miniature pigs expressing human <Emphasis Type="Italic">CD39</Emphasis>

Production of transgenic pigs for use as xenotransplant donors is a solution to the severe shortage of human organs for transplantation. The first barrier to successful xenotransplantation is hyperacute rejection, a rapid, massive humoral immune response directed against the pig carbohydrate GGTA1 epitope. Platelet activation, adherence, and clumping, all major features of thrombotic microangiopathy, are inevitable results of immune-mediated transplant rejection. Human CD39 rapidly hydrolyzes ATP and ADP to AMP; AMP is hydrolyzed by ecto-5′-nucleotidase (CD73) to adenosine, an anti-thrombotic and cardiovascular protective mediator. In this study, we developed a vector-based strategy for ablation of GGTA1 function and concurrent expression of human CD39 (hCD39). An hCD39 expression cassette was constructed to target exon 4 of GGTA1. We established heterozygous GGTA1 knock-out cell lines expressing hCD39 from pig ear fibroblasts for somatic cell nuclear transfer (SCNT). We also described production of heterozygous GGTA1 knock-out piglets expressing hCD39 and analyzed expression and function of the transgene. Human CD39 was expressed in heart, kidney and aorta. Human CD39 knock-in heterozygous ear fibroblast from transgenic cloned pigs, but not in non-transgenic pig’s cells. Expression of GGTA1 gene was lower in the knock-in heterozygous ear fibroblast from transgenic pigs compared to the non-transgenic pig’s cell. The peripheral blood mononuclear cells (PBMC) from the transgenic pigs were more resistant to lysis by pooled complement-preserved normal human serum than that from wild type (WT) pig. Accordingly, GGTA1 mutated piglets expressing hCD39 will provide a new organ source for xenotransplantation research.     

Targeted disruption of the alpha1,3-galactosyltransferase gene in cloned pigs

Galactose-alpha1,3-galactose (alpha1,3Gal) is the major xenoantigen causing hyperacute rejection in pig-to-human xenotransplantation. Disruption of the gene encoding pig alpha1,3-galactosyltransferase (alpha1,3GT) by homologous recombination is a means to completely remove the alpha1,3Gal epitopes from xenografts. Here we report the disruption of one allele of the pig alpha1,3GT gene in both male and female porcine primary fetal fibroblasts. Targeting was confirmed in 17 colonies by Southern blot analysis, and 7 of them were used for nuclear transfer. Using cells from one colony, we produced six cloned female piglets, of which five were of normal weight and apparently healthy. Southern blot analysis confirmed that these five piglets contain one disrupted pig alpha1,3GT allele.     

Human recombinant IL-10 reduces xenogenic cytotoxicity via macrophage M2 polarization

Xenotransplantation has been considered an alternative to the moderate shortage of donor organs for transplantation. To achieve successful xenotransplatation, there is the need to overcome immune rejection. Although, hyperacute rejection has been overcome by α1,3-galactosyltransferase knockout pig, cellular immune rejection remains as a subsequent barrier. Interleukin-10 (IL-10) is known as an anti-inflammatory and immunomodulatory cytokine which has been shown to limit inflammatory responses by inhibiting macrophage activation in several animal experiments. To study the effect of human IL-10 (hIL-10) on pig-to-human xenotransplantation, porcine kidney epithelial cell line (PK(15)) expressing hIL-10 was established. The cytotoxicity of macrophages decreased by hIL-10 from transgenic cells. Furthermore, there is a decreased production of pro-inflammatory cytokines, tumor necrosis factor-α and interleukin-23, and increased anti-inflammatory cytokines like IL-10, but not transforming growth factor beta, in the presence of hIL-10. Also, macrophage polarization toward M2-like phenotype were induced by hIL-10 from transgenic PK(15) cells. Finally, we suggest that the cytotoxicity of human macrophages was reduced by hIL-10 from transgenic cells, inducing M2-like macrophage polarization. Therefore, these results show that hIL-10 transgenic pig can be used as a model to overcome acute immune rejection in pig-to-human xenotransplantation.     

A peptide mimetic of Gal-alpha 1,3-Gal is able to block human natural antibodies

The carbohydrate of Gal-alpha1,3-Gal is thought to be the major antigenic epitope present on pig vascular endothelium. The peptides that mimic the binding of antigenic epitope (Gal-alpha1,3-Gal) to lectin BS-I-B4 were identified from screening a filamentous phage-displayed random library. A phage bearing the peptide NCVSPYWCEPLAPSARA has been identified to bind the lectin strongly. Melibiose was able to inhibit the binding of the human natural anti-alpha Gal antibody to the peptide competitively. Our experiments show that the peptide mimetic of Gal-alpha1,3-Gal is able to inhibit the agglutination of pig RBCs by human natural antibody or lectin BS-I-B4. The peptide inhibitor of human natural antibodies may prove useful in pig-to-human xenotransplantation.     

Evaluation of the Galα1-3Gal Epitope as a Host Modification Factor Eliciting Natural Humoral Immunity to Enveloped Viruses

Human sera contain high levels of natural antibody (Ab) to Galα1-3Gal, a terminal glycosidic structure expressed on the surface of cells of mammals other than Old World primates. Incorporation of this determinant onto retroviral membranes by passage of viruses in cells encoding α-1-3-galactosyltransferase (GT) renders retroviruses sensitive to lysis by natural Ab and complement in normal human serum (NHS). Plasma membrane-budding viruses representing four additional virus groups were examined for their sensitivities to serum inactivation after passage through human cell lines that lack a functional GT or human cells expressing recombinant porcine GT. The inactivation of lymphocytic choriomeningitis virus (LCMV) by NHS directly correlated with host modification of the virus via expression of Galα1-3Gal and was blocked by incorporation of soluble Galα1-3Gal disaccharide into the inactivation assay. GT-deficient mice immunized to make high levels of Ab to Galα1-3Gal (anti-Gal Ab) were tested for resistance to LCMV passaged in GT-expressing cells. Resistance was not observed, but in vitro analyses of the mouse immune sera revealed that the antiviral activity of the sera was insufficient to eliminate LCMV infectivity on its natural targets of infection, macrophages, which express receptors for Ab and complement. Newcastle disease virus and vesicular stomatitis virus (VSV) were inactivated by NHS regardless of cell passage history, whereas Sindbis virus (SV) passaged in human cells resisted inactivation. Both VSV and SV passaged in Galα1-3Gal-expressing human cells incorporated this sugar moiety onto their major envelope glycoproteins. SV passaged in mouse cells expressing Galα1-3Gal was moderately sensitive to inactivation by NHS. These results indicate that enveloped viruses expressing Galα1-3Gal differ in their sensitivities to NHS and that a potent complement source, such as that in NHS, is required for efficient inactivation of sensitive viruses in vitro and in vivo.     

Binding of Griffonia simplicifolia 1 isolectin B4 (GS1 B4) to alpha-galactose antigens

Glycoconjugates with terminal Galalpha1-3Galbeta1-4GlcNAc sequences (alpha-galactosyl epitopes, natural xenoreactive antigens) are present on various tissues in pigs and are recognized by human anti-alphagalactosyl (alphaGal) antibodies1. Hence xenotransplantation (pig-to-human) would trigger immune reactions involving complement activation and lead to the hyperactute rejection of the graft. Xenoreactive antigens are often studied by using the lectin Griffonia simplicifolia 1 isolectin B4 (GS1 B4), which shows high affinity to galactose. We here estimate the specificity of GS1 B4 for detecting various galactosyl epitopes by measuring lectin binding to neoglycoproteins, thyroglobulin and pig skeletal muscle. Enzyme linked lectin assays confirmed that GS1 B4 was highly specific to alpha-galactosylated neoglycoproteins while the lectin did not detect a beta-galactosylated ligand. The length of the sugar chains influenced the lectin-carbohydrate interaction. A monosaccharide linked to serum albumin showed higher lectin affinity than did neoglycoproteins with di- and tri-alpha-galactosyl epitopes. When the carbohydrate was extended, as in the xenoreactive pentasaccharide (Galalpha1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc), the carbohydrate- lectin interaction was meagre. Not only the terminal, but also the subterminal sugar affected the lectin binding because the GS1 B4 affinity to Galalpha1-3Gal was much stronger than to Galalpha1-3GalNAc. In bovine and porcine thyroglobulin most alphaGal epitopes appear to be cryptic, but are unmasked by a heat denaturation. In pig skeletal muscle there was lectin reaction not only in the muscle capillaries, but also in the connective tissue and intracellularly in muscle fibres. In Western blots of isolated proteins from pig muscle at least three bands were strongly stained after incubation with lectin.     

Production of alpha 1,3-galactosyltransferase-knockout cloned pigs expressing human alpha 1,2-fucosylosyltransferase

The production of genetically engineered pigs as xenotransplant donors aims to solve the severe shortage of organs for transplantation in humans. The first barrier to successful xenotransplantation is hyperacute rejection (HAR). HAR is a rapid and massive humoral immune response directed against the pig carbohydrate Galalpha 1,3-Gal epitope, which is synthesized by alpha 1,3-galactosyltransferase (alpha1,3-GT). The Galalpha 1,3-Gal antigen also contributes to subsequent acute vascular rejection events. Genetic modifications of donor pigs transgenic for human complement regulatory proteins or different glycosyltransferases to downregulate Galalpha 1,3-Gal expression have been shown to significantly delay xenograft rejection. However, the complete removal of the Galalpha 1,3-Gal antigen is the most attractive option. In this study, the 5' end of the alpha 1,3-GT gene was efficiently targeted with a nonisogenic DNA construct containing predominantly intron sequences and a Kozak translation initiation site to initiate translation of the neomycin resistance reporter gene. We developed two novel polymerase chain reaction screening methods to detect and confirm the targeted G418-resistant clones. This is the first study to use Southern blot analysis to demonstrate the disruption of the alpha 1,3-GT gene in somatic HT-transgenic pig cells before they were used for nuclear transfer. Transgenic male pigs were produced that possess an alpha 1,3-GT knockout allele and express a randomly inserted human alpha 1,2-fucosylosyltransferase (HT) transgene. The generation of homozygous alpha 1,3-GT knockout pigs with the HT-transgenic background is underway and will be unique. This approach intends to combine the alpha 1,3-GT knockout genotype with a ubiquitously expressed fucosyltransferase transgene producing the universally tolerated H antigen. This approach may prove to be more effective than the null phenotype alone in overcoming HAR and delayed xenograft rejection.     

Adipose-derived mesenchymal stromal cells from genetically modified pigs: immunogenicity and immune modulatory properties

Background aimsThe immunomodulatory and anti-inflammatory effects of mesenchymal stromal cells (MSC) could prove to be a potential therapeutic approach for prolongation of survival of cell xenotransplantation. Adipose (Ad) MSC from genetically modified pigs could be an abundant source of pig donor-specific MSC.MethodsPig (p) MSC were isolated from adipose tissue of α1,3-galactosyltransferase gene knock-out pigs transgenic for human (h) CD46 (GTKO/hCD46), a potential source of islets. After characterization with differentiation and flow cytometry (FCM), AdMSC were compared with bone marrow (BM) MSC of the same pig and human adipose-derived (hAd) MSC. The modulation of human peripheral blood mononuclear cell (hPBMC) responses to GTKO pig aortic endothelial cells (pAEC) by different MSC was compared by measuring 3H-thymidine uptake. The supernatants from the AdMSC cultures were used to determine the role of soluble factors.ResultsGTKO/hCD46 pAdMSC (i) did not express galactose-α1,3-galactose (Gal) but expressed hCD46, (ii) differentiated into chondroblasts, osteocytes and adipocytes, (iii) expressed stem cell markers, (iv) expressed lower levels of Swine Leucocyte Antigen I (SLAI), Swine Leucocyte Antigen II DR (SLAIIDR) and CD80 than pAEC before and after pig interferon (IFN)-γ stimulation. The proliferative responses of hPBMC to GTKO/hCD46 pAdMSC and hAdMSC stimulators were similar, and both were significantly lower than to GTKO pAEC (P < 0.05). The proliferation of hPBMC to GTKO pAEC was equally suppressed by GTKO/hCD46 pAdMSC and hAdMSC (P > 0.05). The supernatant from GTKO/hCD46 pAdMSC did not suppress the human xenoresponse to GTKO pAEC, which was cell–cell contact-dependent.ConclusionsInitial evidence suggests that genetically modified pAdMSC function across the xenogeneic barrier and may have a role in cellular xenotransplantation.     

Effect of selective complement deficiency on the rate of neutralization of enveloped viruses by human sera.

The capacity of human sera genetically deficient in selective complement (C) components to enhance neutralization of enveloped viruses was examined by kinetic plaque reduction assays. Vaccinia virus, a DNA virus, and vesicular stomatitis virus (VSV), an RNA virus, were studied. Exogenous rabbit: or human antibody to vaccinia virus, and guinea pig or human antibody to VSV were provided in limiting, C-dependent concentrations. IgG antibodies predominated in most of the antisera employed. C5-deficient and C6-deficient human sera consistently supported normal rates of neutralization of either virus; this effect was heat-labile. C4-deficient human serum did hot exceed heat-inactivated serum in any neutralization assay. C1r-deficient serum displayed slight heat-labile neutralizing capacity against vaccinia but none against VSV. C2- and C3-deficient sera consistently exhibited measurable but clearly subnormal rates of neutralization. Two fresh agammaglobulinemic sera failed to inactivate either virus in the absence of added antibody. These results confirm and extend earlier evidence, based on neutralization of herpes simplex and Newcastle disease viruses in the presence of early (IgM) antibody and functionally pure guinea pig C components or C-deficient animal sera, that the late-acting components C5-C9 are not required for C-dependent neutralization. Data on four enveloped viruses now agree that this function is mediated by C1-C3, although C1 plus C4 appear to have some neutralizing capacity. This requirement for C1-C3 is overcome, however, in the presence of higher antibody cohcentrations, suggesting that the contribution of the C system to viral neutralization in vivo may be chiefly in the early phase of infection when antibody is limited.     

Inactivation of a human norovirus surrogate, human norovirus virus-like particles, and vesicular stomatitis virus by gamma irradiation

Gamma irradiation is a nonthermal processing technology that has been used for the preservation of a variety of food products. This technology has been shown to effectively inactivate bacterial pathogens. Currently, the FDA has approved doses of up to 4.0 kGy to control food-borne pathogens in fresh iceberg lettuce and spinach. However, whether this dose range effectively inactivates food-borne viruses is less understood. We have performed a systematic study on the inactivation of a human norovirus surrogate (murine norovirus 1 [MNV-1]), human norovirus virus-like particles (VLPs), and vesicular stomatitis virus (VSV) by gamma irradiation. We demonstrated that MNV-1 and human norovirus VLPs were resistant to gamma irradiation. For MNV-1, only a 1.7- to 2.4-log virus reduction in fresh produce at the dose of 5.6 kGy was observed. However, VSV was more susceptible to gamma irradiation, and a 3.3-log virus reduction at a dose of 5.6 kGy in Dulbecco's modified Eagle medium (DMEM) was achieved. We further demonstrated that gamma irradiation disrupted virion structure and degraded viral proteins and genomic RNA, which resulted in virus inactivation. Using human norovirus VLPs as a model, we provide the first evidence that the capsid of human norovirus has stability similar to that of MNV-1 after exposure to gamma irradiation. Overall, our results suggest that viruses are much more resistant to irradiation than bacterial pathogens. Although gamma irradiation used to eliminate the virus contaminants in fresh produce by the FDA-approved irradiation dose limits seems impractical, this technology may be practical to inactivate viruses for other purposes, such as sterilization of medical equipment.     

The synthesis of deoxy-alpha-Gal epitope derivatives for the evaluation of an anti-alpha-Gal antibody binding

Alpha-Gal epitopes (also termed as alpha-Gal) are carbohydrate structures bearing the alpha-D-Gal-(1-->3)-beta-D-Gal terminus 1 and are known to be the antigen responsible for antibody-mediated hyperacute rejection in xenotransplantation. Terminal 2-, 3-, 4-, and 6-deoxy-Gal derivatives of alpha-Gal were synthesized. Inhibition ELISA using mouse laminin was established to determine the binding affinity of the synthesized alpha-Gal derivatives. 4-Deoxy-alpha-Gal derivative 7 showed a significant reduction in antibody recognition. The IC(50) value was 15-fold poorer than the standard alpha-Gal epitopes alpha-D-Gal-(1-->3)-beta-D-Gal-(1-->4)-beta-D-Glc-NHAc (39) and alpha-D-Gal-(1-->3)-beta-D-Gal-(1-->4)-beta-D-Glc-OBn (40). A similar observation was seen with 2-deoxy-alpha-Gal derivative 5, whose IC(50) value was nearly tenfold higher than the standards. Interestingly, substitution at the terminal 3-position resulted in only a fourfold decrease in antibody recognition, suggesting a possible point of future derivation. Finally, 6-deoxy-alpha-Gal derivative 8 exhibited similar antibody recognition to both alpha-Gal epitope 39 and alpha-Gal epitope 40. This strongly suggests that derivatization at the 6-position can be accomplished without loss of antibody recognition. These findings can be utilized for the future design of other alpha-Gal derivatives.