Genetic engineering strategies to prevent the effects of antibody and complement on xenogeneic chondrocytes.

Advances in animal transgenesis may allow using xenogeneic chondrocytes in tissue-engineering applications for clinical cartilage repair. Porcine cartilage is rejected by humoral and cellular mechanisms that could be overcome by identifying key molecules triggering rejection and developing effective genetic-engineering strategies. Accordingly, high expression of α1,2-fucosyltransferase (HT) in xenogeneic cartilage protects from galactose α1,3-galactose (Gal)-mediated antibody responses. Now, we studied whether expression of a complement inhibitor provides further protection. First, porcine articular chondrocytes (PAC) were isolated from non-transgenic, single and double transgenic pigs expressing HT and moderate levels of human CD59 (hCD59) and their response to human serum was assessed. High recombinant expression of human complement regulatory molecules hCD59 and hDAF was also attained by retroviral transduction of PAC for further analyses. Complement activation on PAC after exposure to 20 % human serum for 24 hours mainly triggered the release of pro-inflammatory cytokines IL-6 and IL-8. Transgenic expression of HT and hCD59 did not suffice to fully counteract this effect. Nevertheless, the combination of blocking anti-Gal antibodies (or C5a) and high hCD59 levels conferred very high protection. On the contrary, high hDAF expression attained the most dramatic reduction in IL-6/IL-8 secretion by a single strategy, but the additional inhibition of anti-Gal antibodies or C5a did not provide further improvement. Notably, we demonstrate that both hCD59 and hDAF inhibit anaphylatoxin release in this setting. In conclusion, our study identifies genetic-engineering approaches to prevent humoral rejection of xenogeneic chondrocytes for use in cartilage repair.

In Contrast to Anti-C5 Therapy, Cobra Venom Factor Does Not Prevent Rejection of Xenogeneic Cartilage in Mice.

BACKGROUND: The use of xenogeneic chondrocytes may benefit the development of clinical tissue-engineering applications for cartilage repair. However, cartilage xenografts are slowly rejected by humoral and cellular mechanisms to which galactose α1,3-galactose (Gal) antigen and complement contribute. Accordingly, transgenic expression of human α1,2-fucosyltransferase (HT) in porcine cartilage helps to protect from the Gal-mediated immune response. Here, we aimed to assess the effect of the broadly used complement inhibitor cobra venom factor (CVF) in comparison with anti-C5 therapy in α1,3-galactosyltransferase knockout (Gal KO) mice transplanted with porcine cartilage. METHODS: Gal KO mice grafted with control or HT-transgenic cartilage were left untreated or treated systemically with either anti-C5 antibody or CVF for 5 weeks. The degree of rejection was evaluated by use of histopathological analysis, and serum anti-Gal antibodies were measured in all cohorts. RESULTS: The rejection process of control cartilage was well advanced by 5 weeks after transplantation in untreated Gal KO mice, whereas enhanced graft survival characterized by reduced cellular immune infiltrate was found in mice grafted with HT cartilage and/or treated with anti-C5. In contrast, CVF administration led to inconsistent results, with some grafts showing no improvement or even increased amounts of granulocytes. Regarding antibody titers, the anti-Gal immunoglobulin (Ig)M increased in the control transplant cohort and remained unchanged in the HT-graft recipients at 5 weeks after transplantation. Notably, a strong anti-Gal IgM response was readily detected in CVF-treated mice of both transplanted cohorts. CONCLUSIONS: CVF does not present advantages over anti-C5 therapy for preventing rejection of xenogeneic porcine cartilage.

Inhibition of complement component C5 protects porcine chondrocytes from xenogeneic rejection.

OBJECTIVE: Tissue-based xenografts such as cartilage are rejected within weeks by humoral and cellular mechanisms that preclude its clinical application in regenerative medicine. The problem could be overcome by identifying key molecules triggering rejection and the development of genetic-engineering strategies to counteract them. Accordingly, high expression of α1,2-fucosyltransferase (HT) in xenogeneic cartilage reduces the galactose α1,3-galactose (Gal) antigen and delays rejection. Yet, the role of complement activation in this setting is unknown. DESIGN: To determine its contribution, we assessed the effect of inhibiting C5 complement component in α1,3-galactosyltransferase-knockout (Gal KO) mice transplanted with porcine cartilage and studied the effect of human complement on porcine articular chondrocytes (PAC). RESULTS: Treatment with an anti-mouse C5 blocking antibody for 5 weeks enhanced graft survival by reducing cellular rejection. Moreover, PAC were highly resistant to complement-mediated lysis and primarily responded to human complement by releasing IL-6 and IL-8. This occurred even in the absence of anti-Gal antibody and was mediated by both C5a and C5b-9. Indeed, C5a directly triggered IL-6 and IL-8 secretion and up-regulated expression of swine leukocyte antigen I (SLA-I) and adhesion molecules on chondrocytes, all processes that enhance cellular rejection. Finally, the use of anti-human C5/C5a antibodies and/or recombinant expression of human complement regulatory molecule CD59 (hCD59) conferred protection in correspondence with their specific functions. CONCLUSIONS: Our study demonstrates that complement activation contributes to rejection of xenogeneic cartilage and provides valuable information for selecting approaches for complement inhibition.

Characterization of cartilage from H-transferase transgenic pigs.

Cartilage engineering is the object of intense research as a result of major medical needs and therapeutic prospects. Porcine xenogeneic cells/tissues may help in the development of clinical applications such as articular cartilage repair. However, unmodified porcine cartilage is rejected in primates by humoral and cellular mechanisms. We previously showed that porcine articular chondrocytes (PAC) isolated from H-transferase (HT) transgenic pigs show markedly reduced expression of the Galalpha1,3Gal antigen (alphaGal) and prolonged survival when transplanted into alpha1,3galactosyltransferase-deficient mice. In this work, we further studied the protective mechanisms of HT transgenic expression in cartilage, particularly its effects on monocyte adhesion. To this end, PAC isolated from control and HT transgenic pigs were assayed for human complement deposition and adhesion to the human monoblastic cell line U937. Consistent with a reduction in complement activation by the classical pathway, the HT transgenic PAC showed a 2-fold reduction in the deposition of complement components C4 and C3 relative to controls. Adhesion of U937 cells to HT PAC was also diminished under various conditions. This reduction was more dramatic at high effector:target ratios and especially observed when combined with anti-alphaGal antibodies (5-fold difference). Nevertheless, this effect was also observed in the absence of anti-alphaGal. antibodies and after tumor necrosis factor treatment. These results suggest that HT expression on porcine chondrocytes protects them from both humoral and cellular rejection.

Amino acids critical for the functions of the bovine papillomavirus type 1 E2 transactivator.

The N-terminal domain of the bovine papillomavirus type 1 E2 protein is important for viral DNA replication, for transcriptional transactivation, and for interaction with the E1 protein. To determine which residues of this 200-amino-acid domain are important for these activities, single conservative amino acid substitutions have been generated in 17 residues that are invariant among all papillomavirus E2 proteins. The resulting mutated E2 proteins were tested for the ability to support viral DNA replication, activate transcription, and cooperatively bind to the origin of replication with the E1 protein. We identified five mutated proteins that were completely defective for transcriptional activation and either were defective or could support viral DNA replication at only low levels. However, several of these proteins could still interact efficiently with the E1 protein. In addition, we identified several mutated proteins that were unable to efficiently cooperatively bind to the origin with the E1 protein. Although a number of the mutated proteins demonstrated wild-type activity in all of the functions tested, only 3 out of 17 mutated viral genomes were able to induce foci in a C127 focus formation assay when the mutations were generated in the background of the entire bovine papillomavirus type 1 genome. This finding suggests that the E2 protein may have additional activities that are important for the viral life cycle.

A mutational analysis of the amino terminal domain of the human papillomavirus type 16 E7 oncoprotein.

The human papillomavirus type 16 (HPV-16) E7 oncoprotein shares structural and functional similarity with the adenovirus (Ad) E1A protein and the SV40 large tumor antigen (TAg). Like these other DNA tumor virus oncoproteins, HPV-16 E7 interacts with the "pocket proteins," a family of host cellular proteins that include the retinoblastoma tumor suppressor protein and can cooperate with the ras oncogene to transform primary rodent cells. Mutational analyses have indicated that amino acid sequences outside of the pRB binding region are also important for the cellular transformation property of HPV-16 E7. These sequences include an amino terminal domain of the E7 protein that is similar to a portion of conserved region 1 of Ad E1A. In this study it is shown that the homologous amino acid sequences in Ad E1A and SV40 TAg are functionally interchangeable with the amino terminal HPV-16 E7 domain in transformation assays. Deletion analysis across the amino terminus of HPV-16 E7 indicated that the overall integrity of the entire CR1 homology domain is important for the biological activity of the HPV E7 oncoprotein.

Conserved patterns of somatic mutation and secondary VH gene rearrangement create aberrant Ig-encoding genes in Epstein-Barr virus-transformed and normal human B lymphocytes.

Expression of N-terminal-truncated Ig heavy chains without normal light chain expression has been shown to occur in human B cell tumor lines, and to be due to diverse types of structural alteration within the expressed Ig heavy and light chain genes. Due to the tumor cell origin of these lines, generation of aberrant Ig-encoding genes may only occur after malignant transformation, reflecting the release of the tumor B cell from the need to express functional Ig for continued clonal proliferation. The genetic basis for expression of VH-truncated mu chains without light chains in several Epstein-Barr virus (EBV)-transformed human B cell lines was investigated with the aim that this information would lead to detection of similarly aberrant Ig genes in normal human B lymphocytes. Analysis of the productive mu genes in three truncated mu-only human B cell lines showed a consistent structural change where a secondary VH-VHDJH gene rearrangement had occurred. The site of VH-VH joining was suggestive of a V(D)J recombinase-mediated event. A consistent pattern of mutation was also observed in the normal-sized, but non-functional kappa light chain transcripts, making them incapable of coding for a functional kappa chain. Using genomic DNA from peripheral blood lymphocytes of a donor whose B cells were originally used to make one of the truncated mu-only EBV B cell lines, a similarly mutated V kappa gene was detected and a similar composite VH-VH gene was cloned. The lack of such aberrant VH and V kappa genes in non-lymphoid cells of this individual showed that the structural abnormalities in the expressed Ig genes arose somatically during development of that B cell clone. The presence of these altered Ig heavy and light chain genes in the genomic DNA of untransformed lymphocytes also shows that generation of such variant B cell clones can be a pre-neoplastic event and occurs by genetic mechanisms active during normal B cell development.