This review describes a novel method developed for processing porcine tendon and other ligament implants that enables in situ remodeling into autologous ligaments in humans. The method differs from methods using extracellular matrices (ECMs) that provide postoperative orthobiological support (i.e., augmentation grafts) for healing of injured ligaments, in that the porcine bone-patellar-tendon-bone itself serves as the graft replacing ruptured anterior cruciate ligament (ACL). The method allows for gradual remodeling of porcine tendon into autologous human ACL while maintaining the biomechanical integrity. The method was first evaluated in a preclinical model of monkeys and subsequently in patients. The method overcomes detrimental effects of the natural anti-Gal antibody and harnesses anti-non-gal antibodies for the remodeling process in two steps: Step 1. Elimination of alpha-gal epitopes-this epitope that is abundant in pigs (as in other nonprimate mammals) binds the natural anti-Gal antibody, which is the most abundant natural antibody in humans. This interaction, which can induce fast resorption of the porcine implant, is avoided by enzymatic elimination of alpha-gal epitopes from the implant with recombinant alpha-galactosidase. Step 2. Partial cross-linking of porcine tendon with glutaraldehyde-this cross-linking generates covalent bonds in the ECM, which slow infiltration of macrophages into the implant. Anti-non-gal antibodies are produced in recipients against the multiple porcine antigenic proteins and proteoglycans because of sequence differences between human and porcine homologous proteins. Anti-non-gal antibodies bind to the implant ECM, recruit macrophages, and induce the implant destruction by directing proteolytic activity of macrophages. Partial cross-linking of the tendon ECM decreases the extent of macrophage infiltration and degradation of the implant and enables concomitant infiltration of fibroblasts that follow the infiltrating macrophages. These fibroblasts align with the implant collagen fibers and secrete their own collagen and other ECM proteins, which gradually remodel the porcine tendon into human ACL. This ligamentization process lasts approximately 2 years and the biomechanical integrity of the graft is maintained throughout the whole period. These studies are the first, and so far the only, to demonstrate remodeling of porcine tendon implants into permanently functional autologous ACL in humans.

Significance: Rapid recruitment and activation of macrophages may accelerate wound healing. Such accelerated healing was observed in wounds and burns of experimental animals treated with alpha-gal nanoparticles. Recent Advances: alpha-Gal nanoparticles present multiple alpha-gal epitopes (Galalpha1-3Galbeta1-4GlcNAc-R). alpha-Gal nanoparticles applied to wounds bind anti-Gal (the most abundant antibody in humans) and generate chemotactic complement peptides, which rapidly recruit macrophages. Fc/Fc receptor interaction between anti-Gal coating the alpha-gal nanoparticles and recruited macrophages activates macrophages to produce cytokines that accelerate healing. alpha-Gal nanoparticles applied to burns and wounds in mice and pigs producing anti-Gal, decreased healing time by 40-60%. In mice, this accelerated healing avoided scar formation. alpha-Gal nanoparticle-treated wounds, in diabetic mice producing anti-Gal, healed within 12 days, whereas saline-treated wounds became chronic wounds. alpha-Gal nanoparticles are stable for years and may be applied dried, in suspension, aerosol, ointments, or within biodegradable materials. Critical Issues: alpha-Gal nanoparticle therapy can be evaluated only in mammalian models producing anti-Gal, including alpha1,3-galactosyltransferase knockout mice and pigs or Old World primates. Traditional experimental animal models synthesize alpha-gal epitopes and lack anti-Gal. Future Directions: Since anti-Gal is naturally produced in all humans, it is of interest to determine safety and efficacy of alpha-gal nanoparticles in accelerating wound and burn healing in healthy individuals and in patients with impaired wound healing such as diabetic patients and elderly individuals. In addition, efficacy of alpha-gal nanoparticle therapy should be studied in healing and regeneration of internal injuries such as surgical incisions, ischemic myocardium following myocardial infarction, and injured nerves.

Application of alpha-gal nanoparticles to wounds and burns induces accelerated healing by harnessing the natural anti-Gal antibody which constitutes ~1% of human immunoglobulins. alpha-gal nanoparticles present multiple alpha-gal epitopes (Galalpha1-3Galbeta1-4GlcNAc-R), the carbohydrate ligand of anti-Gal. Studied alpha-gal nanoparticles were comprised of glycolipids with alpha-gal epitopes, phospholipids, and cholesterol. Binding of anti-Gal to alpha-gal nanoparticles in wounds activates the complement cascade, resulting in formation of chemotactic complement cleavage peptides that induce rapid recruitment of many macrophages. The Fc/Fcgamma receptors interaction between anti-Gal coating alpha-gal nanoparticles and the recruited macrophages activates macrophages to produce cytokines/growth factors that promote wound healing and recruit stem cells. Studies of wound healing by alpha-gal nanoparticles were feasible in alpha1,3galactosyltransferase knockout mice and pigs. In contrast to other nonprimate mammals, these mice and pigs lack the alpha-gal epitope, and thus they are not immunotolerant to it and produce anti-Gal. Treatment of skin wounds and burns with alpha-gal nanoparticles resulted in 40-60% decrease in healing time in comparison with control wounds treated with saline. This accelerated healing is associated with increased recruitment of macrophages and extensive angiogenesis in wounds, faster regrowth of epidermis, and regeneration of the dermis. The accelerated healing further decreases and may completely eliminate fibrosis and scar formation in wounds. Since healing of internal injuries is mediated by mechanisms similar to those in external wound healing, it is suggested that alpha-gal nanoparticles treatment may also improve regeneration and restoration of biological function following internal injuries such as surgical incisions, myocardial ischemia following infarction, and nerve injuries.

BACKGROUND: Anti-Gal is the most abundant natural antibody in humans and Old World primates (apes and Old World monkeys). Its ligand, the alpha-gal epitope (Galalpha1-3Galbeta1-4GlcNAc-R), is abundant in nonprimate mammals, prosimians and New World monkeys whereas it is absent in humans and Old World primates as a result of inactivation of the alpha1,3galactosyltransferase (alpha1,3GT) gene in ancestral Old World primates, as recent as 20-28 million years ago. Since anti-Gal has been a "forbidden" autoantibody for greater than 140 million years of evolution in mammals producing alpha-gal epitopes it was of interest to determine whether ancestral Old World primates could produce anti-Gal once alpha-gal epitopes were eliminated, i.e. did they carry anti-Gal encoding immunoglobulin genes, or did evolutionary selection eliminate these genes that may be detrimental in mammals synthesizing alpha-gal epitopes. This question was studied by evaluating anti-Gal prodution in alpha1,3GT knockout (GT-KO) pigs recently generated from wild-type pigs in which the alpha-gal epitope is a major self-antigen. METHODS: Anti-Gal antibody activity in pig sera was assessed by ELISA, flow cytometry and complement mediated cytolysis and compared to that in human sera. RESULTS: The study demonstrates abundant production of the natural anti-Gal antibody in GT-KO pigs at titers even higher than in humans. The fine specificity of GT-KO pig anti-Gal is identical to that of human anti-Gal. CONCLUSIONS: Pigs and probably other mammals producing alpha-gal epitopes carry immunoglobulin genes encoding anti-Gal as an autoantibody. Once the alpha-gal epitope is eliminated in GT-KO pigs, they produce anti-Gal. These findings strongly suggest that similar to GT-KO pigs, inactivation of the alpha1,3GT gene in ancestral Old World primates enabled the immediate production of anti-Gal, possibly as a protective antibody against detrimental microbial agents carrying alpha-gal epitopes.

This is a personal account of the discovery of the natural anti-Gal antibody, the most abundant natural antibody in humans, the reciprocal distribution of this antibody and its ligand the alpha-gal epitope in mammals and the immunological barrier this antibody has formed in porcine to human xenotransplantation. This barrier has been overcome in the recent decade with the generation of alpha1,3-galactosyltransferase gene-knockout pigs. However, anti-Gal continues to be relevant in medicine as it can be harnessed for various therapeutic effects. Anti-Gal converts tumor lesions injected with alpha-gal glycolipids into vaccines that elicit a protective anti-tumor immune response by in situ targeting of tumor cells for uptake by antigen-presenting cells. This antibody further accelerates wound and burn healing by interaction with alpha-gal nanoparticles applied to injured areas and induction of rapid recruitment and activation of macrophages. Anti-Gal/alpha-gal nanoparticle immune complexes may further induce rapid recruitment and activation of macrophages in ischemic myocardium and injured nerves, thereby inducing tissue regeneration and prevention of fibrosis.

Anti-Gal is the most abundant natural antibody in humans constituting ~1% of immunoglobulins. Anti-Gal is naturally produced also in apes and Old World monkeys. The ligand of anti-Gal is a carbohydrate antigen called the "alpha-gal epitope" with the structure Galalpha1-3Galbeta1-4GlcNAc-R. The alpha-gal epitope is present as a major carbohydrate antigen in nonprimate mammals, prosimians and New World monkeys. Anti-Gal can contribute to several immunological pathogeneses. Anti-Gal IgE produced in some individuals causes allergies to meat and to the therapeutic monoclonal antibody cetuximab, all presenting alpha-gal epitopes. Aberrant expression of the alpha-gal epitope or of antigens mimicking it in humans may result in autoimmune processes, as in Graves' Disease. alpha-Gal epitopes produced by Trypanosoma cruzi interact with anti-Gal and induce "autoimmune like" inflammatory reactions in Chagas Disease. Anti-Gal IgM and IgG further mediate rejection of xenografts expressing alpha-gal epitopes. Because of its abundance, anti-Gal may be exploited for various clinical uses. It increases immunogenicity of microbial vaccines (e.g., flu vaccine) presenting alpha-gal epitopes by targeting them for effective uptake by APC. Tumor lesions are converted into vaccines against autologous tumor associated antigens by intratumoral injection of alpha-gal glycolipids which insert into tumor cell membranes. Anti-Gal binding to alpha-gal epitopes on tumor cells targets them for uptake by APC. Accelerated wound healing is achieved by application of alpha-gal nanoparticles which bind anti-Gal, activate complement, recruit and activate macrophages that induce tissue regeneration. This therapy may be of further significance in regeneration of internally injured tissues such as ischemic myocardium and injured nerves. This article is protected by copyright. All rights reserved.

Anti-Gal is the most abundant antibody in humans, constituting 1% of immunoglobulins. Anti-Gal binds specifically alpha-gal epitopes (Galalpha1-3Galbeta1-4GlcNAc-R). Immunogenicity of autologous tumor associated antigens (TAA) is greatly increased by manipulating tumor cells to express alpha-gal epitopes and bind anti-Gal. Glycolipids with alphagal epitopes (alpha-gal glycolipids) injected into tumors insert into the tumor cell membrane. Anti-Gal binding to the multiple alpha-gal epitopes de novo presented on the tumor cells results in targeting of these cells to APC via the interaction between the Fc portion of the bound anti-Gal and Fcgamma; receptors on APC. The APC process and present immunogenic TAA peptides and thus, effectively activate tumor specific CD4+ helper T cells and CD8+ cytotoxic T cells which destroy tumor cells in micrometastases. The induced immune response is potent enough to overcome immunosuppression by Treg cells. A phase I clinical trial indicated that alpha-gal glycolipid treatment has no adverse effects. In addition to achieving destruction of micrometastases in cancer patients with advance disease, alpha-gal glycolipid treatment may be effective as neo-adjuvant immunotherapy. Injection of alpha-gal glycolipids into primary tumors few weeks prior to resection can induce a protective immune response capable of destroying micrometastases expressing autologous TAA, long after primary tumor resection.

This study describes a method for increasing the immunogenicity of influenza virus vaccines by exploiting the natural anti-Gal antibody to effectively target vaccines to antigen-presenting cells (APC). This method is based on enzymatic engineering of carbohydrate chains on virus envelope hemagglutinin to carry the alpha-Gal epitope (Gal alpha 1-3Gal beta 1-4GlcNAc-R). This epitope interacts with anti-Gal, the most abundant antibody in humans (1% of immunoglobulins). Influenza virus vaccine expressing alpha-Gal epitopes is opsonized in situ by anti-Gal immunoglobulin G. The Fc portion of opsonizing anti-Gal interacts with Fc gamma receptors on APC and induces effective uptake of the vaccine virus by APC. APC internalizes the opsonized virus to transport it to draining lymph nodes for stimulation of influenza virus-specific T cells, thereby eliciting a protective immune response. The efficacy of such an influenza vaccine was demonstrated in alpha 1,3galactosyltransferase (alpha 1,3GT) knockout mice, which produce anti-Gal, using the influenza virus strain A/Puerto Rico/8/34-H1N1 (PR8). Synthesis of alpha-Gal epitopes on carbohydrate chains of PR8 virus (PR8(alpha gal)) was catalyzed by recombinant alpha1,3GT, the glycosylation enzyme that synthesizes alpha-Gal epitopes in cells of nonprimate mammals. Mice immunized with PR8(alpha gal) displayed much higher numbers of PR8-specific CD8(+) and CD4(+) T cells (determined by intracellular cytokine staining and enzyme-linked immunospot assay) and produced anti-PR8 antibodies with much higher titers than mice immunized with PR8 lacking alpha-Gal epitopes. Mice immunized with PR8(alpha gal) also displayed a much higher level of protection than PR8 immunized mice after being challenged with lethal doses of live PR8 virus. We suggest that a similar method for increasing immunogenicity may be applicable to avian influenza vaccines.

This study describes a novel cancer immunotherapy treatment that exploits the natural anti-Gal Ab to destroy tumor lesions and convert them into an endogenous vaccine targeted to APC via FcgammaR. Anti-Gal constitutes 1% of immunoglobulins in humans and interacts specifically with alpha-gal epitopes (Galalpha1-3Galbeta1-4GlcNAc-R). The binding of anti-Gal to alpha-gal epitopes on pig cells mediates xenograft rejection. The proposed method uses glycolipid micelles with multiple alpha-gal epitopes (alpha-gal glycolipids). These glycolipids are extracted from rabbit red cell membranes and are comprised of ceramides with carbohydrate chains containing 5-25 carbohydrates, all capped with alpha-gal epitopes. Efficacy of this treatment was demonstrated in alpha1,3-galactosyltransferase knockout mice producing anti-Gal and bearing B16 melanoma or B16/OVA producing OVA as a surrogate tumor Ag. These mice are unique among nonprimate mammals in that, similar to humans, they lack alpha-gal epitopes and can produce the anti-Gal Ab. Intratumoral injection of alpha-gal glycolipids results in local inflammation mediated by anti-Gal binding to the multiple alpha-gal epitopes and activation of complement. These glycolipids spontaneously insert into tumor cell membranes. The binding of anti-Gal to alpha-gal expressing tumor cells induces the destruction of treated lesions as in anti-Gal-mediated xenograft rejection. Anti-Gal further opsonizes tumor cells within the lesion and, thus, targets them for effective uptake by APC that transport the tumor Ags to draining lymph nodes. APC further cross-present immunogenic tumor Ag peptides and elicit a systemic anti-tumor immune response. Similar intratumoral injection of alpha-gal glycolipids in humans is likely to induce the destruction of treated lesions and elicit a protective immune response against micrometastases.