An ancient evolutionary regenerative mechanism of injured myocardium in vertebrates has been conserved in zebrafish, urodeles (salamander, newt, and axolotl) and neonatal mice. This innate regenerative mechanism is characterized by extensive migration of pro-regenerative macrophages into the injured myocardium and non-immune activation of parts of the complement system. Loss of regenerative activity in neonatal mice within a few days after birth implies that it is suppressed and replaced by fibrotic repair and scar formation. Fibrosis prevents ventricular wall rupture following myocardial infarction (MI), but it compromises contractility and can lead to heart failure and premature death. Reactivation of the suppressed regenerative mechanism in post-MI adult mice may be feasible by localized immune activation of the complement system, resulting in extensive recruitment of pro-regenerative macrophages into the injured myocardium, recapitulating neonatal mechanisms. Localized complement activation can be achieved by a new method of harnessing the natural anti-Gal antibody, which constitutes ~1% of human immunoglobulins and binds the carbohydrate antigen "α-gal epitope". α-Gal nanoparticles (small liposomes presenting multiple α-gal epitopes) bind anti-Gal when administered into reperfused myocardium post-MI in anti-Gal-producing mice, thereby inducing localized complement activation. In this novel approach, macrophages recruited into the ischemic myocardium by complement cleavage chemotactic peptides, and binding anti-Gal-coated α-gal nanoparticles, polarize to become pro-regenerative macrophages that produce pro-regenerative cytokines and recruit stem cells. This process results in near-complete regeneration of the injured myocardium within 14 days. Future evaluation of this novel approach in larger animal models will help in determining whether trans-endocardial delivery by catheter of α-gal nanoparticles into ischemic myocardium warrants clinical application in acute MI.
The human natural anti-carbohydrate antibodies anti-Gal, anti-Neu5Gc, and anti-Forssman are "living-fossils" that appeared in ancestral apes, monkeys and hominins millions of years ago. These antibodies appeared at various evolutionary periods in few mutated-offspring that lost the ability to synthesize the corresponding dispensable (i.e., nonessential) carbohydrate-antigens, α-gal epitope, Neu5Gc (N-glycolyl neuraminic acid) and Forssman-antigen, respectively. Production of these antibodies is stimulated by environmental antigens such as those of the human microbiota. Elimination of carbohydrate-antigens in the few mutated-offspring was caused by accidental nonsense or missense mutations that inactivated genes encoding enzymes involved in their biosynthesis, while most individuals in parental-populations continued synthesizing these carbohydrate-antigens. It has been suggested that dispensable carbohydrate-antigens which are absent in some mammalian species were evolutionary eliminated due to selective pressure by lethal viruses using these carbohydrate-antigens as "docking" receptors. An alternative selective mechanism which is based on the distribution of anti-Gal, anti-Neu5Gc and anti-Forssman in mammals, is presented in this review and is associated with the protective effects of these natural antibodies. It is suggested that epidemics of lethal enveloped-viruses caused the extinction of parental-populations synthesizing the corresponding carbohydrate-antigens of these antibodies,  independent of the cell adhesion mechanisms of such viruses. However, the few mutated offspring were protected by these natural antibodies which bound to carbohydrate-antigens synthesized on viruses as a result of their replication in individuals of the parental-populations. Recent studies suggest that these antibodies continue to contribute to the immune protection of humans against zoonotic infections by viruses presenting α-gal, Neu5Gc or Forssman antigens.
The healing of skin wounds, myocardial, and spinal cord injuries in salamander, newt, and axolotl amphibians, and in mouse neonates, results in scar-free regeneration, whereas injuries in adult mice heal by fibrosis and scar formation. Although both types of healing are mediated by macrophages, regeneration in these amphibians and in mouse neonates also involves innate activation of the complement system. These differences suggest that localized complement activation in adult mouse injuries might induce regeneration instead of the default fibrosis and scar formation. Localized complement activation is feasible by antigen/antibody interaction between biodegradable nanoparticles presenting α-gal epitopes (α-gal nanoparticles) and the natural anti-Gal antibody which is abundant in humans. Administration of α-gal nanoparticles into injuries of anti-Gal-producing adult mice results in localized complement activation which induces rapid and extensive macrophage recruitment. These macrophages bind anti-Gal-coated α-gal nanoparticles and polarize into M2 pro-regenerative macrophages that orchestrate accelerated scar-free regeneration of skin wounds and regeneration of myocardium injured by myocardial infarction (MI). Furthermore, injection of α-gal nanoparticles into spinal cord injuries of anti-Gal-producing adult mice induces recruitment of M2 macrophages, that mediate extensive angiogenesis and axonal sprouting, which reconnects between proximal and distal severed axons. Thus, α-gal nanoparticle treatment in adult mice mimics physiologic regeneration in amphibians. These studies further suggest that α-gal nanoparticles may be of significance in the treatment of human injuries.
Gopalakrishnan B, Galili U, Saenger M, Burket NJ, Koss W, et al.
Tissue engineering and regenerative medicine •
Previous investigations have shown that local application of nanoparticles presenting the carbohydrate moiety galactose-α-1,3-galactose (α-gal epitopes) enhance wound healing by activating the complement system and recruiting pro-healing macrophages to the injury site. Our companion in vitro paper suggest α-gal epitopes can similarly recruit and polarize human microglia toward a pro-healing phenotype. In this continuation study, we investigate the in vivo implications of α-gal nanoparticle administration directly to the injured spinal cord. α-Gal knock-out (KO) mice subjected to spinal cord crush were injected either with saline (control) or with α-gal nanoparticles immediately following injury. Animals were assessed longitudinally with neurobehavioral and histological endpoints. Mice injected with α-gal nanoparticles showed increased recruitment of anti-inflammatory macrophages to the injection site in conjunction with increased production of anti-inflammatory markers and a reduction in apoptosis. Further, the treated group showed increased axonal infiltration into the lesion, a reduction in reactive astrocyte populations and increased angiogenesis. These results translated into improved sensorimotor metrics versus the control group. Application of α-gal nanoparticles after spinal cord injury (SCI) induces a pro-healing inflammatory response resulting in neuroprotection, improved axonal ingrowth into the lesion and enhanced sensorimotor recovery. The data shows α-gal nanoparticles may be a promising avenue for further study in CNS trauma.
Gopalakrishnan B, Galili U, Dunbar A, Solorio L, Shi R, et al.
Tissue engineering and regenerative medicine •
Macrophages and microglia play critical roles after spinal cord injury (SCI), with the pro-healing, anti-inflammatory (M2) subtype being implicated in tissue repair. We hypothesize that promoting this phenotype within the post-injured cord microenvironment may provide beneficial effects for mitigating tissue damage. As a proof of concept, we propose the use of nanoparticles incorporating the carbohydrate antigen, galactose-α-1,3-galactose (α-gal epitope) as an immunomodulator to transition human microglia (HMC3) cells toward a pro-healing state. Quiescent HMC3 cells were acutely exposed to α-gal nanoparticles in the presence of human serum and subsequently characterized for changes in cell shape, expression of anti or pro-inflammatory markers, and secretion of phenotype-specific cytokines. HMC3 cells treated with serum activated α-gal nanoparticles exhibited rapid enlargement and shape change in addition to expressing CD68. Moreover, these activated cells showed increased expression of anti-inflammatory markers like Arginase-1 and CD206 without increasing production of pro-inflammatory cytokines TNF-α or IL-6. This study is the first to show that resting human microglia exposed to a complex of α-gal nanoparticles and anti-Gal (from human serum) can be activated and polarized toward a putative M2 state. The data suggests that α-gal nanoparticles may have therapeutic relevance to the CNS microenvironment, in both recruiting and polarizing macrophages/microglia at the application site. The immunomodulatory activity of these α-gal nanoparticles post-SCI is further described in the companion work (Part II).
Macrophages play a pivotal role in the process of healing burns. One of the major risks in the course of burn healing, in the absence of regenerating epidermis, is infections, which greatly contribute to morbidity and mortality in such patients. Therefore, it is widely agreed that accelerating the recruitment of macrophages into burns may contribute to faster regeneration of the epidermis, thus decreasing the risk of infections. This review describes a unique method for the rapid recruitment of macrophages into burns and the activation of these macrophages to mediate accelerated regrowth of the epidermis and healing of burns. The method is based on the application of bio-degradable "α-gal" nanoparticles to burns. These nanoparticles present multiple α-gal epitopes (Galα1-3Galβ1-4GlcNAc-R), which bind the abundant natural anti-Gal antibody that constitutes ~1% of immunoglobulins in humans. Anti-Gal/α-gal nanoparticle interaction activates the complement system, resulting in localized production of the complement cleavage peptides C5a and C3a, which are highly effective chemotactic factors for monocyte-derived macrophages. The macrophages recruited into the α-gal nanoparticle-treated burns are activated following interaction between the Fc portion of anti-Gal coating the nanoparticles and the multiple Fc receptors on macrophage cell membranes. The activated macrophages secrete a variety of cytokines/growth factors that accelerate the regrowth of the epidermis and regeneration of the injured skin, thereby cutting the healing time by half. Studies on the healing of thermal injuries in the skin of anti-Gal-producing mice demonstrated a much faster recruitment of macrophages into burns treated with α-gal nanoparticles than in control burns treated with saline and healing of the burns within 6 days, whereas healing of control burns took ~12 days. α-Gal nanoparticles are non-toxic and do not cause chronic granulomas. These findings suggest that α-gal nanoparticles treatment may harness anti-Gal for inducing similar accelerated burn healing effects also in humans.
Two human natural anti-carbohydrate antibodies appeared in critical evolutionary events that brought primates and hominins to brink of extinction. The first is the anti-Gal antibody, produced in Old-World monkeys (OWM), apes and humans. It binds the carbohydrate-antigen 'α-gal epitope' (Galα1-3Galβ1-4GlcNAc-R) on carbohydrate-chains (glycans) synthesized by non-primate mammals, lemurs and New-World monkeys (NWM). The second is anti-N-glycolylneuraminic-acid (anti-Neu5Gc) antibody binding Neu5Gc on glycans synthesized by OWM, apes and most non-primate mammals. Ancestral OWM and apes synthesized α-gal epitopes and were eliminated ~20-30 million-years-ago (mya). Only few accidentally mutated offspring lacking α-gal epitopes, produced anti-Gal and survived. Hominin-populations living ~3 mya synthesized Neu5Gc and were eliminated, but few mutated offspring that accidently lost their ability to synthesize Neu5Gc, produced natural anti-Neu5Gc antibody. These hominins survived and ultimately evolved into present-day humans. It is argued that these two near-extinction events were likely to be the result of epidemics caused by highly virulent and lethal enveloped viruses that killed parental-populations. These viruses presented α-gal epitopes or Neu5Gc synthesized in host-cells of the parental-populations. Mutated offspring survived the epidemics because they were protected from the lethal virus by the natural anti-Gal or anti-Neu5Gc antibodies they produced due to loss of immune-tolerance to α-gal epitopes or to Neu5Gc, respectively.
This review discusses a novel experimental approach for the regeneration of original tissue structure by recruitment of endogenous stem-cells to injured sites following administration of α-gal nanoparticles, which harness the natural anti-Gal antibody. Anti-Gal is produced in large amounts in all humans, and it binds the multiple α-gal epitopes (Galα1-3Galβ1-4GlcNAc-R) presented on α-gal nanoparticles. In situ binding of anti-Gal to α-gal nanoparticles activates the complement system and generates complement cleavage chemotactic-peptides that rapidly recruit macrophages. Macrophages reaching anti-Gal coated α-gal nanoparticles bind them via Fc/Fc receptor interaction and polarize into M2 pro-reparative macrophages. These macrophages secrete various cytokines that orchestrate regeneration of the injured tissue, including VEGF inducing neo-vascularization and cytokines directing homing of stem-cells to injury sites. Homing of stem-cells is also directed by interaction of complement cleavage peptides with their corresponding receptors on the stem-cells. Application of α-gal nanoparticles to skin wounds of anti-Gal producing mice results in decrease in healing time by half. Furthermore, α-gal nanoparticles treated wounds restore the normal structure of the injured skin without fibrosis or scar formation. Similarly, in a mouse model of occlusion/reperfusion myocardial-infarction, near complete regeneration after intramyocardial injection of α-gal nanoparticles was demonstrated, whereas hearts injected with saline display ~20% fibrosis and scar formation of the left ventricular wall. It is suggested that recruitment of stem-cells following anti-Gal/α-gal nanoparticles interaction in injured tissues may result in induction of localized regeneration facilitated by conducive microenvironments generated by pro-reparative macrophage secretions and "cues" provided by the extracellular matrix in the injury site.
Anaphylaxis is a potentially deadly condition that requires emergent therapy. While frequently treated in the emergency department (ED), recent evidence updates may improve the diagnosis and management of this condition. This paper evaluates key evidence-based updates concerning the diagnosis and management of anaphylaxis for the emergency clinician. The presentation of anaphylaxis can vary. Current diagnostic criteria can be helpful when evaluating patients for anaphylaxis, though multiple criteria exist. While the most common causes of anaphylaxis include medications, insect venom, and foods, recent literature has identified an IgE antibody response to mammalian galactose alpha-1,3-galactose, known as alpha-gal anaphylaxis. Epinephrine is the first-line therapy and is given in doses of 0.01 mg/kg (up to 0.5 mg in adults) intramuscularly (IM) in the anterolateral thigh. Intravenous (IV) epinephrine administration is recommended in patients refractory to IM epinephrine and IV fluids, or those with cardiovascular collapse. Antihistamines and glucocorticoids should not delay administration of epinephrine and do not demonstrate a significant reduction in risk of biphasic reactions. Biphasic reactions may affect 1-7% of patients with anaphylaxis. Risk factors for biphasic reaction include severe initial presentation and repeated doses of epinephrine. Disposition of patients with anaphylaxis requires consideration of several factors. Emergency clinicians must be aware of current updates in the evaluation and management of this disease.
This review describes the first studies on successful conversion of porcine soft-tissue bioprostheses into viable permanently functional tissue in humans. This process includes gradual degradation of the porcine tissue, with concomitant neo-vascularization and reconstruction of the implanted bioprosthesis with human cells and extracellular matrix. Such a reconstruction process is referred to in this review as "humanization". Humanization was achieved with porcine bone-patellar-tendon-bone (BTB), replacing torn anterior-cruciate-ligament (ACL) in patients. In addition to its possible use in orthopedic surgery, it is suggested that this humanization method should be studied as a possible mechanism for converting implanted porcine bioprosthetic heart-valves (BHV) into viable tissue valves in young patients. Presently, these patients are only implanted with mechanical heart-valves, which require constant anticoagulation therapy. The processing of porcine bioprostheses, which enables humanization, includes elimination of α-gal epitopes and partial (incomplete) crosslinking with glutaraldehyde. Studies on implantation of porcine BTB bioprostheses indicated that enzymatic elimination of α-gal epitopes prevents subsequent accelerated destruction of implanted tissues by the natural anti-Gal antibody, whereas the partial crosslinking by glutaraldehyde molecules results in their function as "speed bumps" that slow the infiltration of macrophages. Anti-non gal antibodies produced against porcine antigens in implanted bioprostheses recruit macrophages, which infiltrate at a pace that enables slow degradation of the porcine tissue, neo-vascularization, and infiltration of fibroblasts. These fibroblasts align with the porcine collagen-fibers scaffold, secrete their collagen-fibers and other extracellular-matrix (ECM) components, and gradually replace porcine tissues degraded by macrophages with autologous functional viable tissue. Porcine BTB implanted in patients completes humanization into autologous ACL within ~2 years. The similarities in cells and ECM comprising heart-valves and tendons, raises the possibility that porcine BHV undergoing a similar processing, may also undergo humanization, resulting in formation of an autologous, viable, permanently functional, non-calcifying heart-valves.
Neonatal mice, but not older mice, can regenerate their hearts after myocardial-infarction (MI), a process mediated by pro-reparative macrophages. α-Gal nanoparticles applied to skin wounds in adult-mice bind the anti-Gal antibody, activate the complement cascade and generate complement chemotactic peptides that recruit pro-reparative macrophages which are further activated by these nanoparticles. The recruited macrophages decrease wound healing time by ~50%, restore the normal skin structure and prevent fibrosis and scar formation in mice. The objective of this study is to determine if α-gal nanoparticles injected into the reperfused myocardium after MI in adult-mice can induce myocardial repair that restores normal structure, similar to that observed in skin injuries. MI was induced by occluding the mid-portion of the left anterior descending (LAD) coronary artery for 30 min. Immediately following reperfusion, each mouse received two 10 μl injections of 100 μg α-gal nanoparticles in saline into the LAD territory ( = 20), or saline for controls ( = 10). Myocardial infarct size was measured by planimetry following Trichrome staining and macrophage recruitment by hematoxylin-eosin staining. Left ventricular (LV) function was measured by echocardiography. Control mice displayed peak macrophage infiltration at 4-days, whereas treated mice had a delayed peak macrophage infiltration at 7-days. At 28-days, control mice demonstrated large transmural infarcts with extensive scar formation and poor contractile function. In contrast, mice treated with α-gal nanoparticles demonstrated after 28-days a marked reduction in infarct size (~10-fold smaller), restoration of normal myocardium structure and contractile function. Intramyocardial injection of α-gal nanoparticles post-MI in anti-Gal producing adult-mice results in near complete repair of the infarcted territory, with restoration of normal LV structure and contractile function. The mechanism responsible for this benefit likely involves alteration of the usual inflammatory response post-MI, as previously observed with regeneration of injured hearts in adult zebrafish, salamanders and neonatal mice.
Kaymakcalan OE, Abadeer A, Goldufsky JW, Galili U, Karinja SJ, et al.
Experimental dermatology •
An inadequate response from macrophages, key orchestrators of the wound healing process, has been implicated in the pathophysiology of impaired healing in diabetes. This study explored the utility of nanoparticles presenting the α-gal (Galα1-3Galβ1-4GlcNAc-R) epitope to induce anti-Gal antibody-mediated local transient recruitment of macrophages to accelerate wound closure and healing in a diabetic murine model. α1,3galactosyltrasferase knockout mice were stimulated to produce anti-Gal antibodies and subsequently diabetes was induced by streptozotocin-induced β-cell destruction. Six mm full-thickness skin wounds were made and α-gal nanoparticles (AGN) were topically applied on postwounding days 0 and 1. Wounds were analysed histologically for macrophage invasion and markers of wound healing, including epithelialization, vascularization and granulation tissue deposition through postoperative day 12. We found that application of AGN transiently but significantly increased macrophage recruitment into the wounds of diabetic mice. Treated wounds demonstrated more rapid closure and enhanced wound healing as demonstrated by significantly accelerated rates of epithelialization, vascularization and granulation tissue deposition. Thus, topical treatment of full-thickness wounds in diabetic mice with α-gal nanoparticles induced a transient but significant increase in macrophage recruitment resulting in an accelerated rate of wound healing. Using α-gal nanoparticles as a topical wound healing adjunct is a simple, safe and effective means of augmenting dysregulated macrophage recruitment present in the diabetic state.
"Catastrophic-selection" is an evolutionary mechanism, by which entire parental-populations are eliminated but very few mutated offspring survive and replace extinct parental-populations. The human natural anti-carbohydrate antibodies, anti-Gal and anti-Neu5Gc suggest the occurrence of catastrophic-selection events in primate evolution. Parental-populations synthesizing corresponding carbohydrate-antigens underwent extinction in viral epidemics, and few offspring survived. These offspring carried accidental mutations that inactivated carbohydrate-antigen synthesis and produced natural-antibody against the lost antigen. Such natural anti-carbohydrate antibody was produced against environmental carbohydrate-antigens (e.g., gastrointestinal bacteria). The carbohydrate-antigen in infected parental-populations was also synthesized on viruses by the host glycosylation-machinery. The natural-antibody in the offspring bound to the carbohydrate-antigen on infecting viruses produced in parental-populations, destroyed the viruses and protected these offspring from extinction. This process occurred in ancestral Old-World monkeys and apes synthesizing α-gal epitopes, which were replaced 20-30 million-years-ago by offspring lacking α-gal epitopes and producing natural anti-Gal antibody against this antigen, and later in hominins synthesizing the sialic-acid antigen Neu5Gc, which were replaced by offspring lacking Neu5Gc and producing anti-Neu5Gc antibody. A present-day example for accidental mutations in very few humans that lost a common carbohydrate-antigen and produce a natural antibody against it is the rare blood-group "Bombay" individuals. These individuals lack the H-antigen (blood-group O) which is synthesized in all other humans, and produce the natural anti-H antibody against blood-group O. Overall, it is suggested that natural anti-carbohydrate antibodies played a critical role in preventing complete extinction of mammalian species in epidemics of highly virulent viruses and may have similar role in future events.
Expression of the alpha-gal epitope in mice can be completely eliminated by disruption of the alpha1,3 galactosyltransferase gene. As an initial step for assessing the feasibility of this approach in the pig, it was of interest to compare the expression of alpha-gal epitopes in pig and mouse organs. Membranes from pig and mouse organ homogenates were analyzed for alpha-gal epitope expression by Western blots, enzyme-linked immunosorbent assay (ELISA), immunostaining of tissues, and ELISA inhibition assay. Immunostaining of Western blots with human anti-Gal detected alpha-gal epitopes on glycoproteins from pig organs but not on glycoproteins from the corresponding mouse organs. ELISA with membrane homogenates and immunostaining of tissue sections demonstrated a much higher binding of human anti-Gal to alpha-gal epitopes on pig membranes than on mouse membranes. ELISA inhibition assay with monoclonal anti-Gal indicated that alpha-gal epitope expression in pig organs is up to 500-fold higher than in mouse organs. Expression of alpha-gal epitopes in pig organs is many fold higher than in mouse organs. The abundance of these epitopes in pigs raises the question of whether pigs can properly develop without expression of alpha-gal epitopes.
