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Anti-Gal: An abundant human natural antibody of multiple pathogeneses and clinical benefits
Abstract
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 "α-gal epitope" with the structure Galα1-3Galβ1-4GlcNAc-R. The α-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 α-gal epitopes. Aberrant expression of the α-gal epitope or of antigens mimicking it in humans may result in autoimmune processes, as in Graves' Disease. α-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 α-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 α-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 α-gal glycolipids which insert into tumor cell membranes. Anti-Gal binding to α-gal epitopes on tumor cells targets them for uptake by APC. Accelerated wound healing is achieved by application of α-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.
... The -gal glycolipids comprising these micelles spontaneously insert into the cell membrane of tumor cells in treated lesions. These glycolipids bind the abundant natural anti-Gal antibody [15] which targets tumor cells and cell membranes expressing the full range of TA for effective uptake by APC and their transport to regional lymph-nodes. The processing and presentation of TA peptides by these APC within lymph-nodes induces a protective immune response against metastatic tumor cells. ...
... Anti-Gal is an antibody that is naturally produced throughout life in humans [15,16] in response to antigenic stimulation by gastrointestinal bacteria [17][18][19]. Anti-Gal is produced in all humans who are not severely immunocompromised. ...
... (www.preprints.org) | NOT PEER-REVIEWED | Posted: 13 August 2024 doi:10.20944/preprints202408.0938.v115 ...
A major reason for failure of the immune system to detect tumor antigens (TA) is the insufficient uptake, processing, and presentation of TA by antigen-presenting-cells (APC). Immunogenicity of public and private TA of the individual patient can be markedly increased by in situ targeting of tumor cells for uptake by APC, without the need to identify and characterize the TA. This is feasible by intra-tumoral administration of alpha-gal micelles comprised of glycolipids presenting the carbohydrate-antigen “alpha-gal epitope” (Galalpha1-3Gal beta1-4GlcNAc-R). Humans produce a natural antibody called “anti-Gal” (constituting ~1% of immunoglobulins) which binds alpha-gal epitopes. Tumor injected alpha-gal micelles spontaneously insert into tumor cell membranes, so that multiple alpha-gal epitopes present on tumor cells. Anti-Gal binding to these epitopes activates the complement system, resulting in killing of tumor cells, and recruitment of multiple APC (dendritic cells and macrophages) into treated tumors by chemotactic complement cleavage peptides C5a and C3a. In this process of converting the treated tumor into personalized TA vaccine, recruited APC internalize anti-Gal opsonized tumor cells and cell membranes, process the internalized TA and transport them to regional lymph-nodes. TA peptides presented on APC activate TA specific T and B cells to proliferate and destroy metastatic tumor cells presenting the TA. Studies in anti-Gal producing mice demonstrated induction of effective protection against distant metastases of the highly tumorigenic B16 melanoma following injection of natural and synthetic alpha-gal micelles into primary tumors. This treatment was further found to synergize with anti-PD1 antibody. Phase-1 clinical-trials indicated that this immunotherapy is safe and can induce infiltration of CD4+ and CD8+ T cells into untreated distant metastases. It is suggested that in addition to converting treated metastases into autologous TA vaccine, this treatment should be considered as neo-adjuvant therapy administering alpha-gal micelles into primary tumors immediately following their detection. Such immunotherapy will convert tumors into personalized anti-TA vaccine for the period prior to their resection.
... These observations have led to the assumption that the regeneration-inducing capacity of macrophages, observed in urodeles, has been evolutionarily conserved in the early stages of mammalian life, but it is suppressed within few days after birth. Thus, it was suggested that identifying the causes for this suppression may restore the regenerative potential in the healing of injuries in adult mice [27,28]. ...
... Therefore, we hypothesized that extensive localized activation of the complement system by antigen/antibody interactions within injuries of adult mice may reactivate the suppressed ability of macrophages to become "pro-regenerative" cells which induce regenerative processes such as those observed in urodeles and neonatal mice. This assumption was studied Nanomaterials 2024, 14, 730 3 of 23 by performing in situ interaction between the anti-Gal antibody and α-gal nanoparticles presenting a carbohydrate antigen called the "α-gal epitope" [28,[33][34][35]. ...
... We hypothesized that the natural macrophage-mediated regeneration observed in injured tissues of urodeles and of mouse neonates can be reactivated in adult mice by the treatment of the injuries with α-gal nanoparticles [28,[33][34][35]. This hypothesis is illustrated in Figure 1B in which the α-gal nanoparticle-induced regenerative process consists of several steps. ...
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.
... The α-gal glycolipids comprising these micelles spontaneously insert into the cell membrane of tumor cells in treated lesions. These glycolipids bind the abundant natural anti-Gal antibody [19], which targets tumor cells and cell membranes expressing the full range of TAs for robust uptake by APCs and their transport to regional lymph nodes. The processing and presentation of TA peptides by these APCs within lymph nodes induces a protective immune response against metastatic tumor cells. ...
... Anti-Gal is an antibody that is naturally produced throughout life in humans [19,20] in response to antigenic stimulation by gastrointestinal bacteria [21][22][23]. Anti-Gal is produced in all humans who are not severely immunocompromised. ...
A major reason for the failure of the immune system to detect tumor antigens (TAs) is the insufficient uptake, processing, and presentation of TAs by antigen-presenting cells (APCs). The immunogenicity of TAs in the individual patient can be markedly increased by the in situ targeting of tumor cells for robust uptake by APCs, without the need to identify and characterize the TAs. This is feasible by the intra-tumoral injection of α-gal micelles comprised of glycolipids presenting the carbohydrate-antigen “α-gal epitope” (Galα1-3Galβ1-4GlcNAc-R). Humans produce a natural antibody called “anti-Gal” (constituting ~1% of immunoglobulins), which binds to α-gal epitopes. Tumor-injected α-gal micelles spontaneously insert into tumor cell membranes, so that multiple α-gal epitopes are presented on tumor cells. Anti-Gal binding to these epitopes activates the complement system, resulting in the killing of tumor cells, and the recruitment of multiple APCs (dendritic cells and macrophages) into treated tumors by the chemotactic complement cleavage peptides C5a and C3a. In this process of converting the treated tumor into a personalized TA vaccine, the recruited APC phagocytose anti-Gal opsonized tumor cells and cell membranes, process the internalized TAs and transport them to regional lymph-nodes. TA peptides presented on APCs activate TA-specific T cells to proliferate and destroy the metastatic tumor cells presenting the TAs. Studies in anti-Gal-producing mice demonstrated the induction of effective protection against distant metastases of the highly tumorigenic B16 melanoma following injection of natural and synthetic α-gal micelles into primary tumors. This treatment was further found to synergize with checkpoint inhibitor therapy by the anti-PD1 antibody. Phase-1 clinical trials indicated that α-gal micelle immunotherapy is safe and can induce the infiltration of CD4+ and CD8+ T cells into untreated distant metastases. It is suggested that, in addition to converting treated metastases into an autologous TA vaccine, this treatment should be considered as a neoadjuvant therapy, administering α-gal micelles into primary tumors immediately following their detection. Such an immunotherapy will convert tumors into a personalized anti-TA vaccine for the period prior to their resection.
... Plasma samples that were incubated with the mock immunosorbent assay showed reduced levels of T. cruzi-specific Ab. This is probably due to the elimination of Abs recognizing galactosyl residues of Sepharose ® 4B (a resin composed of agarose beads and a polymer of Gal β1,4 [3,6]-anhydro-L-galactose), since the production of anti-GAL Abs is strongly stimulated in T. cruzi infection [21,22]. ...
... Plasma samples that were incubated with the mock immunosorbent assay showed reduced levels of T. cruzi-specific Ab. This is probably due to the elimination of Abs recognizing galactosyl residues of Sepharose ® 4B (a resin composed of agarose beads and a polymer of Gal β1,4 [3,6]-anhydro-L-galactose), since the production of anti-GAL Abs is strongly stimulated in T. cruzi infection [21,22]. Figure 6 shows that, after 96 h of culture, the lower the level of T. cruzi-specific Ab were, the higher were the number of intracellular amastigotes. ...
Activated monocytes/macrophages that produce inflammatory cytokines and nitric oxide are crucial for controlling Trypanosoma cruzi infection. We previously showed that uninfected newborns from T. cruzi infected mothers (M+B- newborns) were sensitized to produce higher levels of inflammatory cytokines than newborns from uninfected mothers (M-B- newborns), suggesting that their monocytes were more activated. Thus, we wondered whether these cells might help limit congenital infection. We investigated this possibility by studying the activation status of M+B- cord blood monocytes and their ability to control T. cruzi in vitro infection. We showed that M+B- monocytes have an upregulated capacity to produce the inflammatory cytokine TNF-α and a better ability to control T. cruzi infection than M-B- monocytes. Our study also showed that T. cruzi-specific Abs transferred from the mother play a dual role by favoring trypomastigote entry into M+B- monocytes and inhibiting intracellular amastigote multiplication. These results support the possibility that some M+B- fetuses may eliminate the parasite transmitted in utero from their mothers, thus being uninfected at birth.
... Complete elimination of α-gal epitopes was achieved after the third decellularization cycle, as indicated by histological and quantification analysis results. α-Gal epitopes have been considered main immunogenic antigens and have been related to hyper-acute rejection when xeno-transplantation was performed in the past [63][64][65]. Currently, there is a great amount of effort to produce α-Gal-epitope-free knockout pigs, which can offer a solution for a global shortage of organs; however, this attempt is costly [66,67]. ...
... In this way, SDS has been shown to have the potential to interfere with α-gal epitopes, forcing their elimination by the tissue's ECM. Although efforts to remove animal-derived antigens from biological scaffolds have been made, α-gal epitopes have been found in transplants, such as heart valves, SIS-ECM, and vessel grafts, impairing the graft longevity after transplantation [63][64][65][66][67][68][69][70]. In this study, the proposed decellularization protocol successfully eliminated α-gal epitopes from the produced scaffolds, thus decreasing their potential immunogenicity upon implantation [68][69][70]. ...
Background: Functional restoration of abdominal wall defects represents one of the fundamental challenges of reconstructive surgery. Synthetic grafts or crosslinked animal-derived biological grafts are characterized by significant adverse reactions, which are mostly observed after their implantation. The aim of this study was to evaluate the efficacy of the decellularization protocol to produce a completely acellular full-thickness abdominal wall scaffold. Methods: Full-thickness abdominal wall samples were harvested from Wistar rats and submitted to a three-cycle decellularization process. Histological, biochemical, and DNA quantification analyses were applied to evaluate the effect of the decellularization protocol. Mechanical testing and immunogenicity assessment were also performed. Results: Histological, biochemical, and DNA analysis results showed efficient decellularization of the abdominal wall samples after the third cycle. Decellularized abdominal wall scaffolds were characterized by good biochemical and mechanical properties. Conclusion: The data presented herein confirm the effective production of a rat-derived full-thickness abdominal wall scaffold. Expanding this approach will allow the exploitation of the capacity of the proposed decellularization protocol in producing acellular abdominal wall scaffolds from larger animal models or human cadaveric donors. In this way, the utility of biological scaffolds with preserved in vivo remodeling properties may be one step closer to its application in clinical studies.
... Complete elimination of α-gal epitope was achieved after the performance of the 3 rd decellularization cycle, as it was indicated by histological and quantification analysis results. A-gal epitopes are considered as main immunogenic antigens and related to hyper-acute rejection when xeno-transplantation was performed in the past [59][60][61]. Currently, there is a great amount of performing efforts to produce α-Gal epitope free knockout pigs, which can offer a solution to a global shortage of organs, however, this attempt is costly [62,63]. ...
... In such a way, SDS has been shown that potential may interfere with the α-gal epitopes, forcing their elimination by the tissue's ECM. Although efforts in removing animal-derived antigens from biological scaffolds were performed, α-Gal epitopes have been found in transplants such as heart valves, SIS-ECM and vessel grafts, impairing the graft longevity after transplantation [59][60][61][62][63][64][65][66]. In the present study, the proposed decellularization protocol successfully eliminated the α-gal epitopes from the produced scaffolds, thus decreasing their potential immunogenicity upon implantation [64][65][66]. ...
Background: Functional restoration of abdominal wall defects represent one of the fundamental challenges of reconstructive surgery. Synthetic grafts or crosslinked animal-derived biological grafts are characterized by significant adverse reactions, which are mostly observed after their implantation. The aim of this study was to evaluate the efficacy of the decellularization protocol to produce a completely acellular full-thickness abdominal wall scaffold. Methods: Full-thickness abdominal wall samples were harvested from Wistar rats and submitted to a 3 cylce decellularization process. Histological, biochemical and DNA quantification analyses were applied to evaluate the effect of the decellularization protocol. Mechanical testing and immunogenicity assessment were also performed. Results: Histological, biochemical and DNA analysis results showed the efficient decellularization abdominal wall samples after the 3rd cycle. Decellularized abdominal wall scaffolds were characterized by good biochemical and mechanical properties Conclusion: The data presented herein confirm the effective production of rat-derived full-thickness abdominal wall scaffold. Expanding this approach will allow the exploitation of the capacity of the proposed decellularization protocol in producing acellular abdominal wall scaffolds from larger animal models or human cadaveric donors. In this way, the utility of biological scaffolds with preserved in vivo remodelling properties may be one step closer to its application in clinical studies.
... As it is evident from the fossils that the process of development of red meat allergy due to tick bites started 28 million years ago. The enzyme responsible for the production of alpha-gal was inactivated in our ancestors at that time, this is why the human body gets alpha-gal as a pathogenic particle, and alpha-gal IgE antibodies are produced, hence giving defense to the pathogenic bacteria, coated viruses and protozoa that contain alpha-gal (Galili 2013). As per available literature, alpha-gal is an external particle for humans that prepares them after bites from ticks and initiates the pro-allergy Th2 cells cytokines in the humans that starts the preparation of anti-alpha-gal antibodies (Abs) by the IgG and ultimately the IgE Abs from B cells (Ferreira and Silva 1999). ...
... Alpha-gal is a component of many drugs and medicines and can be dangerous in some new therapies for the persons who are allergic to alpha-gal (Galili 2013). ◆ Because of the alpha-gal present in cetuximab, dangerous reactions appeared by its intake. ...
... As it is evident from the fossils that the process of development of red meat allergy due to tick bites started 28 million years ago. The enzyme responsible for the production of alpha-gal was inactivated in our ancestors at that time, this is why the human body gets alpha-gal as a pathogenic particle, and alpha-gal IgE antibodies are produced, hence giving defense to the pathogenic bacteria, coated viruses and protozoa that contain alpha-gal (Galili 2013). As per available literature, alpha-gal is an external particle for humans that prepares them after bites from ticks and initiates the pro-allergy Th2 cells cytokines in the humans that starts the preparation of anti-alpha-gal antibodies (Abs) by the IgG and ultimately the IgE Abs from B cells (Ferreira and Silva 1999). ...
... Alpha-gal is a component of many drugs and medicines and can be dangerous in some new therapies for the persons who are allergic to alpha-gal (Galili 2013). ◆ Because of the alpha-gal present in cetuximab, dangerous reactions appeared by its intake. ...
... As it is evident from the fossils that the process of development of red meat allergy due to tick bites started 28 million years ago. The enzyme responsible for the production of alpha-gal was inactivated in our ancestors at that time, this is why the human body gets alpha-gal as a pathogenic particle, and alpha-gal IgE antibodies are produced, hence giving defense to the pathogenic bacteria, coated viruses and protozoa that contain alpha-gal (Galili 2013). As per available literature, alpha-gal is an external particle for humans that prepares them after bites from ticks and initiates the pro-allergy Th2 cells cytokines in the humans that starts the preparation of anti-alpha-gal antibodies (Abs) by the IgG and ultimately the IgE Abs from B cells (Ferreira and Silva 1999). ...
... Alpha-gal is a component of many drugs and medicines and can be dangerous in some new therapies for the persons who are allergic to alpha-gal (Galili 2013). ◆ Because of the alpha-gal present in cetuximab, dangerous reactions appeared by its intake. ...
Red meat allergy is different from other conventional food
allergies. Tick bites play a role in triggering this disease, but
this association is not fully proven yet. It has been
diagnosed across the world but is more prevalent in areas
abundant with ticks’ population. The role of alpha-gal in the
development of mammalian meat allergy after tick bites has
strong scientific evidence. The reactions might appear
immediately when the medicines (containing alpha-gal) are
given via the parenteral route, and there is a delay in the
appearance of the symptoms from 3-6 hours if meat from
mammals, dairy, and other mammal-derived products are
consumed via the oral route. The best management of this
syndrome is to avoid further tick bites, mammalian meat,
and other mammal-derived products.
... Anti-Gal is one of the most abundant natural antibodies in humans, constituting 1% of immunoglobulins [26][27][28][29][30]. It is produced throughout life, starting few months after birth [26,31], in response to antigenic stimulation by gastrointestinal bacteria [32][33][34][35][36][37]. ...
... It is produced throughout life, starting few months after birth [26,31], in response to antigenic stimulation by gastrointestinal bacteria [32][33][34][35][36][37]. Anti-Gal binds specifically to a carbohydrate antigen called the "α-gal epitope" with the structure Galα1-3Galβ1-4GlcNAc-R [29,[38][39][40][41]. Whereas anti-Gal is naturally produced in all humans, apes, and Old-World monkeys (monkeys of Asia and Africa), the α-gal epitope is synthesized as~10 5 -10 7 epitopes/cell in nonprimate mammals (marsupials and placentals), lemurs (living in Madagascar) and New-World monkeys (monkeys of South-America), all of which lack the anti-Gal antibody [42][43][44]. ...
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.
... 61,67 Healthy individuals also produce anti-gal Abs against the Gram-negative enterobacteria of the human flora. 15 However, the normal human serum (NHS) anti-α-Gal Abs have less affinity for the parasite antigens than the CD anti-α-gal antibodies. 67,68 On a molar basis, a 100-fold difference was reported for the binding of CD anti-α-gal to a mucin fraction, in comparison to that of NHS anti-α-gal Abs. ...
... 69,70 The affinity of NHS anti-α-gal antibodies to the free trisaccharide α-Galp-(1 → 3)-β-D-Galp-(1 → 4)-D-GlcNAc (1) is approximately 7-fold higher than that to the disaccharide Galp-(1 → 3)-β-D-Galp (2). 12,15 The enterobacteria predominantly express terminal α-D-Galp linked 1 → 2, 1 → 4, and 1 → 6 to the rest of the glycan in the lipopolysaccharide of the enterobacteria. 71 These diverse structures may explain the different responses of the natural human antibodies to CD antigens. ...
The α-gal epitope, which refers to the carbohydrate α-d-Galp-(1 → 3)-β-d-Galp-(1 → 4)-d-GlcNAc-R, was first described in the glycoconjugates of mammals other than humans. Evolution caused a mutation that resulted in the inactivation of the α-1,3-galactosyltransferase gene. For that reason, humans produce antibodies against α-d-Galp containing glycoproteins and glycolipids of other species. We summarize here the glycoconjugates with α-d-Galp structures in Trypanosoma, Leishmania, and Plasmodium pathogenic protozoa. These were identified in infective stages of Trypanosoma cruzi and in Plasmodium sporozoites. In Leishmania, α-d-Galp is linked differently in the glycans of glycoinositolphospholipids (GIPLs). Chemically synthesized neoglycoconjugates have been proposed as diagnostic tools and as antigens for vaccines. Several syntheses reported for the α-gal trisaccharide, also called the Galili epitope, and the glycans of GIPLs found in Leishmania, the preparation of neoglycoconjugates, and the studies in which they were involved are also included in this Review.
... Alpha-1,3-galactosyltransferase, encoded by GGTA1, is an enzyme that synthesizes galactose-alpha-1,3-galactose (α-gal), a common glycosylation structure in mammals and bacteria. Due to a mutation in GGTA1, humans lack expression of α-gal and instead anti-α-gal antibodies naturally occur in substantial titers, making α-gal one of the most potent immune activators known in humans [7][8][9]. ...
Oncolytic adenovirus has been widely evaluated as a cancer treatment agent with tolerable toxicity profile. We have recently developed a new oncolytic adenovirus Adf35(OGN) with two immunostimulatory transgenes alpha-1,3-galactosyltransferase (GGTA1) from Sus scrofa and neutrophil-activating protein (NAP) from Helicobacter pylori. Adf35(OGN) can kill tumor cells and trigger a strong immune response against tumor antigens. Here, we report the toxicity and biodistribution of Adf35(OGN) in Syrian hamster and GGTA1-knockout mouse. The virus was delivered subcutaneously in naïve hamsters and intratumorally in GGTA1-knockout mouse in multiple doses at dosages of 1–5 × 10¹¹ viral particles (VP)/kg. The virus did not replicate in any tissues, evidenced as low or no viral copies detected by qPCR. The virus was also found at low levels in biofluids (saliva, urine, and feces), indicating that spread to the environment is low with a low risk of secondary infections via shedding. The virus did not cause any biochemical, hematological, or histopathological alterations. In summary, Adf35(OGN) has a good safety profile in these animal models and these results support future clinical evaluation for Adf35(OGN).
... 65 A subclass of XNAs known as natural anti-Gal antibodies is particularly important for rejection responses. 66 They specifically target the terminal carbohydrate portion of Gal, a sugar molecule abundantly produced in the cell membranes of most animals, including pigs. 67 Interestingly, a genetic mutation in the a1,3-galactosyltransferase gene (GGTA1) causes Gal absence in humans. ...
Corneal diseases represent a significant cause of blindness worldwide, with corneal transplantation being an effective treatment to prevent vision loss. Despite substantial advances in transplantation techniques, the demand for donor corneas exceeds the available supply, particularly in developing countries. Cornea xenotransplantation has emerged as a promising strategy to address the worldwide scarcity, notably using porcine corneas. In addition to the inherent immune privilege of the cornea, the low cost of porcine breeding and the anatomical and physiological similarities between humans and pigs have made porcine corneas a viable alternative. Nonetheless, ethical concerns, specifically the risk of xenozoonotic transmission and the necessity for stringent biosafety measures, remain significant obstacles. Moreover, the success of xenotransplantation is compromised by innate and adaptive immune responses, which requires meticulous consideration and further studies. Despite these challenges, recent breakthroughs have further contributed to reducing immunogenicity while preserving the corneal architecture. Advances in genetic engineering, such as the use of CRISPR-Cas9 to eliminate critical porcine antigens, have shown promise for mitigating immune reactions. Additionally, new immunosuppressive protocols, such as have techniques like decellularization and the use of porcine-derived acellular matrices, have greatly increased graft survival in preclinical models. Future research must focus on refining immunomodulatory strategies and improving graft preparation techniques to ensure the long-term survival and safety of porcine corneal xenotransplantation in clinical trials in humans.
... 5 Consequently, anti-α-Gal IgG, IgM, and IgA antibodies have been identified in human serum 6 and IgA in secretions such as saliva, milk, vaginal secretion etc.. 7 These antibodies are present in abundant quantities; in addition to their protective role they can also be detrimental in certain cases such as rejection of mammalian grafts in humans. 8 Clinical observation in humans as well as tick feeding on α-GALT-KO mice has clearly shown induction of α-Gal-specific IgE by tick bites. [9][10][11][12] Therefore, frequent exposure to tick bites can possibly increase the chances of sensitization. ...
Background
The α‐Gal syndrome (AGS) is characterized by the presence of specific IgE‐antibodies to the carbohydrate galactose‐α‐1,3‐galactose (α‐Gal). Sensitization to α‐Gal has been associated with tick bites and individuals exposed to ticks have an elevated risk of sensitization. The aim of this study was to analyze IgG and IgE antibody responses to α‐Gal in a high‐risk cohort of forestry employees (FE) in Luxembourg.
Methods
Questionnaires and serum samples of FE from Luxembourg (n = 219) were retrospectively analyzed. α‐Gal specific IgE was quantified by ImmunoCAP, α‐Gal specific IgG and subclasses IgG1–4 were determined by ELISA. Additionally, sera from population‐based controls (n = 150) and two groups of food‐allergic patients, patients with AGS (n = 45) and fish‐allergic patients (n = 22) were assessed for IgG antibody responses to α‐Gal and cod extract.
Results
Twenty‐one percent of FE was sensitized to α‐Gal (sIgE ≥ 0.1 kUA/L). Both sensitized and non‐sensitized FE exhibited high levels of α‐Gal specific IgG, IgG1 and IgG3 compared with controls, indicating a stimulation of IgG responses by recurrent tick bites, independent of the sensitization status. AGS patients had the highest levels of IgG1 and IgG2 antibodies, whereas the profile of fish‐allergic patients was similar to the profile of the controls for which anti‐α‐Gal responses were dominated by IgG2 antibodies. α‐Gal sIgG4 levels were either very low or undetectable in all groups.
Conclusion
Our study provides evidence for a continuous stimulation of α‐Gal related immune responses by repeated tick bites, translating into highly elevated levels of IgG1 antibodies directed against α‐Gal.
... Alpha-Gal epitopes (Galα1-3Galβ1-4GlcNAc-R) are specific carbohydrate structures found on the cell surface of non-primate mammals, such as pigs. These epitopes are absent in humans and Old-World monkeys due to mutations in the α1,3-galactosyl-transferase gene [64,65]. The absence of alpha-Gal epitopes in humans leads to the production of anti-Gal antibodies (approximately represent 1 % of serum antibodies), primarily due to exposure to intestinal bacteria carrying these epitopes [66]. ...
Liver transplantation represents the only definitive treatment for patients with end-stage liver disease. However, the shortage of liver donors provokes a dramatic gap between available grafts and patients on the waiting list. Whole liver bioengineering, an emerging field of tissue engineering, holds great potential to overcome this gap. This approach involves two main steps; the first is liver decellularization and the second is recellularization. Liver decellularization aims to remove cellular and nuclear materials from the organ, leaving behind extracellular matrices containing different structural proteins and growth factors while retaining both the vascular and biliary networks. Recellularization involves repopulating the decellularized liver with appropriate cells, theoretically from the recipient patient, to reconstruct the parenchyma, vascular tree, and biliary network. The aim of this review is to identify the major advances in decellularization and recellularization strategies and investigate obstacles for the clinical application of bioengineered liver, including immunogenicity of the designed liver extracellular matrices, the need for standardization of scaffold fabrication techniques, selection of suitable cell sources for parenchymal repopulation, vascular, and biliary tree reconstruction. In vivo transplantation models are also summarized for evaluating the functionality of bioengineered livers. Finally, the regulatory measures and future directions for confirming the safety and efficacy of bioengineered liver are also discussed. Addressing these challenges in whole liver bioengineering may offer new solutions to meet the demand for liver transplantation and improve patient outcomes.
... In healthy humans, anti-α-Gal antibodies, specifically of the IgG, IgM, and IgA isotypes, are naturally produced as part of the immune response to continuous exposure to Gram-negative bacteria present in the gut flora. These bacteria express a wide range of α-Gal-linked glycans, primarily in Galα1,2-, Galα1,4-, and Galα1,6-R forms [107,108]. Pacheco et al. [107] recently provided evidence demonstrating the protective effect of probiotics with high α-Gal content against mycobacteriosis caused by M. marinum. They identified and isolated native Aeromonas veronii and Pseudomonas entomophila bacteria, both rich in α-Gal, from the gut of zebrafish. ...
Aquaculture is a growing industry worldwide, but it faces challenges related to animal health. These challenges include infections by parasites, bacteria, and viral pathogens. These harmful pathogens have devastating effects on the industry, despite efforts to control them through vaccination and antimicrobial treatments. Unfortunately, these measures have proven insufficient to address the sanitary problems, resulting in greater environmental impact due to the excessive use of antimicrobials. In recent years, probiotics have emerged as a promising solution to enhance the performance of the immune system against parasitic, bacterial, and viral pathogens in various species, including mammals, birds, and fish. Some probiotics have been genetically engineered to express and deliver immunomodulatory molecules. These promote selective therapeutic effects and specific immunization against specific pathogens. This review aims to summarize recent research on the use of probiotics in fish aquaculture, with a particular emphasis on genetically modified probiotics. In particular, we focus on the advantages of using these microorganisms and highlight the main barriers hindering their widespread application in the aquaculture industry.
... However, following exposure to certain tick bites, innate anti-Gal-producing B cells can undergo isotype switching to produce anti-Gal IgE. These newly formed anti-Gal IgE molecules can then bind to alpha-gal, including that found in common perioperative medications, and induce a hypersensitivity reaction [15][16]. These anti-Gal immunoglobulins are the most abundant naturally occurring antibodies (accounting for roughly 1% of immunoglobulins). ...
Galactose-⍺-1, 3-galactose (alpha-gal) is an oligosaccharide found in mammalian tissues that causes allergic reactions in patients with alpha-gal syndrome (AGS). AGS is a hypersensitivity reaction notable for both immediate and delayed allergic and anaphylactic symptoms. As a tick-based disease, AGS has gained increasing prevalence across the United States and can have a significant influence on which medications are safe for patients. Many medications used within the operating room and intensive care units have inactive ingredients that can be mammalian-derived and therefore should be vetted before administering to patients with AGS. Management of patients with AGS involves diligent action in the preoperative and perioperative settings to reduce patient exposure to potentially harmful medications. In conducting a comprehensive risk stratification assessment, the anesthesia team should identify any at-risk patients and determine which medications they have safely tolerated in the past. Despite obtaining a complete history, not all patients with AGS will be identified preoperatively. The perioperative team should understand which common medications pose a risk of containing alpha-gal moieties (e.g., heparins, gelatin capsules, vaccines, lidocaine patches, surgifoam, etc. ). For this reason, this paper includes a compendium of common anesthetic medications that have been cross-referenced for ingredients that have the potential to cause an AGS reaction. Any potentially unsafe medications have been identified such that medical providers can cross-reference with the ingredients listed at their respective institutions.
... Humans naturally acquire antibodies against several porcine carbohydrate antigens that share antigenicity with environmental microbes and food (12,13). Galactose-a1,3-galactose (Gal) and, to a lesser extent, N-glycolylneuraminic acid (Neu5Gc) and Sd(a) are expressed on the surface of porcine cells, and humans naturally acquire antibodies against them (14). ...
The decline in blood donation rates and the ongoing shortage of blood products pose significant challenges to medical societies. One potential solution is to use porcine red blood cells (pRBCs) from genetically modified pigs as an alternative to human red blood cells (hRBCs). However, adverse immunological reactions remain a significant obstacle to their use. This study aimed to evaluate the compatibility of diverse genetically modified pRBCs with human serum. We acquired human complement-competent serum, complement 7 (C7)-deficient serum, and hRBCs from all ABO blood types. Additionally, we used leftover clinical samples from health checkups for further evaluation. pRBCs were collected from wild-type (WT) and genetically modified pigs: triple knockout (TKO), quadruple KO (QKO), and TKO/hCD55.hCD39 knockin (hCD55.hCD39KI). The extent of C3 deposition on RBCs was measured using flow cytometry after incubation in C7-deficient serum diluted in Ca⁺⁺-enriched or Ca⁺⁺-depleted and Mg⁺⁺-enriched buffers. The binding of immunoglobulin (Ig) M/IgG antibody to RBCs after incubation in ABO-type human serum was evaluated using flow cytometry. Naïve human serum- or sensitized monkey serum-mediated hemolysis was also evaluated. Phagocytosis was assessed by incubating labeled RBCs with the human monocytic cell line THP-1 and measurement by flow cytometry. All three genetic modifications significantly improved the compatibility of pRBCs with human serum relative to that of WT pRBCs. The extent of IgM/IgG binding to genetically modified pRBCs was lower than that of WT pRBCs and similar to that of O-type hRBCs. Total and alternative pathway complement activation in all three genetically modified pRBCs was significantly weaker than that in WT pRBCs and did not differ from that in O-type hRBCs. The extent of serum-mediated hemolysis and phagocytosis of these genetically modified pRBCs was low and similar to that of O-type hRBCs. Sensitized monkey serum-mediated hemolysis in QKO and TKO/hCD55.hCD39KI pRBCs was higher than in O-type hRBCs but lower than in TKO pRBCs. The elimination of porcine carbohydrate antigens in genetically modified pigs significantly enhanced pRBC compatibility with naïve human sera, which was comparable to that of O-type hRBCs. These findings provide valuable insights into the development of pRBCs as potential alternatives to hRBCs.
... As before, Cmas −/− TSC revealed a massively reduced viability in complement active bovine serum, while complement active murine serum supported growth of Cmas −/− TSC under differentiation conditions (Fig. S4A). Since immunoglobulins (Ig) are a known activator of complement, especially in xenogenic conditions [11], we examined whether bovine Ig recognize murine cell surface components. Immunofluorescence analysis of cell surface bound bovine Ig revealed that Cmas −/− TSC cultivated in complement active FCS indeed show reactivity for bovine Ig (Fig. S4B). ...
The complement system is a part of the innate immune system in the fluid phase and efficiently eliminates pathogens. However, its activation requires tight regulation on the host cell surface in order not to compromise cellular viability. Previously, we showed that loss of placental cell surface sialylation in mice in vivo leads to a maternal complement attack at the fetal-maternal interface, ultimately resulting in loss of pregnancy. To gain insight into the regulatory function of sialylation in complement activation, we here generated trophoblast stem cells (TSC) devoid of sialylation, which also revealed complement sensitivity and cell death in vitro. Glycolipid-analysis by multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection (xCGE-LIF) allowed us to identify the monosialoganglioside GM1a as a key element of cell surface complement regulation. Exogenously administered GM1a integrated into the plasma membrane of trophoblasts, substantially increased binding of complement factor H (FH) and was sufficient to protect the cells from complement attack and cell death. GM1a treatment also rescued human endothelial cells and erythrocytes from complement attack in a concentration dependent manner. Furthermore, GM1a significantly reduced complement mediated hemolysis of erythrocytes from a patient with Paroxysmal nocturnal hemoglobinuria (PNH). This study demonstrates the complement regulatory potential of exogenously administered gangliosides and paves the way for sialoglycotherapeutics as a novel substance class for membrane-targeted complement regulators.
... Therefore, alpha-Gal epitopes are not present on human glycoproteins and glycolipids, and this allows humans to mount an immune response against foreign alpha-Gal-containing antigens, and is suggested to be an evolutionarily selected process for gaining resistance to pathogens such as Plasmodium sporozoites that contain these glycan modifications [34,35]. Alpha-Gal-containing glycoproteins presented on outer membranes of bacteria that are part of the intestinal microbiome also elicit a humoral response [31,36] and anti-alpha-Gal antibodies circulate in different forms, including IgM, IgA, and IgG in diverse human secretions and in mucosa [31,37,38]. By contrast, AGS is caused by a specific IgE (sIgE) antibody response targeting alpha-Gal antigens [30,39,40]. ...
... Humans have a high frequency of memory B cells producing antibodies to alpha-Gal epitopes 36 . Thus, we assumed that polyclonal in vitro B cell activation by CpG might induce detectable levels of anti-pig antibodies. ...
Transgenic expression of protective molecules in porcine cells and tissues is a promising approach to prevent xenograft rejection. Viruses have developed various strategies to escape the host’s immune system. We generated porcine B cells (B cell line L23) expressing the human adenovirus protein E3/49K or the human cytomegalovirus protein pUL11 and investigated how human T, NK and B cell responses are affected by the expression of the viral proteins. Binding studies revealed that E3/49K and pUL11 interact with CD45 on human but not porcine peripheral blood mononuclear cells. T cell proliferation in response to L23-E3/49K cells was significantly reduced and accompanied by development of an anti-inflammatory cytokine milieu (low: TNF-alpha, IFN-gamma, IL-6; high: IL-4, IL-10). Human peripheral blood mononuclear cells which had been primed for four weeks by L23-E3/49K cells included an extended population of regulatory T cells. Cytotoxicity of effector T and natural killer cells against L23 cells was significantly reduced (40 to 50%) by E3/49K expression. B cell activation and antibody production to E3/49K expressing cells was also diminished. Surprisingly, pUL11 expression showed no effects. Reduction of human anti-pig immune responses by transgenic expression of selected viral genes may be a novel approach for protection of porcine xenografts.
... Due to the mutation of the alpha-1,3-galactosyltransferase gene in humans and Old World Monkeys over 20 million years ago, alpha-gal became immunogenic to humans allowing the production of anti-alpha-gal antibodies, usually of the IgG type [20,21]. Factors contributing to sIgE formation are of higher interest because there are patients with alpha-gal syndrome. ...
Background: Tick bite-induced IgE-mediated reactions to the oligosaccharide galactose α-1,3-galactose (alpha-gal) are increasingly recognized. This study investigated alpha-gal sensitization in three groups with different tick bite exposure. Materials and methods: Specific IgE antibodies to alpha-gal and total IgE were investigated in 485 patients with Lyme borreliosis with different disease manifestations and compared to a control group of 200 randomly selected patients without increased exposure to tick bites. A group of 232 hunters and forest workers served as a model for multiple tick bites. Results: Specific IgE (sIgE) antibodies to alpha-gal (> 0.1 kU/L) were found in 12.6% of all borreliosis samples compared to the control group with 9% (relative risk 1.4; 95% CI 0.85 – 2.3; not significant (n.s.). The highest prevalence of sIgE to alpha-gal was observed in hunters and forest service employees (22.8%, relative risk 2.5; 95% CI 1.5 – 4.2; p < 0.001). Higher age and elevated total IgE were also associated with alpha-gal sensitization. Conclusion: IgE sensitization to alpha-gal tends to be more frequent in tick-exposed patients with borreliosis than in controls (n.s.). Moreover, hunters and forest workers show an even higher rate of elevated IgE to alpha-gal. Thus, frequent tick contact may result in alpha-gal sensitization. In the area of Munich, the prevalence of alpha-gal sensitization appears lower than in the state of Baden-Württemberg and lower than in the USA, which may be due to the difference in tick species or the frequency of tick exposure. This study could show that alpha-gal sensitization and presumably alpha-gal syndrome does not seem to be a modern problem but existed already more than 30 years ago.
... However, the establishment of CD also depends on a favorable environment for intracellular parasite proliferation, avoiding immune elimination (Chain et al., 2020). The persistence of the protozoan in the indeterminate and chronic phases yields antigens (Ags) and Pattern Recognition Receptor (PRR) ligands that induce extensive localized inflammatory reactions, characterized by macrophage and T lymphocyte (TL) infiltration, contributing to the pathogenesis of the disease (Galili, 2013;Macaluso et al., 2023). This has been associated with immune cell dysfunction (Duran-Rehbein et al., 2017). ...
... These immunizing bacteria comprise~25% of the fecal material (Gerritsen et al., 2011). Among the most known natural anti-carbohydrate antibodies are anti-blood-group A (anti-A), anti-blood-group B (anti-B) (Springer, 1971;Watkins, 1980), anti-Gal (Galili et al., 1984;McMorrow et al., 1997a;Parker et al., 1999;Galili, 2013a), anti-Forssman (Young et al., 1979Kijimoto-Ochiai et al., 1981), and anti-N-glycolyl neuraminic acid (anti-Neu5Gc) antibodies (Merrick et al., 1978;Zhu and Hurst, 2002;Nguyen et al., 2005;Padler-Karavani et al., 2008). 2) A second important difference is that immunogenic protein antigens can activate both cytotoxic and helper T cells. ...
This review describes the significance of the α-gal epitope (Galα-3Galβ1-4GlcNAc-R) as the core of human blood-group A and B antigens (A and B antigens), determines in mouse models the principles underlying the immune response to these antigens, and suggests future strategies for the induction of immune tolerance to incompatible A and B antigens in human allografts. Carbohydrate antigens, such as ABO antigens and the α-gal epitope, differ from protein antigens in that they do not interact with T cells, but B cells interacting with them require T-cell help for their activation. The α-gal epitope is the core of both A and B antigens and is the ligand of the natural anti-Gal antibody, which is abundant in all humans. In A and O individuals, anti-Gal clones (called anti-Gal/B) comprise >85% of the so-called anti-B activity and bind to the B antigen in facets that do not include fucose-linked α1–2 to the core α-gal. As many as 1% of B cells are anti-Gal B cells. Activation of quiescent anti-Gal B cells upon exposure to α-gal epitopes on xenografts and some protozoa can increase the titer of anti-Gal by 100-fold. α1,3-Galactosyltransferase knockout (GT-KO) mice lack α-gal epitopes and can produce anti-Gal. These mice simulate human recipients of ABO-incompatible human allografts. Exposure for 2–4 weeks of naïve and memory mouse anti-Gal B cells to α-gal epitopes in the heterotopically grafted wild-type (WT) mouse heart results in the elimination of these cells and immune tolerance to this epitope. Shorter exposures of 7 days of anti-Gal B cells to α-gal epitopes in the WT heart result in the production of accommodating anti-Gal antibodies that bind to α-gal epitopes but do not lyse cells or reject the graft. Tolerance to α-gal epitopes due to the elimination of naïve and memory anti-Gal B cells can be further induced by 2 weeks in vivo exposure to WT lymphocytes or autologous lymphocytes engineered to present α-gal epitopes by transduction of the α1,3-galactosyltransferase gene. These mouse studies suggest that autologous human lymphocytes similarly engineered to present the A or B antigen may induce corresponding tolerance in recipients of ABO-incompatible allografts. The review further summarizes experimental works demonstrating the efficacy of α-gal therapies in amplifying anti-viral and anti-tumor immune-protection and regeneration of injured tissues.
... Therefore an effective removal of DNA and partial deglycosylation can overcome rapid destruction of xenogeneic tissues, and protocols can be developed intensely investigating immune response [27][28][29]. Immune reactions against xenoantigens are driven by preformed IgM antibodies, activating the complement system, leading to an immediate response [30,31]. These rapid reactions make in vitro incubation assays with human whole blood a powerful tool for first-line evaluation of xenograft developments [32][33][34]. ...
Hemocompatibility tuning was adopted to explore and refine an innovative, GA-free preparation strategy combining decellularization, riboflavin/UV crosslinking, and low-energy electron irradiation (SULEEI) procedure. A SULEEI-protocol was established to avoid GA-dependent deterioration that results in insufficient long-term aortic valve bioprosthesis durability. Final SULEEI-pericardium, intermediate steps and GA-fixed reference pericardium were exposed in vitro to fresh human whole blood to elucidate effects of preparation parameters on coagulation and inflammation activation and tissue histology. The riboflavin/UV crosslinking step showed to be less efficient in inactivating extracellular matrix (ECM) protein activity than the GA fixation, leading to tissue-factor mediated blood clotting. Intensifying the riboflavin/UV crosslinking with elevated riboflavin concentration and dextran caused an enhanced activation of the complement system. Yet activation processes induced by the previous protocol steps were quenched with the final electron beam treatment step. An optimized SULEEI protocol was developed using an intense and extended, trypsin-containing decellularization step to inactivate tissue factor and a dextran-free, low riboflavin, high UV crosslinking step. The innovative and improved GA-free SULEEI-preparation protocol results in low coagulant and low inflammatory bovine pericardium for surgical application.
... In transfers made using this method, immunological tissue compatibility is important for the tissue to survive. Especially in vascularized organ studies, materials taken from primates cannot be used due to the risk of hyperacute rejection caused by anti-GAL antibodies generated in mammals against the a-1,3-galactosyl transferase (ABO blood group) enzyme, which is only present in primates [9]. When xenogenic material's tissue compatibility is required in animal experiments, athymic animals are selected to suppress immune reactions. ...
The platelet-rich plasma (PRP) has become popular in the medical world due to its content of growth factors and numerous studies are experimental. In experimental studies, the preparation and application of PRP are problematic and allogenic PRP transfers have been preffered, because of the difficulties in preparation of autogenic PRP in animal experiments. Xenogenic transfers and their effects have not been studied in this topic. This study aimed to investigate the effect of autogenic and xenogenic use of PRP on composite graft viability.
Methods: Two composite grafts are prepared for each ear of nine rabbits. Each ear was randomly divided into three groups. After the procedure, the wound edges and base were injected with 1 cc serum physiologic, autogenic PRP or 1 cc human-derived xenogenic PRP. At 3 weeks, samples were taken, photographic and histopathological evaluations were made.
Results: The graft viability was better in autogenic and xenogenic group compared to the control group. In comprasion of autogenic and xenogenic groups, although the macroscopic evaluation revealed better graft viability and less necrosis in the group which had been treated with autogenic PRP, the difference was not statistically significant. The three groups did not significantly differ in terms of inflammation. Vascularization examined histopathologically. CD31 staining, which was used to evaluate angiogenesis, was significantly higher in the autogenic PRP group than the remaining two groups.
Conclusion: Although autogenic PRP has better results histopathologically, the xenogenic use of PRP may be an alternative for studies, when macroscopic evaluation is necessary.
... The carbohydrate epitope Gala1-3Galb1-4GlcNAc-R (α-gal) is a heterologous xenoantigen that causes hyperacute rejection. It is widely present in new world monkeys and non-primate mammalian species, but not in humans because the α-(1,3)-galactosyltransferase (α-1,3GT) that catalyses the synthesis of α-gal was inactivated during evolution [13][14][15]. The incubation of the cancer cells engineered to express the α-1,3GT gene with human serum, containing abundant naturally existing anti-α-gal epitope antibodies, results in immediate initiation of the complement system through the classical pathway and the subsequent lysis of tumour cells due to CDC. ...
The overexpression of membrane-bound complement regulatory proteins (mCRPs) on tumour cells helps them survive complement attacks by suppressing antibody-mediated complement-dependent cytotoxicity (CDC). Consequently, mCRP overexpression limits monoclonal antibody drug immune efficacy. CD55, an mCRP, plays an important role in inhibiting antibody-mediated CDC. However, the mechanisms regulating CD55 expression in tumour cells remain unclear. Here, the aim was to explore CD55-targeting miRNAs. We previously constructed an in vitro model comprising cancer cell lines expressing α-gal and serum containing natural antibodies against α-gal and complement. This was used to simulate antibody-mediated CDC in colon cancer cells. We screened microRNAs that directly target CD55 using LoVo and Ls-174T colon cell lines, which express CD55 at low and high levels, respectively. miR-132-3p expression was dramatically lower in Ls-174T cells than in LoVo cells. miR-132-3p overexpression or inhibition transcriptionally regulated CD55 expression by specifically targeting its mRNA 3'-untranslated regions. Further, miR-132-3p modulation regulated colon cancer cell sensitivity to antibody-mediated CDC through C5a release and C5b-9 deposition. Moreover, miR-132-3p expression was significantly reduced, whereas CD55 expression was increased, in colon cancer tissues compared to levels in adjacent normal tissues. CD55 protein levels were negatively correlated with miR-132-3p expression in colon cancer tissues. Our results indicate that miR-132-3p regulates colon cancer cell sensitivity to antibody-mediated CDC by directly targeting CD55. In addition, incubating the LoVo human tumour cell line, stably transfected with the xenoantigen α-gal, with human serum containing natural antibodies comprises a stable and cheap in vitro model to explore the mechanisms underlying antibody-mediated CDC.
... The indicated triggers might include native xenogeneic epitopes such as galactoseα-1,3-galactose and Nglycolylneuraminic acid as well as neoepitopes formed by citrullination. Previously, it has been shown that galactoseα-1,3-galactose and N-glycolylneuraminic acid cause immune rejection of xenogeneic transplants, 25,26 including glutaraldehyde-treated BHVs. 27,28 However, Russell-Movat's pentachrome staining did not detect any glycosaminoglycans in the control leaflets of XPB-BHVs that have not been implanted. ...
Background
Whereas the risk factors for structural valve degeneration (SVD) of glutaraldehyde‐treated bioprosthetic heart valves (BHVs) are well studied, those responsible for the failure of BHVs fixed with alternative next‐generation chemicals remain largely unknown. This study aimed to investigate the reasons behind the development of SVD in ethylene glycol diglycidyl ether–treated BHVs.
Methods and Results
Ten ethylene glycol diglycidyl ether–treated BHVs excised because of SVD, and 5 calcified aortic valves (AVs) replaced with BHVs because of calcific AV disease were collected and their proteomic profile was deciphered. Then, BHVs and AVs were interrogated for immune cell infiltration, microbial contamination, distribution of matrix‐degrading enzymes and their tissue inhibitors, lipid deposition, and calcification. In contrast with dysfunctional AVs, failing BHVs suffered from complement‐driven neutrophil invasion, excessive proteolysis, unwanted coagulation, and lipid deposition. Neutrophil infiltration was triggered by an asymptomatic bacterial colonization of the prosthetic tissue. Neutrophil elastase, myeloblastin/proteinase 3, cathepsin G, and matrix metalloproteinases (MMPs; neutrophil‐derived MMP‐8 and plasma‐derived MMP‐9), were significantly overexpressed, while tissue inhibitors of metalloproteinases 1/2 were downregulated in the BHVs as compared with AVs, together indicative of unbalanced proteolysis in the failing BHVs. As opposed to other proteases, MMP‐9 was mostly expressed in the disorganized prosthetic extracellular matrix, suggesting plasma‐derived proteases as the primary culprit of SVD in ethylene glycol diglycidyl ether–treated BHVs. Hence, hemodynamic stress and progressive accumulation of proteases led to the extracellular matrix degeneration and dystrophic calcification, ultimately resulting in SVD.
Conclusions
Neutrophil‐ and plasma‐derived proteases are responsible for the loss of BHV mechanical competence and need to be thwarted to prevent SVD.
... Unlike the B glycosyltransferase, however, it does not require the H antigen as a substrate (Lanté ri et al., 2002). In humans, anti-a-gal IgM, IgG, and IgA antibodies are naturally occurring and in some individuals represent $1% of their immunoglobulins, which means they may be the most abundant antibody in humans (Galili, 2013). These antibodies may protect humans from pathogens that express a-Gal epitopes, including viruses, bacteria, and protozoa (Galili, 2020). ...
Enzymes catalyze biochemical reactions and play critical roles in human health and disease. Enzyme variants and deficiencies lead to variable expression of glycans, which can affect physiology, influence predilection for disease, and/or directly contribute to disease pathogenesis. While certain well-characterized enzyme deficiencies result in overt disease, some of the most common enzyme deficiencies in humans form the basis of blood groups. These carbohydrate blood groups impact fundamental areas of clinical medicine, including the risk of infection and severity of the infectious disease, bleeding risk, transfusion medicine, and tissue / organ transplantation. In this review we examine the enzymes responsible for carbohydrate-based blood group antigen biosynthesis and their expression within the human population. We also consider the evolutionary selective pressures, e.g., malaria, that may account for the variation in carbohydrate structures on human cells and proteins and the implications of this biology for human disease.
... Humans naturally acquire antibodies against some porcine carbohydrate antigens, which share antigenicity with ubiquitous environmental microbes or food content (11,12). Among them, anti-Gal IgM antibodies are the most frequently and abundantly detected in humans. ...
Background
Triple knockout (TKO) donor pigs lacking alpha-1,3-galactose (Gal), N-glycolylneuraminic acid (Neu5Gc), and Sd(a) expressions were developed to improve the clinical success of xenotransplantation. Neu5Gc, a sialic acid expressed on cell surfaces, recruits factor H to protect cells from attack by the complement system. Lack of Neu5Gc expression may cause unwanted complement activation, abrogating the potential benefit of gene-modified donor pigs. To investigate whether TKO porcine cells display increased susceptibility to complement activation in human serum, pathway-specific complement activation, apoptosis, and human platelet aggregation by porcine cells were compared between alpha-1,3-galactosyltransferase gene-knockout (GTKO) and TKO porcine cells.
Methods
Primary porcine peripheral blood mononuclear cells (pPBMCs) and endothelial cells (pECs) from GTKO and TKO pigs were used. Cells were incubated in human serum diluted in gelatin veronal buffer (GVB⁺⁺) or Mg⁺⁺-EGTA GVB, and C3 deposition and apoptotic changes in these cells were measured by flow cytometry. C3 deposition levels were also measured after incubating these cells in 10% human serum supplemented with human factor H. Platelet aggregation in human platelet-rich plasma containing GTKO or TKO pECs was analyzed.
Results
The C3 deposition level in GTKO pPBMCs or pECs in GVB⁺⁺ was significantly higher than that of TKO pPBMCs or pECs, respectively, but C3 deposition levels in Mg⁺⁺-EGTA-GVB were comparable between them. The addition of factor H into the porcine cell suspension in 10% serum in Mg⁺⁺ -EGTA-GVB inhibited C3 deposition in a dose-dependent manner, and the extent of inhibition by factor H was similar between GTKO and TKO porcine cells. The percentage of late apoptotic cells in porcine cell suspension in GVB⁺⁺ increased with the addition of human serum, of which the net increase was significantly less in TKO pPBMCs than in GTKO pPBMCs. Finally, the lag time of platelet aggregation in recalcified human plasma was significantly prolonged in the presence of TKO pECs compared to that in the presence of GTKO pECs.
Conclusion
TKO genetic modification protects porcine cells from serum-induced complement activation and apoptotic changes, and delays recalcification-induced human platelet aggregation. It does not hamper factor H recruitment on cell surfaces, allowing the suppression of alternative complement pathway activation.
... Therefore, the animal origin cardiac tissue, especially those derived from pigs that are free from α-Gal, can be an attractive source for tissue engineered animal origin prosthesis. Decellularization protocols may differ by the type of the elaborated tissue [23][24][25][26][27][28], the amount of the removed cells, or preservation level of extracellular matrix [29]. Unfortunately, using these protocols, it is impossible to remove all cells within the sample tissue. ...
The goal of this work was to create a bioactive tissue-based scaffold using multi-disciplinary engineering materials and tissue engineering techniques. Materials & methods: Physical techniques such as direct laser interference lithography and proton radiation were selected as alternative methods of enzymatic and chemical decellularization to remove cells from a tissue without degradation of the extracellular matrix nor its protein structure. This study was an attempt to prepare a functional scaffold for cell culture from tissue of animal origin using new physical methods that have not been considered before. The work was carried out under full control of the histological and molecular analysis. Results & conclusions: The most important finding was that the physical methods used to obtain the decellularized tissue scaffold differed in the efficiency of cell removal from the tissue in favour of the laser method. Both the laser method and the proton method exhibited a destructive effect on tissue structure and the genetic material in cell nuclei. This effect was visible on histology images as blurred areas within the cell nucleus. The finite element 3D simulation of decellularization process of the three-layer tissue of animal origin sample reflected well the mechanical response of tissue described by hyperelastic material models and provided results comparable to the experimental ones.
Isoglobotrihexosylceramide (iGb3), a well-characterized natural killer T cell ligand found in mammalian tissues, is also known as a glycosphingolipid that contains the human IgE epitope α-Gal (Galα1,3Gal) structure. Here, we analyzed the reactivity of several mice and human serum immunoglobulins against iGb3. Additionally, we isolated and characterized the variable region sequences of a monoclonal antibody that specifically recognizes iGb3. No IgE reactive with iGb3 was detected in sera from MRL/lpr mice, which are known to produce autoreactive antibodies, or in sera from healthy human donors. Furthermore, no induction of IgE and IgG was observed in the sera of mice immunized with iGb3; only IgM reactivity to iGb3 was detected. Further analysis of an anti-iGb3 monoclonal antibody generated from the splenocytes of an iGb3-immunized mouse revealed that the nucleotide sequences of the variable regions exhibited high homology to those of antibodies recognizing glycoconjugates containing Galα1,3 or Galα1,4 structures. These results indicate that the mouse genome harbors genes capable of encoding antibodies that recognize α-linked galactose-containing glycans, including iGb3, but that iGb3 is not sufficiently immunogenic to induce IgE in mammalian lymphocytes.
The distinct molecules composing the biological fluids lead to different coronas on NMs, altering their physicochemical properties and affect their biological fate.
Type 1 diabetes mellitus (T1DM) is characterized by absolute insulin deficiency primarily due to autoimmune destruction of pancreatic β‐cells. The prevailing treatment for T1DM involves daily subcutaneous insulin injections, but a substantial proportion of patients face challenges such as severe hypoglycemic episodes and poorly controlled hyperglycemia. For T1DM patients, a more effective therapeutic option involves the replacement of β‐cells through allogeneic transplantation of either the entire pancreas or isolated pancreatic islets. Unfortunately, the scarcity of transplantable human organs has led to a growing list of patients waiting for an islet transplant. One potential alternative is xenotransplantation of porcine pancreatic islets. However, due to inter‐species molecular incompatibilities, porcine tissues trigger a robust immune response in humans, leading to xenograft rejection. Several promising strategies aim to overcome this challenge and enhance the long‐term survival and functionality of xenogeneic islet grafts. These strategies include the use of islets derived from genetically modified pigs, immunoisolation of islets by encapsulation in biocompatible materials, and the creation of an immunomodulatory microenvironment by co‐transplanting islets with accessory cells or utilizing immunomodulatory biomaterials. This review concentrates on delineating the primary obstacles in islet xenotransplantation and elucidates the fundamental principles and recent breakthroughs aimed at addressing these challenges.
Background:
Immunoassay measurements of serum alpha-gal (AG) specific IgE (sIgE) enable antibody detection and quantification with high sensitivity and specificity and are essential for AG syndrome diagnosis and patient management. We here present and analyze results from over 15 000 patient serum samples tested using the ImmunoCAP (Thermo/Phadia) assay.
Methods:
AG-sIgE levels and positivity rates were correlated to patient age, gender, geographic location, repeat testing results, sIgE levels to co-tested red meat whole allergen extracts, and Rocky Mountain spotted fever (RMSF) serology performed on a subset of patient samples.
Results:
Of the tested samples, 36.7% contained detectable (>0.1 KUA/L) AG-sIgE. Antibody levels were higher in patients of older age, in samples submitted from lower midwestern and southern states, and during the June-December period of the year. Specific IgE to co-tested red meat whole allergens showed moderate to strong correlation to AG-sIgE and were of lower levels. Samples with positive RMSF IgG titers (≥1:64) were of overall higher AG-IgE levels.
Conclusion:
Findings are consistent with the role of lone star ticks in AG syndrome pathogenesis. Levels of measured sIgE to AG are higher than co-tested sIgE to red meat whole allergen, consistent with the improved diagnostic performance of component-resolved testing.
Hyper acute rejection (HAR) occurs 48 h immediately after transplantation. HAR is caused by cytotoxic antibody against donor’s HLA or ABO antigen. Its symptoms include high fever, edema, complement activation, and blood coagulation. Blood coagulation is also caused by bleeding from endothelial cells, and its symptoms are heart attack, stroke, pulmonary embolism, and thrombosis. In blood vessel, blood clot (thrombus) formation induces endothelial cell injury and abnormal blood stream. During HAR, antibody-mediated rejection (AMR) occurs by donor antigen-induced Abs, which were directed against ABO antigenic carbohydrates, donor-specific molecule HLA proteins, antigens of endothelial cells, or surfaced antigens on pig cells. Despite technology development, there are still obstacles and hurdles of HAR and cellular xenogeneic rejection (CXR). First, as a representative xenoantigen, GGTA1 (α1,3-galactosyltransferase) generates a major α1,3-Gal epitope as xenoantigen. Its synthesized α1,3-Gal residue-linked epitopes are present on surfaces of most mammal cells in synthetic mechanism of the donor substrates α1,3-Gal+Galβ1,4GlcNAc (N-acetyllactosamine; LacNAc) to yield Galα1,3Galβ1,4GlcNAc-R carbohydrate structures, known as α1,3-Gal antigenic epitope. For specific aspects of α1,3-Gal epitope, first, the α1,3-Gal antigenic epitope is not existed in humans and several species including Old World monkeys and apes, indicating that α1,3-Gal epitope is a major xenoantigen. Second, cytidine monophosphate (CMP)-N-acetylneuraminic acid (NeuAc) hydroxylase (CMAH) hydroxylates sialic acid Z(Sia), Neu5Ac. The CMAH gene is widely expressed in endothelial cells of mammals, except highly evolved species, humans. Neu5Gc is one of non-Gal xenoantigens. Third, β4GalNT2 (β1,4N-acetylgalactosaminyl transferase) synthesizes the Sia-α2,3-(GalNAc-β1,4)Gal-β1,4-GlcNAc structure known as Sd(a) antigen, transferring a donor substrate of β1,4-GalNAc residue to the Gal residue of an α2,3-sialylglycan structure. Sd(a) is one of non-α1,3Gal xenoantigens (Fig. 10.1). Fourth, human HLA-compatible swine leukocyte antigen (SLA) is a swine type MHC protein, which is highly polymorphic. SLA of pigs, like HLA of humans, is directly associated with the immune responses of pigs to microbial and viral infections as well as vaccinations.
Highlights
Immune processes and mechanisms underlying bioprosthetic heart valve degeneration and rejection of allografts and xenografts are similar.
Manufacturers and surgeons can implement effective approaches to prevent immune rejection in the process of production and implantation of prosthetic heart valves in order to delay the process of structural valve degeneration.
Abstract
Bioprosthetic heart valves (BHV) are characterized by low thrombogenicity, thus circumventing the need for long‐term anticoagulation. However, BHV lifespan is limited to 10–15 years because its tissue components are subject to degeneration. Recent research data indicate that immune responses forming the basis of humoral and cellular rejection of allografts and xenografts play a major role in the development of structural valve degeneration (SVD). This review summarizes up-to-date data on immune processes involved in SVD pathogenesis. Moreover, the latest achievements in the development of strategies to reduce the immunogenicity of BHV, such as data on immune compatibility of allogeneic material and the process of deriving low immunogenic biomaterial from genetically modified animals, decellularization of BHV, and the ways of slowing the process of degeneration are analyzed.
The galactose-α-1,3-galactose (α-Gal) epitope is the cause of a global allergic disease, the α-Gal syndrome (AGS). It is a severe form of allergy to food and products of mammalian origin where IgE against the mammalian carbohydrate, α-Gal, is the cause of the allergic reactions. Allergic reactions triggered by parenterally administered α-Gal sources appear immediately, but those triggered via the oral route appear with a latency of several hours. The α-Gal epitope is highly immunogenic to humans, apes and old-world monkeys, all of which produce anti-α-Gal antibodies of the IgM, IgA and IgG subclasses. Strong evidence suggests that in susceptible individuals, class switch to IgE occurs after several tick bites. In this review, we discuss the strong immunogenic role of the α-Gal epitope and its structural resemblance to the blood type B antigen. We emphasize the broad abundance of α-Gal in different foods and pharmaceuticals and the allergenicity of various α-Gal containing molecules. We give an overview of the association of tick bites with the development of AGS and describe innate and adaptive immune response to tick saliva that possibly leads to sensitization to α-Gal. We further discuss a currently favored hypothesis explaining the mechanisms of the delayed effector phase of the allergic reaction to α-Gal. We highlight AGS from a clinical point of view. We review the different clinical manifestations of the disease and the prevalence of sensitization to α-Gal and AGS. The usefulness of various diagnostic tests is discussed. Finally, we provide different aspects of the management of AGS. With climate change and global warming, the tick density is increasing, and their geographic range is expanding. Thus, more people will be affected by AGS which requires more knowledge of the disease.
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.
Putative mechanism of therapeutic action by α-gal nanoparticles. A. Nanoparticles injected into the injured cord bind to anti-Gal antibodies leaked from ruptured capillaries. The binding of anti-Gal to α-gal epitopes on the α-gal nanoparticles activates the complement system to release complement cleavage chemotactic peptides such as C5a, C3a that recruit macrophages and microglia. These recruited cells bind to the anti-Gal coated α-gal nanoparticles and are further polarized into the M2 state. B. Recruited M2 macrophages and microglia secrete neuroprotective and pro-healing factors to promote tissue repair, neovascularization and axonal regeneration (C.).
Considering the extensive and widespread impact on individuals, cancer can presently be categorized as a pandemic. In many instances, the development of tumors has been linked to endemic microbe infections. Among parasitic infections, Trypanosoma cruzi stands out as one of the most extensively discussed protozoans in the literature that explores the association between diseases of parasite origin and cancer. However, the effective association remains an unsolved paradox. Both the parasite, along with protozoan-derived molecules, and the associated antiparasitic immune response can induce alterations in various host cell pathways, leading to modifications in cell cycle, metabolism, glycosylation, DNA mutations, or changes in neuronal signaling. Furthermore, the presence of the parasite can trigger cell death or a senescent phenotype and modulate the immune system, the metastatic cascade, and the formation of new blood vessels. The interaction among the parasite (and its molecules), the host, and cancer undoubtedly encompasses various mechanisms that operate differentially depending on the context. Remarkably, contrary to expectations, the evidence tilts the balance toward inhibiting tumor growth or resisting tumor development. This effect is primarily observed in malignant cells, rather than normal cells, indicating a selective or specific component. Nevertheless, nonspecific bystander mechanisms, such as T. cruzi’s adjuvancy or the presence of proinflammatory cytokines, may also play a significant role in this phenomenon. This work aims to elucidate this complex scenario by synthesizing the main findings presented in the literature and by proposing new questions and answers, thereby adding pieces to this challenging puzzle.
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).
Resting microglia subjected to α-gal nanoparticle treatment in the presence of anti-Gal (found in serum) become activated and exhibit pro-healing phenotypic markers (Arginase-1, CD206) and secrete VEGF. Expression of pro-inflammatory markers (IL-6, TNF-α) was concomitantly reduced.
Despite the advent of tissue engineering (TE) for the remodeling, restoring, and replacing damaged cardiovascular tissues, the progress is hindered by the optimal mechanical and chemical properties required to induce cardiac tissue-specific cellular behaviors including migration, adhesion, proliferation, and differentiation. Cardiac extracellular matrix (ECM) consists of numerous structural and functional molecules and tissue-specific cells, therefore it plays an important role in stimulating cell proliferation and differentiation, guiding cell migration, and activating regulatory signaling pathways. With the improvement and modification of cell removal methods, decellularized ECM (dECM) preserves biochemical complexity, and bio-inductive properties of the native matrix and improves the process of generating functional tissue. In this review, we first provide an overview of the latest advancements in the utilization of dECM in in vitro model systems for disease and tissue modeling, as well as drug screening. Then, we explore the role of dECM-based biomaterials in cardiovascular regenerative medicine (RM), including both invasive and non-invasive methods. In the next step, we elucidate the engineering and material considerations in the preparation of dECM-based biomaterials, namely various decellularization techniques, dECM sources, modulation, characterizations, and fabrication approaches. Finally, we discuss the limitations and future directions in fabrication of dECM-based biomaterials for cardiovascular modeling, RM, and clinical translation.
Carrageenans are linear sulfated galactans synthesized in the Gigartinales, Rhodophyceae species with a varied range of biological properties that are of value to the pharmaceutical and cosmetic sectors. It is unknown how the fine structure of carrageenans dictates their capacity to affect molecular and cellular responses important to wound healing, or the ability to mitigate oxidative, hemostatic and inflammatory processes. Here we use specific endo-carrageenases from the marine bacterium Zobellia galactanivorans, to produce enzymatically defined neo-series oligosaccharides from carrageenans with 3,6-anhydro-D-galactose on the non-reducing end. Further enzymatic modification of the oligosaccharides was done by treating with the 3,6-anhydro-D-galactosidases from the same bacterium which hydrolyze non-reducing end 3,6-anhydro-D-galactose moieties from neo-carrageenan oligosaccharides. Using the enzymatically produced oligosaccharides, we demonstrate binding to natural human serum antibodies and a monoclonal anti-αGal Ab (m86). The significant interactions with the Galα(1,3)Gal reactive antibodies produced by humans makes them potential potent inducers of complement-dependent reactions and attractive for therapeutic applications. We also demonstrate modulation of the galectin selectivity for the Gal-3 Carbohydrate Recognition Domain (CRD) relative to Gal-1 which has implications to targeting specific biological pathways regulated by the galectins
Congenital heart disease (CHD) affects around 1.35 million neonates worldwide per annum, and surgical repair is necessary in approximately 25% of cases. Xenografts, usually of bovine or porcine origin, are often used for the surgical reconstruction. These xenografts elicit an immune response due to significant immunological incompatibilities between host and donor. Current techniques to dampen the initial hyperacute rejection response involve aldehyde fixation to crosslink xenoantigens, such as galactose-α1,3-galactose and N-glycolylneuraminic acid. While this temporarily masks the epitopes, aldehyde fixation is a suboptimal solution, degrading over time, resulting in cytotoxicity and rejection. The immune response to foreign tissue eventually leads to chronic inflammation and subsequent graft failure, necessitating reintervention to replace the defective bioprosthetic. Decellularisation to remove immunoincompatible material has been suggested as an alternative to fixation and may prove a superior solution. However, incomplete decellularisation poses a significant challenge, causing a substantial immune rejection response and subsequent graft rejection. This review discusses commercially available grafts used in surgical paediatric CHD intervention, looking specifically at bovine jugular vein conduits as a substitute to cryopreserved homografts, as well as decellularised alternatives to the aldehyde-fixed graft. Mechanisms of biological prosthesis rejection are explored, including the signalling cascades of the innate and adaptive immune response. Lastly, emerging strategies of intervention are examined, including the use of tissue from genetically modified pigs, enhanced crosslinking and decellularisation techniques, and augmentation of grafts through in vitro recellularisation or functionalisation with human surface proteins.
Methicillin-resistant Staphylococcus aureus (MRSA) is a pervasive and persistent threat that requires the development of novel therapies or adjuvants for existing ones. Aptamers, small single-stranded oligonucleotides that form 3D structures and can bind to target molecules, provide one possible therapeutic route, especially when presented in combination with current antibiotic applications. BALB/c α-1, 3-galactosyltransferase (−/−) knockout (GTKO) mice were infected with MRSA via tail vein IV and subsequently treated with the αSA31 aptamer (n = 4), vancomycin (n = 12), or αSA31 plus vancomycin (n = 12), with split doses in the morning and evening. The heart, lungs, liver, spleen, and kidneys were harvested upon necropsy for histological and qPCR analysis. All mice treated with αSA31 alone died, whereas 5/12 mice treated with vancomycin alone and 7/12 mice treated with vancomycin plus αSA31 survived the course of the experiment. The treatment of MRSA-infected mice with Vancomycin and an adjuvant aptamer αSA31 reduced disease persistence and dispersion as compared to treatment with either vancomycin SA31 alone, indicating the combination of antibiotic and specifically targeted αSA31 aptamer could be a novel way to control MRSA infection. The data further indicate that aptamers may serve as a potential therapeutic option for other emerging antibiotic resistant pathogens.
Like any other microorganism, pathogenic protozoan parasites rely heavily on glycoconjugates and glycan binding proteins to protect themselves from the environment and to interact with their diverse hosts. A thorough comprehension of how glycobiology contributes to the survival and virulence of these organisms may reveal unknown aspects of their biology and may open much needed avenues for the design of new strategies against them. In the case of Plasmodium falciparum, which causes the vast majority of malaria cases and deaths, the restricted variety and the simplicity of its glycans seemed to confer limited significance to the role played by glycoconjugates in the parasite. Nonetheless, the last 10 to 15 years of research are revealing a clearer and more defined picture. Thus, the use of new experimental techniques and the results obtained provide new avenues for understanding the biology of the parasite, as well as opportunities for the development of much required new tools against malaria.
Tick bites have been shown to transmit a novel form of severe food allergy, the galactose-α-1,3-galactose (α-Gal) syndrome (AGS). Cellular responses to α-Gal in AGS patients have to date not been thoroughly scrutinized. Therefore, we investigated T and B cell proliferation, activation and cytokine profiles in response to tick protein extract (TE) and α-Gal-free TE in AGS patients and healthy controls. T and B cells from both patients and controls proliferated in response to TE, but significantly more in the patients. B cell proliferation, but not T cell proliferation, in AGS patients was reduced by removing α-Gal from the TE. In addition, TE induced a clear Th2 cytokine profile in AGS patients. Expression of CD23 by B cells correlated only to T cell proliferation. However, both B cell proliferation and CD23 expression were reduced when CD40L and IL-4 were blocked. A large proportion of the IgG1 and IgE antibodies binding TE in AGS patients were directed against the α-Gal epitope. We have for the first time investigated T and B cell responses to α-Gal carrying tick proteins in AGS patients, which will be essential for the understanding of the immune response against an allergenic carbohydrate transmitted by ticks.
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, 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. The second is anti‐N‐glycolylneuraminic‐acid (anti‐Neu5Gc) antibody binding Neu5Gc on glycans synthesized by Old‐World monkeys, apes, and most non‐primate mammals. Ancestral Old‐World monkeys 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.
Anti-Gal is a natural antibody, which constitutes as much as 1% of circulating IgG in humans and displays a distinct specificity for the structure Galalpha 1--> 3Gal. This glycosidic structure has been found on various tissues of many nonprimate mammals. A comparative study of the occurrence of anti-Gal versus the expression of the Galalpha 1--> 3Gal epitope was performed in primates, and a distinct evolutionary pattern was observed. Whereas anti-Gal was found to be present in Old World monkeys and apes in titers comparable to those in humans, its corresponding antigenic epitope is abundantly expressed on erythrocytes of New World monkeys. Immunostaining with anti-Gal of glycolipids from New World monkey erythrocytes indicated that the molecules to which anti-Gal binds are similar to those found in rabbit and bovine erythrocytes. These findings indicate that there is an evolutionary reciprocity between New World and Old World primates in the production of the Galalpha 1--> 3Gal structure and the antibody that recognizes it. The expression of the Galalpha 1--> 3Gal epitope was evolutionarily conserved in New World monkeys, but it was suppressed in ancestral lineages of Old World primates. The suppression of this epitope was accompanied by the production of anti-Gal. The observed in vivo binding of anti-Gal to human normal senescent and some pathologic erythrocytes implies that the Galalpha 1--> 3Gal epitope is present in man in a cryptic form.
The primary function of the skin is to serve as a protective barrier against the environment. Loss of the integrity of large portions of the skin as a result of injury or illness may lead to major disability or even death. Every year in the United States more than 1.25 million people have burns1 and 6.5 million have chronic skin ulcers caused by pressure, venous stasis, or diabetes mellitus.2 The primary goals of the treatment of wounds are rapid wound closure and a functional and aesthetically satisfactory scar. Recent advances in cellular and molecular biology have greatly expanded our understanding . . .
Autologous melanoma associated antigens (MAA) on murine melanoma cells can elicit a protective anti-tumor immune response following a variety of vaccine strategies. Most require effective uptake by antigen presenting cells (APC). APC transport and process internalized MAA for activation of anti-tumor T cells. One potential problem with clinical melanoma vaccines against autologous tumors may be that often tumor cells do not express surface markers that label them for uptake by APC. Effective uptake of melanoma cells by APC might be achieved by exploiting the natural anti-Gal antibody which constitutes ~1% of immunoglobulins in humans. This approach has been developed in a syngeneic mouse model using mice capable of producing anti-Gal. Anti-Gal binds specifically to α-gal epitopes (Galα1-3Galβ1-4GlcNAc-R). Injection of glycolipids carrying α-gal epitopes (α-gal glycolipids) into melanoma lesions results in glycolipid insertion into melanoma cell membranes, expression of α-gal epitopes on the tumor cells and binding of anti-Gal to these epitopes. Interaction between the Fc portions of bound anti-Gal and Fcγ receptors on APC induces effective uptake of tumor cells by APC. The resulting anti-MAA immune response can be potent enough to destroy distant micrometastases. A clinical trial is now open testing effects of intratumoral α-gal glycolipid injections in melanoma patients.
Cancer evolves dynamically as clonal expansions supersede one another driven by shifting selective pressures, mutational processes, and disrupted cancer genes. These processes mark the genome, such that a cancer's life history is encrypted in the somatic mutations present. We developed algorithms to decipher this narrative and applied them to 21 breast cancers. Mutational processes evolve across a cancer's lifespan, with many emerging late but contributing extensive genetic variation. Subclonal diversification is prominent, and most mutations are found in just a fraction of tumor cells. Every tumor has a dominant subclonal lineage, representing more than 50% of tumor cells. Minimal expansion of these subclones occurs until many hundreds to thousands of mutations have accumulated, implying the existence of long-lived, quiescent cell lineages capable of substantial proliferation upon acquisition of enabling genomic changes. Expansion of the dominant subclone to an appreciable mass may therefore represent the final rate-limiting step in a breast cancer's development, triggering diagnosis.
PaperClip
There is definitive biochemical evidence for the presence of terminal α-galactosyl residues (α-gal) in the N-linked oligosaccharides and glycophosphatidylinositol anchors (GPI anchors) of the variant surface glycoprotein of Trypanosoma brucei bloodstream trypomastigotes. Indirect evidence also exists for α-gal in Plasmodium falciparum asexual blood stage glycoproteins and glycolipids. The occurrence of α-gal in glycoproteins and glycolipids of T. brucei bloodstream trypomastigotes and P. falciparum late asexual blood stages was investigated by the binding of α-gal-specific Bandeirea simplicifolia B4 lectin 1 (BSB4), incorporation of [(3)H]galactose from UDP-[(3)H]galactose into glycoproteins and glycolipids in microsomes in vitro, and bioinformatic searches for galactosyl-transferase coding sequences. The findings confirm the presence of α-gal in a spectrum of T. brucei bloodstream trypomastigote glycoproteins and glycolipids and indicate its relative absence from P. falciparum asexual blood stage glycoconjugates.
Macrophages are pivotal in promoting wound healing. We hypothesized that topical application of liposomes with glycolipids that carry Galα1-3Galβ1-4GlcNAc-R epitopes (α-gal liposomes) on wounds may accelerate the healing process by rapid recruitment and activation of macrophages in wounds. Immune complexes of the natural anti-Gal Ab (constituting ∼1% of Ig in humans) bound to its ligand, the α-gal epitope on α-gal liposomes would induce local activation of complement and generation of complement chemotactic factors that rapidly recruit macrophages. Subsequent binding of the Fc portion of anti-Gal coating α-gal liposomes to FcγRs on recruited macrophages may activate macrophage genes encoding cytokines that mediate wound healing. We documented the efficacy of this treatment in α1,3galactosyltrasferase knockout mice. In contrast to wild-type mice, these knockout mice lack α-gal epitopes and can produce the anti-Gal Ab. The healing time of excisional skin wounds treated with α-gal liposomes in these mice is twice as fast as that of control wounds. Moreover, scar formation in α-gal liposome-treated wounds is much lower than in physiologic healing. Additional sonication of α-gal liposomes resulted in their conversion into submicroscopic α-gal nanoparticles. These α-gal nanoparticles diffused more efficiently in wounds and further increased the efficacy of the treatment, resulting in 95-100% regeneration of the epidermis in wounds within 6 d. The study suggests that α-gal liposome and α-gal nanoparticle treatment may enhance wound healing in the clinic because of the presence of high complement activity and high anti-Gal Ab titers in humans.
The prognosis of patients with colorectal cancer has sometimes proved uncertain; thus, the prognostic significance of immune criteria was compared with that of the tumor extension criteria using the American Joint Committee on Cancer/International Union Against Cancer-TNM (AJCC/UICC-TNM) staging system.
We studied the intratumoral immune infiltrates in the center of the tumor and in the invasive margin of 599 specimens of stage I to IV colorectal cancers from two independent cohorts. We analyzed these findings in relation to the degree of tumor extension and to the frequency of recurrence.
Growth of the primary tumor and metastatic spread were associated with decreased intratumoral immune T-cell densities. Sixty percent of patients with high densities of CD8(+) cytotoxic T-lymphocyte infiltrate presented with stage Tis/T1 tumor, whereas no patients with low densities presented with such early-stage tumor. In patients who did not relapse, the density of CD8 infiltrates was inversely correlated with T stage. In contrast, in patients whose tumor recurred, the number of CD8 cells was low regardless of the T stage of the tumor. Univariate analysis showed that the immune score was significantly associated with differences in disease-free, disease-specific, and overall survival (hazard ratio [HR], 0.64, 0.60, and 0.70, respectively; P < .005). Time-dependent receiver operating characteristic curve analysis illustrated the predictive accuracy of the immune parameters (c-index = 65.3%, time-dependent c-index [Cτ] = 66.5%). A final stepwise model for Cox multivariate analysis supports the advantage of the immune score (HR, 0.64; P < .001; Cτ = 67.9%) compared with histopathologic features in predicting recurrence as well as survival.
Assessment of CD8(+) cytotoxic T lymphocytes in combined tumor regions provides an indicator of tumor recurrence beyond that predicted by AJCC/UICC-TNM staging.
Mucin 1 (MUC1), a bound mucin glycoprotein, is overexpressed and aberrantly glycosylated in >80% of human ductal pancreatic carcinoma. Evidence suggests that MUC1 can be used as a tumor marker and is a potential target for immunotherapy of pancreatic cancer. However, vaccination with MUC1 peptides fails to stimulate the immune response against cancer cells because immunity toward tumor-associated antigens (TAA), including MUC1, in cancer patients is relatively weak, and the presentation of these TAAs to the immune system is poor due to their low immunogenicity. We investigated whether vaccination with immunogenetically enhanced MUC1 (by expressing alpha-gal epitopes; Galalpha1-3Galbeta1-4GlcNAc-R) can elicit effective antibody production for MUC1 itself as well as certain TAAs derived from pancreatic cancer cells and induced tumor-specific T-cell responses. We also used alpha1,3galactosyltransferase (alpha1,3GT) knockout mice that were preimmunized with pig kidney and transplanted with B16F10 melanoma cells transfected with MUC1 expression vector. Vaccination of these mice with alpha-gal MUC1 resulted in marked inhibition of tumor growth and significant improvement of overall survival time compared with mice vaccinated with MUC1 alone (P = 0.003). Furthermore, vaccination with pancreatic cancer cells expressing alpha-gal epitopes induced immune responses against not only differentiated cancer cells but also cancer stem cells. The results suggested that vaccination using cells engineered to express alpha-gal epitopes is a novel strategy for treatment of pancreatic cancer.
Understanding the endogenous repair capacity of spinal cord is pivotal to develop strategies to improve it. Here we design a paradigm of spinal cord lesion in the dorsal column using a 2-photon microscopy technique to dynamically and chronically monitor simultaneous changes of vascular and axonal networks in living mice up to 4 months postinjury. High-resolution images showed that early explorative sprouting of surviving injured axons resulted in extensive regrowth until and past the lesion site within 2 months. Blood vessel density was transiently up-regulated and most neurovascular interactions occurred within 2 weeks. Time-lapse analysis showed that neovessels exerted a potent growth stimulating action, but no guidance effect on neighboring sprouts, possibly because of their geometry and plasticity. Nevertheless, if reconnection depends on axon sprout density, stimulation of angiogenesis would probably be beneficial to repair. More generally, this imaging approach is showing promise to aid in monitoring brain diseases and the efficacy of potential treatments.
alpha-Gal glycolipids capable of converting tumors into endogenous vaccines, have alpha-gal epitopes (Gal alpha 1-3 Gal beta 1-4GlcNAc-R) and are extracted from rabbit RBC membranes. alpha-Gal epitopes bind anti-Gal, the most abundant natural antibody in humans constituting 1% of immunoglobulins. alpha-Gal glycolipids insert into tumor cell membranes, bind anti-Gal and activate complement. The complement cleavage peptides C5a and C3a recruit inflammatory cells and APC into the treated lesion. Anti-Gal further opsonizes the tumor cells and targets them for effective uptake by recruited APC, via Fc gamma receptors. These APC transport internalized tumor cells to draining lymph nodes, and present immunogenic tumor antigen peptides for activation of tumor specific T cells. The present study demonstrates the ability of alpha-gal glycolipids treatment to prevent development of metastases at distant sites and to protect against tumor challenge in the treated mice. Adoptive transfer studies indicate that this protective immune response is mediated by CD8+ T cells, activated by tumor lesions turned vaccine. This T cell activation is potent enough to overcome the suppressive activity of Treg cells present in tumor bearing mice, however it does not elicit an autoimmune response against antigens on normal cells. Insertion of alpha-gal glycolipids and subsequent binding of anti-Gal are further demonstrated with human melanoma cells, suggesting that intratumoral injection of alpha-gal glycolipids is likely to elicit a protective immune response against micrometastases also in cancer patients.
The involvement of complement-activation products in promoting tumor growth has not yet been recognized. Here we show that the generation of complement C5a in a tumor microenvironment enhanced tumor growth by suppressing the antitumor CD8(+) T cell-mediated response. This suppression was associated with the recruitment of myeloid-derived suppressor cells into tumors and augmentation of their T cell-directed suppressive abilities. Amplification of the suppressive capacity of myeloid-derived suppressor cells by C5a occurred through regulation of the production of reactive oxygen and nitrogen species. Pharmacological blockade of the C5a receptor considerably impaired tumor growth to a degree similar to the effect produced by the anticancer drug paclitaxel. Thus, our study demonstrates a therapeutic function for complement inhibition in the treatment of cancer.
Antibodies that lyse trypomastigotes in a complement-mediated reaction are believed to be the main participants in the protection against virulent Trypanosoma cruzi. Antibodies with a specificity for alpha-galactosyl-containing determinants--generally called antiGal--were studied to determine their role in the lysis of trypomastigote forms. The titers of antiGal markedly increase in Chagas's disease. In the present study we demonstrate binding of this antibody to T. cruzi and the complement-mediated lysis of trypomastigotes by antiGal. Lysis of metacyclic trypomastigotes by whole Chagasic (Ch) serum or isolated antiGal fractions was equally inhibited by alpha- but not by beta-galactosides. Most of the lytic power of the Ch antiGal as well as of the whole Ch serum was removed by absorption on Synsorb-linked Gal alpha 1, 3Gal beta 1, 4GlcNAc followed by rabbit erythrocyte absorption. The Ch antiGal had a lower affinity for melibiose bound to agarose than for the trisaccharide linked to Synsorb, and was several times more effective in the immunolysis of trypomastigotes than the corresponding antiGal from normal human serum. Lytic antibodies were partly absorbed by Serratia marcescens but not by Escherichia coli O111. A human volunteer immunized with an S. marcescens vaccine elicited a specific antiGal response that was lytic to trypomastigotes (70% lysis). We suggest that in vivo high-affinity antiGal antibody clones, as occur in Ch patients, may significantly contribute to the destruction of the parasite, whereas low-affinity antiGal clones are much less effective in the protection against T. cruzi infection.
The natural anti-Gal antibody constitutes 1% of circulating IgG in humans and interacts specifically with the carbohydrate epitope Gal alpha 1–3Gal beta 1–4GlcNAc-R (the alpha-galactosyl epitope). In view of the unusually large amounts of this antibody in the serum, it was of interest to determine the proportion of circulating B lymphocytes capable of synthesizing anti-Gal. For this purpose, blood B lymphocytes were incubated with Epstein-Barr virus (EBV) and plated in microtiter wells. Proliferation of the EBV transformed B lymphocytes was readily visible after 3 weeks of incubation. The supernatants from wells containing proliferating B-lymphoid clones were assayed for anti-Gal by an agglutination assay with rabbit red blood cells and the specificity of the agglutinating antibodies was further confirmed by their interaction with synthetic oligosaccharides and by enzyme-linked immunosorbent assay with glycoproteins. Approximately 5% of the wells contained anti-Gal antibodies. Limiting dilution studies and IgH gene rearrangement patterns suggested that each well contained an average of five proliferating B-lymphoid clones. Thus, it is concluded that approximately 1% of circulating B lymphocytes are capable of producing anti-Gal. The proportion of anti-Gal--producing lymphoid clones exceeds by fourfold that of clones producing anti-blood group A or anti-blood group B antibodies. Individual anti-Gal clones display fine variations in their combining site, as indicated by their differential interaction with alpha-galactosyl epitopes on glycolipids and on N-linked carbohydrate chains of glycoproteins. The high frequency of precursor B lymphocytes capable of producing anti-Gal, found in every individual and the restricted specificity of this antibody to alpha-galactosyl epitopes, potentially makes anti-Gal--producing lymphocytes an effective system for studying human Ig genes involved in the natural immune response to structurally defined haptens.
A modified antiglobulin test, based on the high affinity between the Fc portion of the red blood cell (RBC) bound IgG and the Fc receptor on the myeloid cell K-562, was utilized for demonstration of immunoglobulins (Ig) on thalassemic RBC. Ig was found on the RBC of 73 out of 80 patients with thalassemia. The immunoglobulins on the thalassemic RBC belonged to the IgG subclass and were autoreactive. Elution studies utilizing various carbohydrates, or by thermal stripping, indicated that at least part of the IgG molecules found on the thalassemic RBC were specifically reactive with terminal galactosyl residues on the RBC membrane. IgG antibodies with similar reactivity were also demonstrated in normal human serum. These natural antigalactosyl IgG antibodies from normal sera could bind to IgG- depleted thalassemic RBC. Thalassemic RBC and normal senescent RBC were previously found to contain reduced amounts of membrane sialic acid (SA). It is suggested that the antigalactosyl IgG antibodies interact with newly exposed galactosyl residues underlying the sialic acid units. Such interaction may lead to the shortened lifespan of thalassemic RBC and may result in sequestration of senescent normal RBC by the reticuloendothelial system.
Human sera contain high levels of natural antibody (Ab) to Galalpha1-3Gal, 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 alpha-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 Galalpha1-3Gal and was blocked by incorporation of soluble Galalpha1-3Gal disaccharide into the inactivation assay. GT-deficient mice immunized to make high levels of Ab to Galalpha1-3Gal (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 Galalpha1-3Gal-expressing human cells incorporated this sugar moiety onto their major envelope glycoproteins. SV passaged in mouse cells expressing Galalpha1-3Gal was moderately sensitive to inactivation by NHS. These results indicate that enveloped viruses expressing Galalpha1-3Gal 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.
A natural IgG antibody (anti-Gal) with alpha-galactosyl binding specificity has been found in large amounts (0.5 - 1.0% of serum IgG) in all individuals tested. It has been purified by affinity chromatography on a column of melibiose-Sepharose. In addition to its affinity for normal and pathological senescent human red cells, the antibody readily interacts with rabbit red blood cell (RRBC) glycolipids with alpha-galactosyl terminal residues. Two types (glycosidic linkages of 1----3 vs. 1----4) of rabbit red cells glycolipids with terminal alpha-galactosyl residues were tested for antibody binding. The antibody specifically bound to glycolipids with Gal alpha 1----3 terminal residues, and treatment of these glycolipids with alpha-galactosidase abolished binding. Hemagglutination inhibition studies with oligosaccharides of known structure also showed that the antibody binds specifically to glycoconjugates with an alpha 1----3 terminal galactose residue. Anti-Gal did not bind to a human B-active glycolipid, indicating that fucose-linked alpha 1----2 to the penultimate galactose prevents anti-Gal binding. The anti-Gal specificity for RRBC glycolipids also paralleled that of the alpha-galactosyl-specific Bandeiraea simplicifolia lectin. The possible reasons for the occurrence of this unique antibody in human serum are discussed.
Sera from patients with American cutaneous leishmaniasis and Chagas disease and from monkeys infected with either Trypanosoma cruzi or Trypanosoma rhodesiense show, in RIAs, strong binding to mouse laminin. A distinct although weaker binding activity is also detected in normal human sera. The antibodies recognize a common carbohydrate epitope present on mouse laminin, which was assigned to a terminal galactosyl(alpha 1-3)-galactose group. Distinct crossreactions were observed with some other basement membrane proteins, rabbit glycosphingolipids, defucosylated human B blood group substance and components produced by some human tumor cells. Only little activity was, however, found on laminin obtained from human placenta. The data indicate that the antibodies arising in infectious diseases are stimulated by similar carbohydrate epitopes present on the surface of parasites. Tissue-specific occurrence of such epitopes may exist and explain the involvement of distinct tissues in autoimmune disorders.
Studies on the distribution of α-galactosyl epitopes with the structure Galα1→3Galβ1→4GlcNac-R on mammalian thyroid cell membranes are of interest, since a natural antibody interacting with this carbohydrate antigen (i.e. the natural anti α-galactosyl IgG antibody) was found to increase in its titre in patients with autoimmune thyroid disorders. By using a radioimmunoassay for quantification of the α-galactosyl epitope, we found variable concentrations of this epitope on thyroid cell membranes of all nonprimate mammals and New World monkeys studied, but not in Old World monkeys and human thyroid. The absence of the identifiable α-galactosyl epitopes on human and Old World monkey thyroid cells correlates with diminished activity of the enzyme, α1-3 galactosyltransferase, which, in other species, synthesizes the α-galactosyl epitopes within the Golgi apparatus. It is argued that a proportion of anti-thyroid reactivity in human normal and pathologic sera, when assayed with mammalian thyroid cells, may be attributed to natural anti α-galactosyl IgG antibody, which interacts with α-galactosyl epitopes on thyroid tissues used for the bioassay.
Background. Expression of the α-gal epitope in mice can be completely eliminated by disruption of the α1,3 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 α-gal epitopes in pig and mouse organs. Methods. Membranes from pig and mouse organ homogenates were analyzed for α-gal epitope expression by Western blots, enzyme-linked immunosorbent assay (ELISA), immunostaining of tissues, and ELISA inhibition assay. Results. Immunostaining of Western blots with human anti-Gal detected α-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 α-gal epitopes on pig membranes than on mouse membranes. ELISA inhibition assay with monoclonal anti-Gal indicated that α-gal epitope expression in pig organs is up to 500-fold higher than in mouse organs. Conclusion. Expression of α-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 α-gal epitopes.
Rejection of pig-to-human or pig-to-primate xenografts is mediated by the natural anti-Gal antibody, which interacts with alpha-galactosyl epitopes (i.e., Gal alpha 1-->3Gal beta 1-4GlcNAc-R) abundantly expressed on porcine cells, The objective of this study was to determine the ability of various synthetic oligosaccharides to inhibit the binding of anti-Gal IgG molecules to porcine endothelial cells in vitro. Such inhibition ultimately may help to reduce or to prevent the in vivo antibody-dependent cell cytotoxicity (ADCC) reaction, In the absence of complement-mediated hyperacute rejection, the ADCC induced by anti-Gal IgG molecules is likely to cause the chronic rejection of xenografts. The synthetic free a galactosyl epitope (Gal alpha 1-3Gal beta 1-4GlcNAc) was found to be 300-fold more effective than melibiose or alpha-methyl galactoside in inhibiting anti-Gal binding to porcine endothelial cells, and to prevent >90% of the antibody binding at a concentration of 1 mM. The disaccharide Gal alpha 1-3Gal was ten-fold less effective than the free alpha-galactosyl epitope, Accordingly, the affinity of the disaccharide to anti-Gal, as measured by equilibrium dialysis, was seven-fold lower than that of the trisaccharide. The effective concentration of oligosaccharides inhibiting anti-Gal is independent of the antibody affinity, but is dependent on the concentration of the antibody, Based on the small difference in affinity between Gal alpha 1-3Gal beta 1-4GlcNAc and Gal alpha 1-3Gal beta 1-4Glc, and the large difference in the price of N-acetyllactosamine vs. lactose, it is suggested that lactose may be considered as an appropriate starting material for synthesizing large amounts of a trisaccharide that effectively neutralizes anti-Gal.
An urgent and steadily increasing need exists world-wide for a greater supply of donor thoracic organs. Xenotransplantation offers the possibility of an unlimited supply of hearts and lungs that could be available electively when required. However, anti-body- mediated mechanisms cause the rejection of pig organs transplanted into non-human primates, and these mechanisms provide major immunologic barriers that have not yet been overcome. Having reviewed the literature on xenotransplantation, we present a number of conclusions on its present status with regard to thoracic organs, and we make a number of recommendations relating to eventual clinical trials. Although pig hearts have functioned in heterotopic sites in non-human primates for periods of several weeks, median survival of orthotopically transplanted hearts is currently ,1 month. No transplanted pig lung has functioned for even 24 hours. Current experimental results indicate that a clinical trial would be premature. A potential risk exists, hitherto undetermined, of transferring infectious organisms along with the donor pig organ to the recipient, and possibly to other members of the community. A clinical trial of xeno-transplantation should not be undertaken until experts in microbiology and the relevant regulatory authorities consider this risk to be minimal. A clinical trial should be considered when approximately 60% survival of life-supporting pig organs in non-human primates has been achieved for a minimum of 3 months, with at least 10 animals surviving for this minimum period. Furthermore, evidence should suggest that longer survival (.6 months) can be achieved. These results should be achieved in the absence of life-threatening complications caused by the immunosuppressive regimen used. The relationship between the presence of anti-HLA antibody and anti-pig antibody and their cross-reactivity, and the outcome of pig-organ xenotransplantation in recipients previously sensitized to HLA antigens require further investigation. We recommend that the patients who initially enter into a clinical trial of cardiac xenotransplantation be unacceptable for allotransplantation, or acceptable for allotransplantation but unlikely to survive until a human cadaveric organ becomes available, and in whom mechanical assist-device bridging is not possible. National bodies that have wide-reaching government-backed control over all aspects of the trials should regulate the initial clinical trial and all subsequent clinical xenotransplantation procedures for the foreseeable future. We recommend coordination and monitoring of these trials through an international body, such as the International Society for Heart and Lung Transplantation, and setting up a registry to record and widely disperse the results of these trials. Xenotransplantation has the potential to solve the problem of donor-organ supply, and therefore research in this field should be actively encouraged and supported.
MAMMALIAN C-type retroviruses are inactivated by human serum1,2, following triggering of the classical complement cascade3. This may have inhibited transmission to humans of C-type oncoviruses from other mammals1. Indeed, the retro-viruses human immunodeficiency virus and human T-cell leukaemia virus are
resistant to human complement4,5. Antibody-independent activation of human Clq, the first component of the classical pathway, by retroviral envelope proteins has been described6. However, retroviruses produced from human cells are resistant to inactivation by human complement7,8 and human serum is known to contain antibodies
directed against carbohydrates on retroviral envelopes9–11. Gal(αl–3)Gal terminal carbohydrates are expressed by most mammals but are absent in humans, which lack a functional (αl–3)galactosyltransferase gene12,13. Here, we demonstrate that anti-Gal(αl–3)Gal antibodies in human serum inactivate
retroviruses produced from animal cells. Expression of porcine (αl–3)galactosyltransferase14–16 in human cells renders the cells and the retroviruses they produce sensitive to human serum.
The repair of wounds is one of the most complex biological processes that occur during human life. After an injury, multiple biological pathways immediately become activated and are synchronized to respond. In human adults, the wound repair process commonly leads to a non-functioning mass of fibrotic tissue known as a scar. By contrast, early in gestation, injured fetal tissues can be completely recreated, without fibrosis, in a process resembling regeneration. Some organisms, however, retain the ability to regenerate tissue throughout adult life. Knowledge gained from studying such organisms might help to unlock latent regenerative pathways in humans, which would change medical practice as much as the introduction of antibiotics did in the twentieth century.
Background. Natural antibodies (NAbs) against a terminal α1-3 galactosyl(αGal) epitope have been identified as the major human anti-pig NAbs.
Methods and Results. We used two synthetic αGal trisaccharides-type 6 (αGal6) and type 2(αGal2)-linked to an inert matrix to remove NAbs from human plasma in vitro. Flow cytometry indicated that an average of 85% of the NAb binding activity was depleted by adsorption with αGal6. By measuring the binding of NAbs to pig peripheral blood mononuclear cells and bone marrow cells, we demonstrated that αGal6 was more effective than αGal2 in removing NAbs, and the combination of αGal6 + αGal2 did not further increase removal of NAbs. The specificity of the removal of NAbs (IgM and IgG) reactive with the αGal epitope by αGal6 matrix was shown by enzyme-linked immunosorbent assay. In vivo studies in nonhuman primates compared plasma perfusion through a αGal6 immunoaffinity column with hemoperfusion through a pig liver for changes in blood pressure, hematocrit, platelets, and NAb adsorption.
Conclusions. Both methods reduced the level of anti-pig IgM and IgG xenoreactive antibodies to nearly background, but column perfusion caused less hypotension and reduction in platelets than liver perfusion. Four pig kidneys transplanted into monkeys after column perfusion did not undergo hyperacute rejection, remaining functional for 2-10 days, with a mean functional period of 7 days, demonstrating that a pig kidney can support renal function in a primate.
Aim/
To determine the feasibility and safety of intratumoral α-gal glycolipids injection for conversion of human tumors into autologous Tumor Associated Antigens (TAA) vaccine. α-Gal glycolipids bind anti-Gal - the most abundant antibody in humans. Pre-clinical studies indicated that injected α-gal glycolipids insert into tumor cell membranes, bind anti-Gal and target tumor cells to Antigen Presenting Cells, thereby converting tumors into autologous TAA vaccines. We hypothesized that α-gal glycolipids might have similar utility in humans.
Eleven patients with advanced solid tumors received one intratumoral injection of 0.1 mg, 1 mg, or 10 mg α-gal glycolipids. The primary endpoint was dose-limiting toxicity (DLT) within 4 weeks. Secondary endpoints included long-term toxicity, autoimmunity, radiological tumor response and survival.
There were no DLT and no clinical or laboratory evidence of autoimmunity, or any other toxicity. Few patients had an unexpectedly long survival.
Intratumoral injection of α-gal glycolipids is feasible and safe for inducing a protective anti-tumor immune response.
Subsets of IgM naturally occurring autoantibodies (NAbs) bind to the cell surface membranes of dying cells. The antibodies predominantly have specificities against lipid antigens or oxidized lipids. Chief among these lipid antigens are phosphorylcholine (PC) and malondialdehyde (MDA). Antibodies to negatively charged phospholipids such as phosphatidylserine (PS) have been described and there is controversy as to whether these antibodies are related to anticardiolipin antibodies observed in disease states. IgM NAbs that bind to apoptotic cells recruit classical complement pathway components and facilitate phagocytosis by both macrophages and dendritic cells, and may block inflammatory pathways. Under these circumstances, pathologic immune responses to self (autoimmunity) are avoided, whereas mice lacking serum IgM develop a lupus-like disease with associated IgG autoantibody responses. Based on these observations, IgM anti-PC NAbs were found to attenuate inflammation in mouse models of arthritis. IgMNAbs antibodies therefore appear to play pivotal roles in the dampening inflammation and maintenance of tolerance.
Almost all Streptococcus pneumoniae (pneumococcus) capsule serotypes employ the Wzy-dependent pathway for their capsular polysaccharide (CPS) biosynthesis. The assembly of the CPS repeating unit (RU) is the first committed step in this pathway. The wciN gene was predicted to encode a galactosyltransferase involved in the RU assembly of pneumococcus type 6B CPS. Herein, we provide the unambiguous in vitro biochemical evidence that wciN encodes an α-1,3-galactosyltransferase catalyzing the transfer of galactosyl from UDP-Gal onto the Glcα-pyrophosphate-lipid (Glcα-PP-lipid) acceptor to form Galα(1-3)Glcα-PP-lipid. A chemically synthesized acceptor (Glcα-PP-O(CH(2))(10)CH(3)) was used to characterize the WciN activity. The disaccharide product, i.e., Galα(1-3)Glcα-PP-O(CH(2))(10)CH(3), was characterized by mass and NMR spectroscopy. Substrate specificity study indicated that the acceptor structural region composed of pyrophosphate and lipid moieties may play an important role in the enzyme-acceptor recognition. Furthermore, divalent metal cations were found indispensable to the WciN activity, suggesting that this glycosyltransferase (GT) belongs to the GT-A superfamily. By analyzing the activities of six WciN mutants, a DXD motif involved in the coordination of a divalent metal cation was identified. This work provides a chemical biology approach to characterize the activities of pneumococcal CPS GTs in vitro and will help to better understand the pneumococcal CPS biosynthetic pathway.
Carbohydrate-specific IgE antibodies present on nonprimate mammalian proteins were incriminated recently in delayed meat anaphylaxis. The aim of this study was to explore whether anaphylaxis to mammalian kidney is also associated with galactose-α-1,3-galactose (αGal)-specific IgE.
Fourteen patients with anaphylaxis to pork or beef kidney underwent prick tests to meat and kidney. Some patients also underwent skin tests to Erbitux(®) (cetuximab). IgE antibodies to αGal, swine urine proteins, beef and pork meat, serum albumin proteins, cat, and rFel d 1 were measured by ImmunoCAP(®). The αGal levels were estimated in meats and kidney by ELISA inhibition assay. Cross-reactivity between αGal and pork kidney was studied with the ImmunoCAP(®) inhibition assay.
Among the 14 patients, 12 presented with anaphylactic shock. Reactions occurred within 2 h from exposure in 67% of patients. Associated risk factors were observed in 10 cases, and alcohol was the main cofactor. Three patients underwent an oral challenge to pork kidney, and anaphylaxis occurred after ingestion of small quantities (1-2 g). Prick tests to kidney were positive in 54% of patients. All tested patients showed positive skin tests to Erbitux(®). All patients tested positive for IgE to αGal, with levels ranging from 0.4 to 294 kU/l. IgE binding to αGal was inhibited by raw pork kidney extract (mean, 77%; range, 55-87%), which showed a high amount of αGal determinants.
Pork or beef kidney anaphylaxis is related to αGal IgE. Its peculiar severity could be due to an elevated content of αGal epitopes in kidney.
Humans, apes and Old World monkeys (catarrhines) differ from other mammals in that they do not synthesize α-galactosyl epitopes (Galα1-3Galβ1-4GlcNAc-R) on cell surface glycoprotein and glycolipid molecules. In contrast these primates produce large amounts of a natural antibody, anti-Gal, which interacts specifically with α-galactosyl epitopes. It is suggested that these differences between catarrhines and other mammals are the result of a major evolutionary selective process which occurred in ancestral Old World primates during the Miocene and which led to the inactivation of the α1, 3galactosyltransferase gene. In New World monkeys, prosimians (lemurs) and non-primate mammals this gene codes for the enzyme synthesizing α-galactosyl epitopes. The potential causes for this gene inactivation, the possible relationship of this genetic event to the fossil record and some of the clinical outcomes of this evolutionary event are discussed.
Primary tissue failure (structural valve deterioration) has been documented as the most prominent complication of porcine bioprostheses. The influence of age on primary tissue failure has received limited consideration. From 1975 to 1986, 1,301 Carpentier-Edwards standard porcine bioprostheses were implanted in 1,183 patients in 1,201 operations. Of the total number of prostheses, 97.7% were implanted prior to 1983. The mean follow-up was 5.6 years and was 97.5% complete. Primary tissue failure was identified in 96 patients (98 operations) at reoperation (95) or autopsy (3). One hundred four (104) prostheses were involved. Thirty-one failed after aortic valve replacement (AVR), 49 after mitral valve replacement (MVR), and 24 after multiple-valve replacement (18 patients). There were 47 male and 49 female patients. The mean age at implantation was 47 years (range, 8 to 72 years). The mean implantation time was 74.0 months. The freedom from primary tissue failure at 10 years is 77.0 ± 2.9% overall; for AVR, 83.1 ± 3.7%; for MVR, 72.1 ± 4.9%; and for multiple-valve replacement, 65.5 ± 7.8%. The freedom from deterioration for patients less than 20 years of age is significantly less than that for other age groups. The freedom from deterioration increased by decades; the greatest freedom was noted in patients 70 to 80 years old and 80 years old or older. The freedom from deterioration at 10 years for patients less than 30 years of age is 26.8 ± 17.2%; 30 to 59 years, 77.4 ± 3.0%; and 60 years and older, 83.1 ± 4.2%. The aortic valve prosthesis performed superiorly in the younger and older age groups while in the intermediate group there was no difference in the performance of aortic and mitral valve prostheses.
The α-gal epitope is a carbohydrate antigen that interacts specifically with the natural anti-Gal antibody--the most abundant antibody in humans. Anti-Gal/α-gal epitope interaction activates complement to generate chemotactic factors that induce rapid recruitment of macrophages. The authors hypothesized that α-gal epitopes on nanoparticles can accelerate wound healing by inducing rapid recruitment and activation of macrophages in wounds.
α-Gal nanoparticles were generated from phospholipids, cholesterol, and α-gal glycolipids. α-Gal nanoparticle treatment of wounds was studied in 12 α1,3galactosyltrasferase knockout pigs. Like humans, these pigs lack α-gal epitopes and produce the natural anti-Gal antibody. Full-thickness wounds (20 × 20 mm) with tattooed borders were created on the back of pigs. α-Gal nanoparticles (10 or 100 mg) were topically applied onto the wounds. Saline-treated wounds served as control. Wound open surface area was measured every 3 to 4 days during dressing changes. Wounds from euthanized pigs were subjected to histological evaluation.
Treated wounds displayed many more macrophages and increased angiogenesis than control wounds in the same pig. On day 10, wounds treated with 10 mg and 100 mg displayed 35 and 60 percent decreased open surface area compared with control wounds, respectively, and 80 and 90 percent less than control wounds on day 13 (p < 0.05). No keloid formation or no increase in scar formation was observed on day 60.
α-Gal nanoparticle treatment of wounds accelerates macrophage recruitment, angiogenesis, and wound healing in pigs producing the anti-Gal antibody. As humans produce high titers of anti-Gal antibodies, this treatment may exhibit a similar beneficial effect in the clinical setting.
Anti-non gal antibodies are produced in xenograft recipients against multiple xenogeneic antigens. Studies in monkeys transplanted with pig organs lacking α-gal epitopes have suggested that anti-non gal antibodies mediate acute and chronic rejection of xenografts. This overview describes studies of these antibodies in patients who received xenografts and includes (1) an ovarian carcinoma patient receiving three intraperitoneal infusions of mouse fibroblasts in a gene therapy study, (2) orthopedic patients with torn anterior cruciate ligament replaced by a ligament made of pig patellar tendon, and (3) diabetic patients receiving fetal pig islet cell clusters xenograft together with a kidney allograft. Anti-non gal antibodies were found to be continuously produced as long as the xenograft was present in the recipient and were directed against a large number of pig proteins. Monitoring the immune response in the recipient of mouse fibroblasts indicated that the production of anti-non gal antibodies is much slower than that of the anti-Gal antibody, suggesting that they are generated by multiple B-cell clones, each initially comprising relatively few cells. Potent immunosuppression to prevent allograft rejection does not fully inhibit the production of anti-non gal antibodies. Much of this antibody response seems to be due to the differences in amino acid sequences between pig and human orthologous proteins as a result of evolutionary mutations. Overcoming the anti-non gal antibody barrier will require immunosuppressive agents that preferentially inhibit this immune response while maintaining protection against pathogens, or alternatively development of methods for induction of immune tolerance to xenogeneic pig antigens.
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.
In 2009, we reported a novel form of delayed anaphylaxis to red meat that is related to serum IgE antibodies to the oligosaccharide galactose-α-1,3-galactose (alpha-gal). Most of these patients had tolerated meat for many years previously. The implication is that some exposure in adult life had stimulated the production of these IgE antibodies.
We sought to investigate possible causes of this IgE antibody response, focusing on evidence related to tick bites, which are common in the region where these reactions occur.
Serum assays were carried out with biotinylated proteins and extracts bound to a streptavidin ImmunoCAP.
Prospective studies on IgE antibodies in 3 subjects after tick bites showed an increase in levels of IgE to alpha-gal of 20-fold or greater. Other evidence included (1) a strong correlation between histories of tick bites and levels of IgE to alpha-gal (χ(2) = 26.8, P < .001), (2) evidence that these IgE antibodies are common in areas where the tick Amblyomma americanum is common, and (3) a significant correlation between IgE antibodies to alpha-gal and IgE antibodies to proteins derived from A americanum (r(s) = 0.75, P < .001).
The results presented here provide evidence that tick bites are a cause, possibly the only cause, of IgE specific for alpha-gal in this area of the United States. Both the number of subjects becoming sensitized and the titer of IgE antibodies to alpha-gal are striking. Here we report the first example of a response to an ectoparasite giving rise to an important form of food allergy.
Human subjects and Old World primates have high levels of antibody to galactose-α-1,3 galactose β-1,4-N-acetylglucosamine (α-Gal). Commercially available bioprosthetic heart valves of porcine and bovine origin retain the Gal antigen despite current processing techniques. Gal-deficient pigs eliminate this xenoantigen. This study tests whether binding of human anti-Gal antibody effects calcification of wild-type and Gal-deficient glutaraldehyde-fixed porcine pericardium by using a standard subcutaneous implant model.
Expression of α-Gal was characterized by lectin Griffonia simplicifolia-IB4 staining. Glutaraldehyde-fixed pericardial disks from Gal-positive and Gal-deficient pigs were implanted into 12-day-old Wistar rats and 1.5-kg rabbits with and without prelabeling with affinity-purified human anti-Gal antibody. Calcification of the implants was determined after 3 weeks by using inductively coupled plasma spectroscopy.
The α-Gal antigen was detected in wild-type but not Gal-deficient porcine pericardium. Wild-type disks prelabeled with human anti-Gal antibody exhibited significantly greater calcification compared with that seen in antibody-free wild-type samples (mean ± standard error of the mean: 111 ± 8.4 and 74 ± 9.6 mg/g, respectively; P = .01). In the presence of anti-Gal antibody, a significantly greater level of calcification was detected in wild-type compared with GTKO porcine pericardium (111 ± 8.4 and 55 ± 11.8 mg/g, respectively; P = .005). Calcification of Gal-deficient pericardium was not affected by the presence of anti-Gal antibody (51 ± 9.1 and 55 ± 11.8 mg/g).
In this model anti-Gal antibody accelerates calcification of wild-type but not Gal-deficient glutaraldehyde-fixed pericardium. This study suggests that preformed anti-Gal antibody present in all patients might contribute to calcification of currently used bioprosthetic heart valves. Gal-deficient pigs might become the preferred source for new, potentially calcium-resistant bioprosthetic heart valves.
Although glutaraldehyde fixation is known to reduce immunogenicity and degeneration of heart valve bioprostheses, some degree of immunogenicity persists, which may trigger calcification. The aims of this study were to: (1) define the role of alpha-1,3-galactosyltransferase (alpha-Gal) antigen in valve calcification by comparing alpha-Gal-positive and alpha-Gal-deficient (GT-KO) pig pericardium; and (2) elucidate the role of human anti-Gal antibodies in the process of calcification and to determine the potential influence of different tissue-fixation techniques.
Glutaraldehyde-treated pericardium from alpha-Gal-positive and GT-KO pigs, with or without pre-labeling with human anti-Gal antibodies, were implanted in rats during 1 month.
In glutaraldehyde-fixed pericardium, calcification levels were significantly lower in GT-KO pig pericardium (132.8 +/- 5.8 microg/mg) as compared with alpha-Gal-positive pig pericardium (155.7 +/- 7.1 microg/mg) (p < 0.015). In glutaraldehyde-fixed pig pericardium followed by a mix of formaldehyde, ethanol and Tween 80 (FET), the calcification levels were lower in GT-KO pig pericardium (0.35 +/- 0.1 microg/mg) as compared with alpha-Gal-positive pig pericardium (4.6 +/- 4.2 microg/mg). In glutaraldehyde-fixed pig pericardium + FET pre-incubated with human anti-Gal antibodies, calcification levels were significantly greater in alpha-Gal-positive pig pericardium (43.8 +/- 8.5 microg/mg) as compared with GT-KO pig pericardium (5.7 +/- 2.9 microg/mg) (p < 0.0001).
This study demonstrates the role of alpha-Gal antigen and human alpha-Gal antibodies in the calcification process of valvular bioprostheses. It is suggested that GT-KO pig pericardium could be beneficial as a new source of material for heart valve bioprostheses.
Developing an effective HIV-1 vaccine will require strategies to enhance antigen presentation to the immune system. In a previous study we demonstrated a marked increase in immunogenicity of the highly glycosylated HIV-1 gp120 protein following enzymatic addition of alpha-gal epitopes to the carbohydrate chains. In the present study we determined whether gp120(alphagal) can also serve as an effective platform for targeting other HIV-1 proteins to APC and thus increase immunogenicity of both proteins. For this purpose we produced a recombinant fusion protein between gp120 and the HIV-1 matrix p24 protein (gp120/p24). Multiple alpha-gal epitopes were synthesized enzymatically on the gp120 portion of the fusion protein to generate a gp120(alphagal)/p24 vaccine. Immune responses to gp120(alphagal)/p24 compared to gp120/p24 vaccine lacking alpha-gal epitopes were evaluated in alpha1,3galactosyltransferase knockout (KO) mice. These mice lack alpha-gal epitopes and, therefore, are capable of producing the anti-Gal antibody. T cell responses to p24, as assessed by ELISPOT and by CD8+ T cells intracellular staining assays for IFNgamma, was on average 12- and 10-fold higher, respectively, in gp120(alphagal)/p24 immunized mice than in mice immunized with gp120/p24. In addition, cellular and humoral immune responses against gp120 were higher by 10-30-fold in mice immunized with gp120(alphagal)/p24 than in gp120/p24 immunized mice. Our data suggest that the alpha-gal epitopes on the gp120 portion of the fusion protein can significantly augment the immunogenicity of gp120, as well as that of the fused viral protein which lacks alpha-gal epitopes. This strategy of anti-Gal mediated targeting to APC may be used for production of effective HIV-1 vaccines comprised of various viral proteins fused to gp120.
Topical application of α-gal liposomes on burns results in rapid local recruitment of neutrophils and macrophages. Recruited macrophages are pivotal for healing of burns because they secrete cytokines/growth factors that induce epidermis regeneration and tissue repair. α-Gal liposomes have glycolipids with α-gal epitopes (Galα1-3Galβ1-4GlcNAc-R) which bind anti-Gal, the most abundant natural antibody in humans constituting ∼1% of immunoglobulins. Interaction of α-gal liposomes with anti-Gal within the fluid film formed on burns, activates complement and generates chemotactic complement cleavage peptides which effectively recruit neutrophils and macrophages. Anti-Gal IgG coating α-gal liposomes further binds to Fcγ receptors on macrophages and activates them to secrete cytokines/growth factors. Efficacy of α-gal liposomes treatment in accelerating burn healing is demonstrated in the experimental model of α1,3galactosyltransferase knockout mice. These mice are the only available nonprimate mammals that can produce anti-Gal in titers similar to those in humans. Pairs of burns in mice were covered either with a spot bandage coated with 10 mg α-gal liposomes, or with a control spot bandage coated with saline. On Day 3 post-treatment, the α-gal liposomes treated burns contained ∼5-fold as many neutrophils as control burns, whereas macrophages were found only in α-gal liposomes treated burns. On Day 6, 50-100% of the surface area of α-gal liposomes treated burns were covered with regenerating epidermis (re-epithelialization), whereas almost no epidermis was found in control burns. The extensive recruitment of macrophages by anti-Gal/α-gal liposomes interaction was further demonstrated in vivo with polyvinyl alcohol (PVA) sponge discs containing α-gal liposomes, implanted subcutaneously. Since anti-Gal is abundant in all humans, it is suggested that treatment with α-gal liposomes will be effective also in patients with burns and other skin wounds.
Anti-Gal constitutes approximately 1% of circulating IgG in humans and interacts specifically with alpha-gal epitopes. We reported previously that expression of alpha-gal epitopes on HIV gp120 and influenza virus vaccines increases immunogenicity by approximately 100-fold. We hypothesize that immunogenicity of any microbial vaccine can be markedly increased by linked alpha-gal epitopes due to in vivo formation of immune complexes with anti-Gal and the effective internalization of such immune complexes by APC, via Fc/FcgammaR interaction. The increased transport to lymph nodes and processing of anti-Gal complexed vaccines internalized by APC, results in effective activation of vaccine specific CD4(+) and CD8(+) T cells, and high cellular and humoral immune response. This universal mechanism for anti-Gal mediated increased immunogenicity is demonstrated in alpha1,3galactosyltransferase knockout mice with ovalbumin as a model vaccine.
All cancers arise as a result of changes that have occurred in the DNA sequence of the genomes of cancer cells. Over the past quarter of a century much has been learnt about these mutations and the abnormal genes that operate in human cancers. We are now, however, moving into an era in which it will be possible to obtain the complete DNA sequence of large numbers of cancer genomes. These studies will provide us with a detailed and comprehensive perspective on how individual cancers have developed.
Carbohydrate moieties are frequently encountered in food and can elicit IgE responses, the clinical significance of which has been unclear. Recent work, however, has shown that IgE antibodies to galactose-alpha-1,3-galactose (alpha-gal), a carbohydrate commonly expressed on nonprimate mammalian proteins, are capable of eliciting serious, even fatal, reactions.
We sought to determine whether IgE antibodies to alpha-gal are present in sera from patients who report anaphylaxis or urticaria after eating beef, pork, or lamb.
Detailed histories were taken from patients presenting to the University of Virginia Allergy Clinic. Skin prick tests (SPTs), intradermal skin tests, and serum IgE antibody analysis were performed for common indoor, outdoor, and food allergens.
Twenty-four patients with IgE antibodies to alpha-gal were identified. These patients described a similar history of anaphylaxis or urticaria 3 to 6 hours after the ingestion of meat and reported fewer or no episodes when following an avoidance diet. SPTs to mammalian meat produced wheals of usually less than 4 mm, whereas intradermal or fresh-food SPTs provided larger and more consistent wheal responses. CAP-RAST testing revealed specific IgE antibodies to beef, pork, lamb, cow's milk, cat, and dog but not turkey, chicken, or fish. Absorption experiments indicated that this pattern of sensitivity was explained by an IgE antibody specific for alpha-gal.
We report a novel and severe food allergy related to IgE antibodies to the carbohydrate epitope alpha-gal. These patients experience delayed symptoms of anaphylaxis, angioedema, or urticaria associated with eating beef, pork, or lamb.
Studies on the distribution of alpha-galactosyl epitopes with the structure Gal alpha 1----3Gal beta 1----4GlcNac-R on mammalian thyroid cell membranes are of interest, since a natural antibody interacting with this carbohydrate antigen (i.e. the natural anti alpha-galactosyl IgG antibody) was found to increase in its titre in patients with autoimmune thyroid disorders. By using a radioimmunoassay for quantification of the alpha-galactosyl epitope, we found variable concentrations of the alpha-galactosyl epitope, we found variable concentrations of this epitope on thyroid cell membranes of all nonprimate mammals and New World monkeys studied, but not in Old World monkeys and human thyroid. The absence of the identifiable alpha-galactosyl epitopes on human and Old World monkey thyroid cells correlates with diminished activity of the enzyme, alpha 1-3 galactosyltransferase, which, in other species, synthesizes the alpha-galactosyl epitopes within the Golgi apparatus. It is argued that a proportion of anti-thyroid reactivity in human normal and pathologic sera, when assayed with mammalian thyroid cells, may be attributed to natural anti alpha-galactosyl IgG antibody, which interacts with alpha-galactosyl epitopes on thyroid tissues used for the bioassay.