α-Gal Antigen Research Articles

Preparation of decellularized bone graft material with supercritical carbon dioxide extraction technique.

To evaluate the immunogenicity and osteogenic ability of animal-derived bone graft material decellularized with supercritical carbon dioxide. Porcine femurs were randomly divided into two groups after preliminary treatment, and decellularized with conventional method (conventional control group) or supercritical carbon dioxide (experimental group). Clearance rate of galactose-α-1, 3-galactose (α-Gal) and residual DNA of the two groups were analyzed to assess the immunogenicity of the xenogenic materials. Clearance rate of α-Gal was determined by enzyme-linked immunosorbent assay and residual DNA was detected by fluorescence method. Nine SPF-grade male athymic nude mice of 6 weeks old were randomly divided into experimental group, conventional control group and positive control group. Samples were implanted over biceps femoris muscle of athymic nude mice, the explants were collected 4 weeks post implantation, hematoxylin and eosin (HE) staining and immunohistochemistry were applied to determine the osteogenic ability and bone tissue-associated protein expressions of the implants. The clearance rates of α-Gal antigen in the experimental group and the conventional control group were (99.09±0.26)% and (30.18±2.02)%, respectively (t=58.67, P<0.01). The residual DNA of the experimental group, the conventional control group and the positive control group were (13.49±0.07) ng/mg, (15.20±0.21) ng/mg and (14.70±0.17) ng/mg, the residual DNA in the experimental group was significantly lower than that in the conventional control group (t=－13.41, P<0.01) and the positive control group (t=－11.30, P<0.01). HE staining showed that multiple bone formation centers with active osteogenesis and rich bone marrow were observed in experimental group 4 weeks after implantation, only a small number of bone formation centers were observed in the conventional control group and the positive control group, with no obvious osteoblasts present. Immunohistochemistry results indicated that the expressions of alkaline phosphatase, Runt-related transcription factor 2, typeⅠcollagen and osteocalcin in the experimental group showed an increasing trend compared with those in the conventional control group and the positive control group. Compared with clinically used demineralized bone matrix and bone graft material decellularized with conventional method, bone graft material decellularized with supercritical carbon dioxide exhibits lower immunogenicity and better osteogenic ability.

A Macrocyclic Tweezers-Shaped Receptor for the Biomimetic Recognition of the Gal(α1-3)Gal Disaccharide of the α-Gal Antigen.

The Gal(α1-3)Gal is the terminal disaccharide unit of the α-Gal epitope [Gal(α1-3)Gal(β1-4)GlcNAc], an exogenous antigenic determinant with several clinical implications, found in all non-primate mammals and in several dangerous pathogens, including certain protozoa and mycobacteria. Its absence in humans makes the α-Gal epitope an interesting target for several infectious diseases. Here we present the development of a macrocyclic tweezers-shaped receptor, resulting from the combination of the structural features of two predecessors belonging to the family of diaminocarbazole receptors, which exhibits binding properties in the low millimolar range toward the Gal(α1-3)Gal disaccharide of the α-Gal antigen.

Preparation and in vivo osteogenesis of acellular dermal matrix/dicalcium phosphate composite scaffold for bone repair

To investigate the physicochemical properties, osteogenic properties, and osteogenic ability in rabbit model of femoral condylar defect of acellular dermal matrix (ADM)/dicalcium phosphate (DCP) composite scaffold. ADM/DCP composite scaffolds were prepared by microfibril technique, and the acellular effect of ADM/DCP composite scaffolds was detected by DNA residue, fat content, and α-1，3-galactosyle (α-Gal) epitopes; the microstructure of scaffolds was characterized by field emission scanning electron microscopy and mercury porosimetry; X-ray diffraction was used to analyze the change of crystal form of scaffold; the solubility of scaffolds was used to detect the pH value and calcium ion content of the solution; the mineralization experiment in vitro was used to observe the surface mineralization. Twelve healthy male New Zealand white rabbits were selected to prepare the femoral condylar defect models, and the left and right defects were implanted with ADM/DCP composite scaffold (experimental group) and skeletal gold ® artificial bone repair material (control group), respectively. Gross observation was performed at 6 and 12 weeks after operation; Micro-CT was used to detect and quantitatively analyze the related indicators [bone volume (BV), bone volume/tissue volume (BV/TV), bone surface/bone volume (BS/BV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), bone mineral density (BMD)], and HE staining and Masson staining were performed to observe the repair of bone defects and the maturation of bone matrix. Gross observation showed that the ADM/DCP composite scaffold was a white spongy solid. Compared with ADM, ADM/DCP composite scaffolds showed a significant decrease in DNA residue, fat content, and α-Gal antigen content ( P<0.05). Field emission scanning electron microscopy showed that the ADM/DCP composite scaffold had a porous structure, and DCP particles were attached to the porcine dermal fibers. The porosity of the ADM/DCP composite scaffold was 76.32%±1.63% measured by mercury porosimetry. X-ray diffraction analysis showed that the crystalline phase of DCP in the ADM/DCP composite scaffolds remained intact. Mineralization results in vitro showed that the hydroxyapatite layer of ADM/DCP composite scaffolds was basically mature. The repair experiment of rabbit femoral condyle defect showed that the incision healed completely after operation without callus or osteophyte. Micro-CT showed that bone healing was complete and a large amount of new bone tissue was generated in the defect site of the two groups, and there was no difference in density between the defect site and the surrounding bone tissue, and the osteogenic properties of the two groups were equivalent. There was no significant difference in BV, BV/TV, BS/BV, Tb.Th, Tb.N, and BMD between the two groups ( P>0.05), except that the Tb.Sp in the experimental group was significantly higher than that in the control group ( P<0.05). At 6 and 12 weeks after operation, HE staining and Masson staining showed that the new bone and autogenous bone fused well in both groups, and the bone tissue tended to be mature. The ADM/DCP composite scaffold has good biocompatibility and osteogenic ability similar to the artificial bone material in repairing rabbit femoral condylar defects. It is a new scaffold material with potential in the field of bone repair.

The α-Gal KO Mouse Animal Model is a Reliable and Predictive Tool for the Immune-Mediated Calcification Assessment of Heart Valve Bioprostheses.

Recent studies highlighted the presence of anti-α-Gal antibodies in patients implanted with commercial bioprosthetic heart valves (BHVs). BHVs expose residual α-Gal xenoantigen and their recognition by the circulating anti-Gal antibodies leads to opsonization of the device's tissue component with the consequent triggering of a deterioration pathway that culminates with calcification. Small animal models such as mice and rats have been broadly involved in the in vivo testing of biomaterials by subcutaneous implantation, especially for the effectiveness of BHVs anti-calcific treatments. However, since models employed for this purpose express α-Gal antigen, the implantation of BHVs' leaflets does not elicit a proper immunological response, so the calcification propensity may be dramatically underestimated. An α-Gal knockout (KO) mouse model has been created, using the CRISP/Cas9 approach, and adopted to assess the calcification potential of commercial BHVs leaflets through the surgical implantation in the back subcutis area. Calcium quantification was performed by inductively coupled plasma analysis; immune response against the BHVs leaflets and α-Gal silencing was evaluated through immunological assays. Two months after the implantation of commercial BHV leaflets, the anti-Gal antibody titers in KO mice doubled when compared with those found in wild-type (WT) ones. Leaflets explanted from KO mice, after one month, showed a four-time increased calcium deposition concerning the ones explanted from WT. The degree of silencing of α-Gal varied, depending on the specific organ that was assessed. In any case, the animal model was suitable for evaluating implanted tissue responses. Such mouse model proved to be an accurate tool for the study of the calcific propensity of commercial BHVs leaflets than those hitherto used. Given its reliability, it could also be successfully used to study even other diseases in which the possible involvement of α-Gal has been observed.

Development of porcine skeletal muscle extracellular matrix–derived hydrogels with improved properties and low immunogenicity

Hydrogels derived from decellularized extracellular matrices (ECM) of animal origin show immense potential for regenerative applications due to their excellent cytocompatibility and biomimetic properties. Despite these benefits, the impact of decellularization protocols on the properties and immunogenicity of these hydrogels remains relatively unexplored. In this study, porcine skeletal muscle ECM (smECM) underwent decellularization using mechanical disruption (MD) and two commonly employed decellularization detergents, sodium deoxycholate (SDC) or Triton X-100. To mitigate immunogenicity associated with animal-derived ECM, all decellularized tissues were enzymatically treated with α-galactosidase to cleave the primary xenoantigen-the α-Gal antigen. Subsequently, the impact of the different decellularization protocols on the resultant hydrogels was thoroughly investigated. All methods significantly reduced total DNA content in hydrogels. Moreover, α-galactosidase treatment was crucial for cleaving α-Gal antigens, suggesting that conventional decellularization methods alone are insufficient. MD preserved total protein, collagen, sulfated glycosaminoglycan, laminin, fibronectin, and growth factors more efficiently than other protocols. The decellularization method impacted hydrogel gelation kinetics and ultrastructure, as confirmed by turbidimetric and scanning electron microscopy analyses. MD hydrogels demonstrated high cytocompatibility, supporting satellite stem cell recruitment, growth, and differentiation into multinucleated myofibers. In contrast, the SDC and Triton X-100 protocols exhibited cytotoxicity. Comprehensive in vivo immunogenicity assessments in a subcutaneous xenotransplantation model revealed MD hydrogels' biocompatibility and low immunogenicity. These findings highlight the significant influence of the decellularization protocol on hydrogel properties. Our results suggest that combining MD with α-galactosidase treatment is an efficient method for preparing low-immunogenic smECM-derived hydrogels with enhanced properties for skeletal muscle regenerative engineering and clinical applications.

Safety and tissue remodeling assay of small intestinal submucosa meshes using a modified porcine surgical hernia model

In studies to date, meshes based on extracellular matrix (ECM) have been extensively used in clinical applications. Unfortunately, little is known about the function of the immunogenic residual, absorbable profile during the tissue repair process. Moreover, there needs to be a recognized preclinical animal model to investigate the safety and efficacy of extracellular matrix meshes. Herein, we designed and fabricated a kind of SIS mesh followed by a scanned electron micrograph characterization and tested α-Gal antigen clearance rate and DNA residual. In order to prove the biocompatibility of the SIS mesh, cell viability, chemotaxis assay and local tissue reaction were assessed by MTT and RTCA cytotoxicity test in vitro as well as implantation and degradation experiments in vivo. Furthermore, we developed a stable preclinical animal model in the porcine ventral hernia repair investigation, which using laparoscopic plus open hybridization method to evaluate tissue adhesion, explant mechanical performance, and histologic analysis after mesh implantation. More importantly, we established a semi-quantitative scoring system to examine the ECM degradation, tissue remodeling and regeneration in the modified porcine surgical hernia model for the first time. Our results highlight the application prospect of the improved porcine ventral hernia model for the safety and efficacy investigation of hernia repair meshes.

Structural and biomechanical characterizations of acellular porcine mitral valve scaffolds: anterior leaflets, posterior leaflets, and chordae tendineae.

Mitral valve (MV) tissue engineering is still in its early stage, and one major challenge in MV tissue engineering is to identify appropriate scaffold materials. With the potential of acellular MV scaffolds being demonstrated recently, it is important to have a full understanding of the biomechanics of the native MV components and their acellular scaffolds. In this study, we have successfully characterized the structural and mechanical properties of porcine MV components, including anterior leaflet (AL), posterior leaflet (PL), strut chordae, and basal chordae, before and after decellularization. Quantitative DNA assay showed more than 90% reduction in DNA content, and Griffonia simplicifolia (GS) lectin immunohistochemistry confirmed the complete lack of porcine α-Gal antigen in the acellular MV components. In the acellular AL and PL, the atrialis, spongiosa, and fibrosa trilayered structure, along with its ECM constitutes, i.e., collagen fibers, elastin fibers, and portion of GAGs, were preserved. Nevertheless, the ECM of both AL and PL experienced a certain degree of disruption, exhibiting a less dense, porous ECM morphology. The overall anatomical morphology of the strut and basal chordae were also maintained after decellularization, with longitudinal morphology experiencing minimum disruption, but the cross-sectional morphology exhibiting evenly-distributed porous structure. In the acellular AL and PL, the nonlinear anisotropic biaxial mechanical behavior was overall preserved; however, uniaxial tensile tests showed that the removal of cellular content and the disruption of structural ECM did result in small decreases in maximum tensile modulus, tissue extensibility, failure stress, and failure strain for both MV leaflets and chordae.

α-Gal antigen-deficient rabbits with GGTA1 gene disruption via CRISPR/Cas9

BackgroundPrevious studies have identified the carbohydrate epitope Galα1–3Galβ1–4GlcNAc-R (termed the α-galactosyl epitope), known as the α-Gal antigen as the primary xenoantigen recognized by the human immune system. The α-Gal antigen is regulated by galactosyltransferase (GGTA1), and α-Gal antigen-deficient mice have been widely used in xenoimmunological studies, as well as for the immunogenic risk evaluation of animal-derived medical devices. The objective of this study was to develop α-Gal antigen-deficient rabbits by GGTA1 gene editing with the CRISPR/Cas9 system.ResultsThe mutation efficiency of GGTA1 gene-editing in rabbits was as high as 92.3% in F0 pups. Phenotype analysis showed that the α-Gal antigen expression in the major organs of F0 rabbits was decreased by more than 99.96% compared with that in wild-type (WT) rabbits, and the specific anti-Gal IgG and IgM antibody levels in F1 rabbits increased with increasing age, peaking at approximately 5 or 6 months. Further study showed that GGTA1 gene expression in F2-edited rabbits was dramatically reduced compared to that in WT rabbits.Conclusionsα-Gal antigen-deficient rabbits were successfully generated by GGTA1 gene editing via the CRISPR/Cas9 system in this study. The feasibility of using these α-Gal antigen-deficient rabbits for the in situ implantation and residual immunogenic risk evaluation of animal tissue-derived medical devices was also preliminarily confirmed.

Comparing the Characteristics of Amniotic Membrane-, Endometrium-, and Urinary-Derived ECMs and Their Effects on Endometrial Regeneration in a Rat Uterine Injury Model

The decellularized extracellular matrices (d-ECMs) currently utilized to repair endometrial injuries are derived from three tissue sources, the endometrium (dE-ECM), placental amniotic membrane (dA-ECM), and urinary (dU-ECM). Notably, the structures of dU-ECM and dE-ECM are similar. These d-ECMs are derived from different tissues, and their specific roles in endometrial injury repair remain unclear. This study aimed to analyse the characteristics of the tissue microstructures and compositions to confirm specific differences among the three ECM types. And using a rat model of endometrial injury, the effects of all the matrices after implantation in vivo on the promotion of endometrial regeneration were analysed. After decellularization, dE-ECM had more residual active factors than the other two ECM types, while dA-ECM had significantly less DNA, α-Gal antigen components and extracellular matrix components than the other two groups. Although the three ECMs had no effect on the proliferation of stromal cells in vitro, dA-ECM may have increased the sensitivity of stromal cells to oestradiol (E2) responses. In vivo experiments confirmed the promotional effect of dA-ECM on endometrial regeneration. For example, the endometrial thickness, collagen deposition, endometrial tissue regeneration, vascular regeneration and pregnancy outcomes were significantly better in this group than in the other two groups. These findings might be associated with the excellent immune tolerance of dA-ECM. Therefore, when selecting a d-ECM for the treatment of endometrial injury, dE-ECM, which has the strongest tissue specificity, is not the preferred choice. Controlling the inflammatory responses in local lesions at the early stage may be a prerequisite for ECMs to exert their functions.

Tick Saliva and the Alpha-Gal Syndrome: Finding a Needle in a Haystack.

Ticks and tick-borne diseases are significant public health concerns. Bioactive molecules in tick saliva facilitate prolonged blood-feeding and transmission of tick-borne pathogens to the vertebrate host. Alpha-gal syndrome (AGS), a newly reported food allergy, is believed to be induced by saliva proteins decorated with a sugar molecule, the oligosaccharide galactose-⍺-1,3-galactose (α-gal). This syndrome is characterized by an IgE antibody-directed hypersensitivity against α-gal. The α-gal antigen was discovered in the salivary glands and saliva of various tick species including, the Lone Star tick (Amblyomma americanum). The underlying immune mechanisms linking tick bites with α-gal-specific IgE production are poorly understood and are crucial to identify and establish novel treatments for this disease. This article reviews the current understanding of AGS and its involvement with tick species.

Immune risk assessment of residual αGal in xenogeneic decellularized cornea using GTKO mice.

The xenogeneic decellularized corneal matrix (DCM) was expected to be used in lamellar keratoplasty in clinic as the substitute of allogeneic cornea. After decellularization treatment, the remaining risk of xenograft rejection needed to be assessed. The galactose-α1,3-galactose, as the most abundant and closely rejection-related xenogeneic antigen, should be one of the important factors concerned in immunological evaluation. In this study, residual αGal in the DCM was first determined by an enzyme-linked immunosorbent assay method with qualified accuracy and specificity. Then the DCM was implanted subcutaneously into the α1,3-galactosyltransferase gene-knockout (GTKO) mice, accompanied by the implantation in the wild-type C57BL/6 mice as a comparison. The total serum antibody levels, anti-Gal antibody levels, inflammatory cytokines and ratios of splenic lymphocyte subtypes were detected and the histopathological analysis of implants were performed to systematically evaluate the immune responses. The experimental result showed the fresh porcine corneal matrix samples had (9.90 ± 1.54) × 1012 αGal epitope per mg while the content of residual αGal in the DCM was (7.90 ± 2.00) × 1012 epitope per mg. The GTKO mice had similar potential of reaction to immune stimulation to that of wild-type C57BL/6 mice. At 4 weeks after implantation of DCM, in WT mice and GTKO mice there were both innate immunity response to the DCM characterized by macrophage infiltration. But the elevations of anti-Gal IgG level and the percentage of splenic natural killer cells were only detected in GTKO mice. These changes were thought to be pertinent to the residual αGal antigen, which could not be detected in WT mice. No further αGal antibody-mediated cellular immunity and significant changes of serum cytokine contents were found in GTKO mice, which perhaps suggested that the immune reactions to the DCM after 4 weeks of implantation were moderate and had minor effect on the survival of the corneal graft.

In vitro and in vivo biocompatibility study on acellular sheep periosteum for guided bone regeneration

This study addresses the fabrication of an extracellular matrix material of the acellular sheep periosteum and the systematic evaluation of its biocompatibility to explore its potential application in guided bone regeneration. Sheep periosteum was harvested and decellularized by a combined decellularization protocol. The effectiveness of cell removal was proved and residual α-Gal antigen was also quantitatively detected. Then, mouse MC3T3-E1 cells were seeded onto the acellular periosteum. A scanning electron microscope (SEM) was used to record the whole process of cell adhesion. The CCK-8 assay suggested that the acellular periosteum not only had zero toxic effect on pre-osteoblasts, but played a positive role in cell proliferation. We also tested whether the acellular periosteum possesses favorable osteogenesis induction activity using an alkaline phosphatase (ALP) assay and a quantitative real-time PCR (Col I, Runx2, OCN) assay. An in vivo study of a subcutaneous implantation test using Sprague Dawley (SD) rats was performed to detect the changes in IL-2, IFN-γ and IL-4 in serum and elucidate the host’s local response to acellular periosteum through hematoxylin and eosin (HE) and immunohistochemical staining. The results show that acellular sheep periosteum did not elicit a severe immunogenic response via the Th1 pathway, unlike fresh sheep periosteum. In conclusion, acellular sheep periosteum possesses favorable biocompatibility to be employed for guided bone regeneration.

Environmental and Molecular Drivers of the α-Gal Syndrome.

The α-Gal syndrome (AGS) is a type of allergy characterized by an IgE antibody (Ab) response against the carbohydrate Galα1-3Galβ1-4GlcNAc-R (α-Gal), which is present in glycoproteins from tick saliva and tissues of non-catarrhine mammals. Recurrent tick bites induce high levels of anti-α-Gal IgE Abs that mediate delayed hypersensitivity to consumed red meat products in humans. This was the first evidence that tick glycoproteins play a major role in allergy development with the potential to cause fatal delayed anaphylaxis to α-Gal-containing foods and drugs and immediate anaphylaxis to tick bites. Initially, it was thought that the origin of tick-derived α-Gal was either residual blood meal mammalian glycoproteins containing α-Gal or tick gut bacteria producing this glycan. However, recently tick galactosyltransferases were shown to be involved in α-Gal synthesis with a role in tick and tick-borne pathogen life cycles. The tick-borne pathogen Anaplasma phagocytophilum increases the level of tick α-Gal, which potentially increases the risk of developing AGS after a bite by a pathogen-infected tick. Two mechanisms might explain the production of anti-α-Gal IgE Abs after tick bites. The first mechanism proposes that the α-Gal antigen on tick salivary proteins is presented to antigen-presenting cells and B-lymphocytes in the context of Th2 cell-mediated immunity induced by tick saliva. The second mechanism is based on the possibility that tick salivary prostaglandin E2 triggers Immunoglobulin class switching to anti-α-Gal IgE-producing B cells from preexisting mature B cells clones producing anti-α-Gal IgM and/or IgG. Importantly, blood group antigens influence the capacity of the immune system to produce anti-α-Gal Abs which in turn impacts individual susceptibility to AGS. The presence of blood type B reduces the capacity of the immune system to produce anti-α-Gal Abs, presumably due to tolerance to α-Gal, which is very similar in structure to blood group B antigen. Therefore, individuals with blood group B and reduced levels of anti-α-Gal Abs have lower risk to develop AGS. Specific immunity to tick α-Gal is linked to host immunity to tick bites. Basophil activation and release of histamine have been implicated in IgE-mediated acquired protective immunity to tick infestations and chronic itch. Basophil reactivity was also found to be higher in patients with AGS when compared to asymptomatic α-Gal sensitized individuals. In addition, host resistance to tick infestation is associated with resistance to tick-borne pathogen infection. Anti-α-Gal IgM and IgG Abs protect humans against vector-borne pathogens and blood group B individuals seem to be more susceptible to vector-borne diseases. The link between blood groups and anti-α-Gal immunity which in turn affects resistance to vector-borne pathogens and susceptibility to AGS, suggests a trade-off between susceptibility to AGS and protection to some infectious diseases. The understanding of the environmental and molecular drivers of the immune mechanisms involved in AGS is essential to developing tools for the diagnosis, control, and prevention of this growing health problem.

Xenogeneic bone matrix immune risk assessment using GGTA1 knockout mice

Homeotransplantation of bones for replacement therapy have been demonstrated reliably in clinical data. However, human donor bones applicable for homeotransplantation are in short supply, which facilitates the search for suitable alternatives, such as xenografts grafts. The α-Gal antigen-related immune risk of xenografts directly affects the safety and effectiveness of the biomaterials and limits their applications in the clinic. The immune risk can be prevented by depletion or breaking anti-Gal antibody prior to transplant. Therefore, how to assess the immune risk of the bone substitutes and select the reliable animal research model become extremely important. In this study, we prepared lyophilized bone substitutes (T1) and Guanghao Biotech bone substitutes (T2, animal-derived biomaterials with α-Gal antigen decreased), aimed to assess the immune risk of xenografts bone substitutes on GGTA1 knockout mice. The α-Gal antigen contents of T1 and T2 were firstly detected by ELISA method in vitro. The bone substitutes were then implanted subcutaneously into GGTA1 knockout mice for 2, 4 and 12 weeks, respectively. The total serum antibody levels, anti-α-Gal antibody levels, inflammatory cytokine and splenic lymphocyte surface molecules were detected and histology analysis of skin and thymus were performed to systematically evaluate the immune response caused by the T1 and T2 bone substitutes in mice. In vitro results showed that the amount of α-Gal epitopes in T1 bone substitutes was significantly higher than T2 bone substitutes, and the clearance rate of α-Gal antigen in T2 bone substitutes achieved about 55.6%. Results of antibody level in vivo showed that the T1 bone substitutes group possessed significantly higher total IgG, IgM, IgA and anti-α-Gal IgG levels than T2 and control group, while T2 group showed no significant changes of these indexes compared with control. In terms of inflammatory cytokines, T1 bone substitutes showed evidently higher levels of IL-4, IL-12P70 and IL-10 than T2 and control, while T2 group was comparable to control. No changes in the levels of splenic lymphocyte surface molecules were found in the three groups (T1, T2 and control group) during the experimental periods. The pathological results demonstrated that the inflammatory response in T2 group was lighter than the T1 group, which was in accordance with the inflammatory cytokines levels. The above results indicated that the process of antigen removal effectively reduced the α-Gal antigens content in T2 bone substitutes, which caused little immune response in vivo and could be used as bone healing materials. This study also demonstrated that GGTA1 knockout mice can be used as a routine tool to assess the immune risk of animal-derived biomaterials.

Effect of membrane-bound complement regulatory proteins on tumor cell sensitivity to complement-dependent cytolysis triggered by heterologous expression of the α-gal xenoantigen.

Engineering malignant cells to express a heterologous α-gal antigen can induce heterograft hyperacute rejection, resulting in complement-dependent cytolysis (CDC) of tumor cells, which has been considered as a novel strategy for antitumor therapy. A549 cells engineered to express Galα1-3Galβ1-4GlcNAc-R (α-gal) epitope exhibited strong resistance to CDC treated by normal human serum (NHS) in a previous study. We hypothesized that the expression of membrane-bound complement regulatory proteins (mCRPs) decay accelerating factor (CD55) and protectin (CD59) influenced the efficacy of the α-gal/NHS-mediated antitumor effect to tumor cells in vitro. The present study confirmed that A549 cells expressed high levels of CD55 and CD59, whereas Lovo cells expressed relatively low levels of these proteins. A549 and Lovo cells transfected with plasmids containing or lacking the α-gal epitope were evaluated for their susceptibility to CDC by NHS and detected using a trypan blue exclusion assay. α-gal-expressing Lovo (Lovo-GT) cells were almost completely killed by α-gal-mediated CDC following incubation with 50% NHS, whereas no cytolysis was observed in α-gal expressing A549 (A549-GT) cells. Abrogating CD55 and CD59 function from A549-GT cells by various concentrations of phosphatidylinositol-specific phospholipase C (PI-PLC) or blocking antibodies increased the susceptibility of cells to CDC, and the survival rate decreased significantly comparing to the controls (P<0.05). The findings of the present study indicated that using the α-gal/NHS system to eliminate tumor cells via inducing the complement cascade reaction might represent a feasible approach for the treatment of cancer. However, high levels of mCRP expression may limit the efficacy of this approach. Therefore, an improved efficacy of cancer cell killing may be achieved by combining strategies of heterologous α-gal expression and mCRP downregulation.

Biomechanical comparison of xenogeneic bone material treated with different methods.

Bone xenografting is considered one of the most effective ways to address the shortage of bone autografts and allografts. Various methods have been employed to minimize the immune rejection issues associated with bone xenografts. However, the side effects of such methods on bone biomechanical properties remain unclear. As such, the objective of this study was to compare the influence of different treatments on the biomechanical properties of porcine bones. Fresh pig ribs were cut into 1.5×0.5×0.4cm specimens, which were randomly divided into four groups and subjected to different modification regimes: group A by degreasing and partial deproteinization, group B by cryopreservation, group C by cryopreservation and enzyme digestion, and group D was a control group using fresh bone. Biomechanical tests and α-Gal antigen detection were performed for all groups. In the axial compression test, the values for maximum load were as follows: group D>group C>group B>group A. The maximum load in group A was significantly less than in the other groups (P<.05). There were no differences between groups D, C, and B in terms of the maximum stress and elastic modulus (P>.05). The maximum stress and elastic modulus values recorded for group A were significantly less than for the other groups (P<.05). There were no significant differences in the maximum load or elastic modulus among groups B, C, and D, in the three-point bending test (P>.05). However, the maximum load and elastic modulus values recorded for group A were significantly lower than the other groups (P<.05). In groups A and C, no α-Gal antigen-positive expression was detected. In group B, there was low level α-Gal antigen-positive expression, while a high level of α-Gal antigen-positive expression was observed in fresh bone samples. The results indicated that xenogeneic bone subjected to degreasing and partial deproteinization had the worst biomechanical properties. Cryopreservation and cryopreservation with enzyme digestion had little effect on the bone biomechanical properties, although enzyme digestion resulted in the complete elimination of the α-Gal antigen. Cryopreservation with enzyme digestion treatment presented the best results in terms of removing the immune-response triggering α-Gal antigen, while preserving the good biomechanical properties of bone xenografts.

Diagnosis of red meat allergy with antigen-specific IgE tests in serum

Red meat allergy is a tick-associated hypersensitivity reaction to galactose-α-1,3-galactose (α-gal) and is characterized by anaphylaxis, angioedema, urticaria and/or gastrointestinal symptoms occurring 3–6 hours after ingesting red meat such as beef, pork, or lamb. Diagnosis of red meat allergy is challenging due to the unusually long delay in symptom onset and poor sensitivity of skin prick tests with commercial meat extracts. The primary diagnostic tools available for this disease are quantification of α-gal-, beef-, pork-, and/or lamb-specific IgE in serum, however the diagnostic performance of these tests has not been reported. Using patient data for n=135 patients with red meat allergy and n=37 controls, we found that measurement of α-gal-specific IgE using the bovine thyroglobulin (bTG) ImmunoCAP method had the best overall sensitivity (100%) and specificity (92.3%) for diagnosis of red meat allergy. Measuring α-gal-specific IgE using the streptavidin (SA)-CAP technique or beef- or pork-specific IgE using ImmunoCAP were also effective tests with high sensitivities (89–92%) and variable specificities (65–82%). Lamb-specific IgE and total IgE had essentially no diagnostic value for red meat allergy. Positive and negative predictive values mirrored these trends. Taken together, these findings indicate that the α-gal-specific IgE test by bTG ImmunoCAP is the most useful for establishing a diagnosis of red meat allergy, although α-gal-specific IgE by SA-CAP and beef- and pork-specific IgE by ImmunoCAP are also effective tests.

Virus-like Particle Display of the α-Gal Epitope for the Diagnostic Assessment of Chagas Disease

The α-Gal antigen [Galα(1,3)Galβ(1,4)GlcNAcα] is an immunodominant epitope displayed by infective trypomastigote forms of Trypanosoma cruzi, the causative agent of Chagas disease. A virus-like particle displaying a high density of α-Gal was found to be a superior reagent for the ELISA-based serological diagnosis of Chagas disease and the assessment of treatment effectiveness. A panel of sera from patients chronically infected with T. cruzi, both untreated and benznidazole-treated, was compared with sera from patients with leishmaniasis and from healthy donors. The nanoparticle-α-Gal construct allowed for perfect discrimination between Chagas patients and the others, avoiding false negative and false positive results obtained with current state-of-the-art reagents. As previously reported with purified α-Gal-containing glycosylphosphatidylinositol-anchored mucins, the current study also showed concentrations of anti-α-Gal IgG to decrease substantially in patients receiving treatment with benznidazole, suggesting that the semiquantitative assessment of serum levels of this highly abundant type of antibody can report on disease status in individual patients.

Quantification of α-Gal Antigen Removal in the Porcine Dermal Tissue by α-Galactosidase.

The α-Gal (Galα1,3-Galβ1-4GlcNAc-R) epitope, the major xenoantigen, is the first barrier in a porcine-to-man tissue and organ xenotransplantation. The elimination or reduction of the α-Gal epitopes is therefore an important step for a successful xenotransplantation. The present study is to evaluate the α-Gal elimination in the porcine skin with α-galactosidase treatment, and to assess two methods (immunohistochemistry and inhibition ELISA) that may be used in quality control for quantifying the extent of the α-Gal elimination. Enzymatic cleavage in a single-step process is extremely efficient and affordable at eliminating the α-Gal epitope even in a tissue as dense as the porcine dermis. The cost of enzymatic cleavage is found to be less than US$7 for a 10 × 10 cm piece of porcine skin (0.5 mm thick) or about US$140 for 100 g of 3-dimensional soft tissues. After enzymatic cleavage, the α-Gal-positive immunostaining was essentially undetectable in enzyme-treated porcine skin. The inhibition rate constant of the monoclonal anti-Gal antibody M86 binding to α-Gal-bovine serum albumin in ELISA was reduced from 15.0 ± 4.3 (n = 10) to 6.1 ± 2.6 (n = 7) after enzyme treatment, in comparison to 4.4 ± 1.8 (n = 9) background inhibition of decellularized human skin (the ultimate negative control), which demonstrates ∼ 84% elimination of α-Gal epitopes in treated porcine skin. To examine the suitability of two detection methods for the routine quality control application, comparative studies were made with control and enzyme-treated porcine skin, porcine skin from the α-Gal knockout animal, as well as decellularized human skin. The data show that the traditional immunohistochemistry and, to a less extent, the inhibition ELISA with further modifications can be used as quality control tools in the production and selection of biocompatible bioprosthetic devices. The biological evaluation of enzyme-treated porcine skin is ongoing with a small animal model and a nonhuman primate model.

Feasibility of pig and human-derived aortic valve interstitial cells seeding on fixative-free decellularized animal pericardium.

Glutaraldehyde-fixed pericardium of animal origin is the elective material for the fabrication of bio-prosthetic valves for surgical replacement of insufficient/stenotic cardiac valves. However, the pericardial tissue employed to this aim undergoes severe calcification due to chronic inflammation resulting from a non-complete immunological compatibility of the animal-derived pericardial tissue resulting from failure to remove animal-derived xeno-antigens. In the mid/long-term, this leads to structural deterioration, mechanical failure, and prosthesis leaflets rupture, with consequent need for re-intervention. In the search for novel procedures to maximize biological compatibility of the pericardial tissue into immunocompetent background, we have recently devised a procedure to decellularize the human pericardium as an alternative to fixation with aldehydes. In the present contribution, we used this procedure to derive sheets of decellularized pig pericardium. The decellularized tissue was first tested for the presence of 1,3 α-galactose (αGal), one of the main xenoantigens involved in prosthetic valve rejection, as well as for mechanical tensile behavior and distensibility, and finally seeded with pig- and human-derived aortic valve interstitial cells. We demonstrate that the decellularization procedure removed the αGAL antigen, maintained the mechanical characteristics of the native pig pericardium, and ensured an efficient surface colonization of the tissue by animal- and human-derived aortic valve interstitial cells. This establishes, for the first time, the feasibility of fixative-free pericardial tissue seeding with valve competent cells for derivation of tissue engineered heart valve leaflets.