Carbohydrate Epitopes Research Articles

Serum immunoglobulin E reactivity to cross-reacting panallergen components in north-eastern Poland patients pollen sensitized

Background: The presence of immunoglobulin E (IgE), which cross-reacts with allergen components, such as profilins, polcalcins, and cross-reacting carbohydrate determinants (CCD), creates a problem when selecting patients for allergen immunotherapy by using conventional methods. The aim of this study was to evaluate the prevalence of sensitization to profilins, polcalcins, and CCDs in patients with seasonal pollen allergic rhinitis. Methods: The study was performed on a group of 112 patients with seasonal pollen allergic rhinitis, ages 14 to 55 years, with sensitization to at least one seasonal allergen (IgE > 0.7 kUA/L). The presence of IgE sensitization to recombinant (r) Bet v 2, rPhl p 12, rBet v 4, rPhl p 7, and CCDs, in addition to rBet v 1, rPhl p 1, rPhl p 5, was evaluated by using a multiparameter immunoblot. Results: Among the studied patients, 64.3, 80.4, and 41.1% were sensitized to birch, timothy grass, and mugwort pollen, respectively. Sensitization to profilins rBet v 2/Phl p 12 was demonstrated in 28.6%, to polcalcins Bet v 4/Phl p 7 in 8.9%, and to CCDs in 25%. In 29.3%, serum IgE reactivity to any of the cross-reactive components could be demonstrated. Serum IgE reactivity to rBet v 2 was always accompanied by IgE reactivity to rPhl p 12, and IgE reactivity to rBet v 4 was always accompanied by IgE reactivity to rPhl p 7. Among the patients with pollinosis co-sensitized to at least two allergen sources according to extract-based diagnosis, possible false-positive results due to sensitization to cross-reactive components were detected in 17.9%. Conclusion: Evaluation of sensitization to cross-reacting components may be useful in evaluation of patients with pollen allergy who are being assessed for allergen immunotherapy to optimize the constitution of their immunotherapy vaccines.

Cross-reactive carbohydrate determinant interference in cellulose-based IgE allergy tests utilizing recombinant allergen components.

BackgroundCross-reactive carbohydrate determinant (CCD) structures found in plant and insect glycoproteins are commonly recognized by IgE antibodies as epitopes that can lead to extensive cross-reactivity and obscure in vitro diagnostic (IVD) serology results. With the introduction of component resolved diagnosis (CRD), recombinant non-glycosylated components have been utilized to mitigate the risk of CCD-specific IgE (sIgE) detection. However, a recent study has shown that CCD-sIgE may bind directly to the cellulose solid phase matrix used in certain in vitro diagnostic assays, eliminating the advantage of CRD over traditional extract-based testing. The aim of this study is to further investigate the prevalence of CCD-sIgE interference on a commonly-used in vitro sIgE automated platform which employs a cellulose-based matrix to immobilize CCD-free recombinant components.MethodsSera from patients sensitized to peanut, silver birch, and/or timothy grass were analyzed for CCD-sIgE reactivity on ImmunoCAP/Phadia and NOVEOS autoanalyzers against the MUXF3 carbohydrate component. Positive CCD-sIgE sera were further analyzed against non-glycosylated recombinant components bound to the ImmunoCAP solid phase in the absence and presence of a soluble CCD inhibitor. For comparison, sera were then analyzed on NOVEOS, a non-cellulose based automated sIgE assay.ResultsSera from 35% of the sensitized population tested in this study were positive (≥0.35 kU/L) for CCD-sIgE. Of those positives, 17% resulted in CCD-sIgE-positive (false positive) results on ImmunoCAP using non-glycosylated allergosorbents that were negative on NOVEOS. Sera producing false-positive results on ImmunoCAP had varying levels of CCD-sIgE from 0.67 kU/L to 36.52 kU/L. The incidence of CCD interference was predominantly delimited to low-positive IgE results (0.35 kUA/L– 3.00 kUA/L).ConclusionFalsely elevated diagnostic allergen-sIgE results can commonly occur due to the presence of CCD-sIgE using assays that employ a carbohydrate matrix-based allergosorbent. Even the use of non-glycosylated recombinant allergenic components coupled to cellulose matrices do not reduce their risk of detection. The risk of CCD interference that compromises quantitative IgE results can be mitigated by the addition of a soluble CCD inhibitor to positive CCD-sIgE containing sera or by alternatively using a non-cellulose based sIgE assay, such as the NOVEOS assay.

Structure of intact IgE and the mechanism of ligelizumab revealed by electron microscopy.

IgE is the central antibody isotype in TH2-biased immunity and allergic diseases. The structure of intact IgE and the impact of IgE-targeting molecules on IgE however remain elusive. In order to obtain insights into IgE biology and the clinical impact, we aimed for structure determination of IgE and the complex of IgE with the anti-IgE antibody ligelizumab. Structures of two distinct intact IgE with specificity for cross-reactive carbohydrate determinants and Der p 2 as well as complexes of ligelizumab-Fab with IgE and IgE Fc were assessed by negative stain electron microscopy and solution scattering. Inhibition of IgE binding and displacement of receptor-bound IgE were assessed using cellular assays, basophil activation testing and ELIFAB assays. Our data reveal that the investigated IgE molecules share an overall rigid conformation. In contrast to the IgE Fc fragment, the IgE Fc in intact IgE is significantly less asymmetrically bent. The proximal and the distal Fabs are rigidly tethered to the Fc. Binding of ligelizumab to IgE in a 2:1 stoichiometry induces an extended and twofold symmetrical conformation of IgE, which retains a rigid Fab-Fc architecture. Analyses of effector cell activation revealed that ligelizumab inhibits IgE binding without displacing receptor-bound IgE. Together with an interference of CD23 binding, the data underline a functional activity similar to omalizumab. Our data reveal the first structures of intact IgE suggesting that the IgE Fab is fixed relative to the Fc. Furthermore, we provide a structural rationale for the inhibitory mechanism of ligelizumab.

Kodecytes: modifying the surface of red blood cells

There are few routine techniques available to modify the antigenic surface of red blood cells (RBC). Primarily, modification techniques involve antigen destruction with enzymes or denaturing chemicals. In contrast, very few antigen additive technologies exist. One antigen additive technology involves the passive insertion of Function‐Spacer‐Lipid constructs (Kode Technology) into the cell membrane. Kode constructs can label any cell (creating kodecytes) with controlled levels of almost any small molecule, including blood group carbohydrate and peptide antigens. They have been used to modify RBCs with a variety of ABH, Lewis and related antigens to study the specificity of monoclonal and polyclonal antibodies. Although kodecytes with carbohydrate epitopes have been extensively evaluated, those with peptide antigens also exist and some can be prepared with cross‐reactive bacterially related peptide antigens, to create IgG‐reactive kodecytes. These so‐called ASKGkodecytes can mimic all serological IgG reactions and are particularly suited for teaching and training. Recently kodecytes have also been used to quantify ABH antibody levels without the requirement for plasma dilution, and in this review the probable mechanism of action is explained. In general, kodecytes allow for the controlled antigen modification of RBCs and have the ability to make almost unlimited quantities of antigen rare or unusual RBCs from common resources. Not only are kodecytes useful research tools to study RBCs in vitro and in vivo but they also have the potential to create standardized diagnostics, quality control systems, advanced serological teaching panels and novel research tools.

Allergie à l’arachide en Afrique sub-saharienne

Peanut was imported from South America into West Africa first during the XVIth century, but its culture and its use as a source of oil and proteins expanded in Africa only in the mid-XIXth century. In rural areas of sub-Saharan Africa, the extent of peanut sensitisation and clinical allergy is very low compared to populations living in Europe and the U.S.A. This could be due to the protective effect of anti-CCD IgE and IgG, which can cross-react between peanut and parasites’ carbohydrate epitopes. These anti-CCD IgE are cause of false positive results for anti-peanut IgE tests, when whole peanut extracts are used in place of non-glycosylated molecular allergens. Several studies in southern Africa have used oral food challenges and show that the low incidence of peanut allergy is limited to rural areas and do not exist among African urban populations. No influence of ethnicity was found. This suggests that environmental factors related to urban living are crucial for the incidence of peanut allergy and probably also for food allergies in general.

Antigenic cross-reactivity between Schistosoma mansoni and allergenic invertebrates putatively due to shared glycanic epitopes

Previous studies have shown that rabbit IgG antibodies against Schistosoma mansoni egg antigens (SmSEA) cross-react with allergens in natural rubber latex, peanuts and grass and tree pollens. Here we describe antigenic molecules that cross-react with rabbit anti-S. mansoni IgG antibodies in extracts of the house dust mite (HDM) Dermatophagoides farinae, the Australian cockroach (ACR) Periplaneta australasiae and in the venom of the honey bee Apis mellifera (HBV). Tandem mass spectrometry identified the cross-reactive allergens as Der f 15 in HDM, two homologues of the Periplaneta americana cockroach allergen Cr-PI/Per a 3 in ACR and two isoforms of the allergen Api m 1 (phospholipase A2: PLA2) in HBV. Cross-reactive rabbit anti-SmSEA IgG antibodies eluted from the three invertebrate allergens reacted with S. mansoni egg antigens and variably with schistosome cercarial and worm antigens. Treatment of the electroblotted allergens with sodium metaperiodate abrogated most of the cross-reactivity of the rabbit anti-SmSEA antibodies, suggesting it was due to cross-reactive carbohydrate determinants (CCDs). Furthermore, analyses of the allergens’ amino acid sequences indicated that they had potential for both N- and O-linked glycosylation. A potential role for the CCDs shared by the schistosome and invertebrates in inducing an allergy-protective effect, as proposed by the hygiene hypothesis, is discussed.

Specificity of human natural antibodies referred to as anti-Tn

To understand the role of human natural IgM known as antibodies against the carbohydrate epitope Tn, the antibodies were isolated using GalNAcα−Sepharose affinity chromatography, and their specificity was profiled using microarrays (a glycan array printed with oligosaccharides and bacterial polysaccharides, as well as a glycopeptide array), flow cytometry, and inhibition ELISA. The antibodies bound a restricted number of GalNAcα-terminated oligosaccharides better than the parent monosaccharide, e.g., 6-O-Su-GalNAcα and GalNAcα1−3Galβ1−3(4)GlcNAcβ. The binding with several bacterial polysaccharides that have no structural resemblance to the affinity ligand GalNAcα was quite unexpected. Given that GalNAcα is considered the key fragment of the Tn antigen, it is surprising that these antibodies bind weakly GalNAcα−OSer and do not bind a wide variety of GalNAcα−OSer/Thr-containing mucin glycopeptides. At the same time, we have observed specific binding to cells having Tn-positive glycoproteins containing similar glycopeptide motifs in a conformationally rigid macromolecule. Thus, specific recognition of the Tn antigen apparently requires that the naturally occurring “anti-Tn” IgM recognize a complex epitope comprising the GalNAcα as an essential component and a fairly long amino acid sequence where the amino acids adjacent to GalNAcα do not contact the antibody paratope; i.e., the antibodies recognize a spatial epitope or a molecular pattern rather than a classical continuous sequence. In addition, we have not found any increase in the binding of natural antibodies when GalNAcα residues were clustered. These results may help in further development of anticancer vaccines based on synthetic Tn constructs.

On the cause and consequences of IgE to galactose-α-1,3-galactose: A report from the National Institute of Allergy and Infectious Diseases Workshop on Understanding IgE-Mediated Mammalian Meat Allergy

The mammalian meat allergy known as the "α-Gal syndrome" relates to IgE specific for galactose-α-1,3-galactose (α-Gal), an oligosaccharide that is present in cells and tissues of nonprimate mammals. The recognition of delayed reactions to food derived from mammals in patients with IgE to α-Gal and also the association with tick bites have been increasing worldwide. In 2018, the National Institute of Allergy and Infectious Diseases, Division of Allergy, Immunology and Transplantation, sponsored a workshop on this emerging tick-related disease. International experts from the fields of tick biology, allergy, immunology, infectious disease, and dermatology discussed the current state of our understanding of this emerging medical condition. The participants provided suggestions for specific research priorities and for the development of resources to advance our knowledge of the mechanisms, diagnosis, management, and prevention of this allergic disease. This publication is a summary of the workshop and the panel's recommendations are presented herein.

Alpha-Gal and Cross-Reactive Carbohydrate Determinants in the N-Glycans of Salivary Glands in the Lone Star Tick, Amblyomma americanum.

Ticks are important ectoparasites and vectors of numerous human and animal pathogens. Ticks secrete saliva that contains various bioactive materials to evade the host defense system, and often facilitates the pathogen transmission. In addition, the Lone star tick saliva is thought to be the sensitizer in red meat allergy that is characterized by an allergic reaction to glycan moieties carrying terminal galactose-alpha-1,3-galactose (aGal). To assess N-glycome of Amblyomma americanum, we examined the N-glycan structures in male and female salivary glands at three different feeding stages and in carcasses of partially fed lone star ticks. We also surveyed the genes involved in the N-glycosylation in the tick species. The aGal epitopes and cross-reactive carbohydrate determinants (CCD) increases over time after the onset of blood feeding in both male and female A. americanum. These CCDs include xylosylation of the core mannose, 1,3-mono and 1,3- and 1,6-difucosylations of the basal GlcNac and mono- or diantennary aGal. Combinations of both xylosylation and aGal and fucosylation and aGal were also found on the N-glycan structures. While the enzymes required for the early steps of the N-glycosylation pathway are quite conserved, the enzymes involved in the later stages of N-glycan maturation in the Golgi apparatus are highly diverged from those of insects. Most of all, we propose that the aGal serves as a molecular mimicry of bioactive proteins during tick feedings on mammalian hosts, while it contributes as a sensitizer of allergy in atypical host human.

N-Glycan Inhibitors in Immunoassay Methods for serum Allergen Specific IgE

Cross-reactive carbohydrate determinants (CCDs) represent fucosylated/xylosylated N-glycans present in pollen and food of plant origin, latex and hymenoptera venoms. IgE antibodies against CCD do not have significant clinical relevance, but represent an important cause of cross-reactivity in the case of in vitro immunoassay methods for determining serum specific IgE. CCD inhibitors play a key role in the laboratory for preventing misinterpretation of such in vitro positive assays and for increasing their specificity.

Evaluation of the anti-Thomsen-Friedenreich antibodies Nemod-TF1 and Nemod-TF2 as prognostic markers in breast cancer.

The TF (Thomsen-Friedenreich, CD176, Galβ1-3GalNAc) carbohydrate moiety is known as a specific oncofetal carbohydrate epitope present in fetal and neoplastic tissue as well as in stem cells. TF was demonstrated to mediate tumor-promoting features and to be highly immunogenic. The current study aimed to evaluate whether presence of the TF antigen is associated with clinico-pathological parameters and prognosis of early breast cancer (BC). Primary BC tissue (n = 226) was stained for TF using two monoclonal anti-TF antibodies (Nemod-TF1, Nemod-TF2). Staining results were correlated to clinical data including survival. Nemod-TF1 staining was positively correlated to lymph node metastasis (p = 0.03) and the presence of tumor-associated MUC1 (TA-MUC1; p = 0.003). Further, the presence of the Nemod-TF1 epitope predicted worse prognosis in TA-MUC1 positive (overall survival: p = 0.026) as well as in triple negative (overall survival: p = 0.002; distant metastasis-free survival: p = 0.012) BC. The data presented here further support a role of TF in BC tumor biology. Whether anti-TF directed treatment approaches may gain clinical relevance in those cases determined as triple negative or TA-MUC1 positive remains to be determined.

Comparative Characteristics of the Diagnostic Potential of Mycobacterial Synthetic Antigens for the Seroriagnosis of Lepra and Tuberculosis

Seven artificial mycobacterial antigens have been synthesized via the conjugation of synthetic carbohydrate epitopes of M. leprae phenolic glycolipid (PGL-1) and M. tuberculosis lipoarabinomannan (LAM) with bovine serum albumin (BSA). Each antigen has a different number of carbohydrate epitopes with a different structure and input sequence of the sugar residues. According to the serum reactivity, only three synthesized antigens can be useful for serodiagnosis of mycobacteriosis: (1) the antigen based on a disaccharide derivative of PGL-1: (DMG–Rha-AEP)-[BSA]; (2) the antigen based on a disaccharide fragment of PGL-1 and a hexasaccharide fragment of LAM: (DMG–Rha-AEP)-[BSA]–(Ara6-EG6); (3) the antigen based on the LAM diarabinofuranosyl fragment (Ara2-AEP)–[BSA].

Identification of carbohydrate peripheral epitopes important for recognition by positive-ion MALDI multistage mass spectrometry

Carbohydrate sequences are important for various biological processes. It has recently been estimated to have 100,000–500,000 carbohydrate structures in mammalian glycome. However, the peripheral carbohydrate determinants on N- and O-glycoproteins, glycolipids, polysaccharides and secreted free sugars are limited in numbers. Among these blood-group-related antigens the ABO(H)- and Lewis-types are particularly important. Negative-ion MS/MS has been successfully used in assignment of these epitopes on free reducing sugars but cannot be applied to reduced sugars, e.g. O-glycans typically released from mucins as alditols, or in positive-ion detection of either reducing or reduced oligosaccharides. In the present study, we investigate the fragmentation features of permethylated reducing and reduced sugars under positive-ion conditions of multi-stage MALDI-MS, and propose the concept of epitope ion and epitope spectrum for determination of peripheral blood-group related epitopes on secreted human milk oligosaccharides and N-glycans as reducing sugars and O-glycans as reduced alditols in conjunction with MALDI-MS glycan profiling.

Immunolocalization of β-(1–4)-D-galactan, xyloglucans and xylans in the reaction xylem fibres of Leucaena leucocephala (Lam.) de Wit

Cell wall architecture of tension wood fibres represents a suitable biological system to study the mechanism of growth and maintenance of posture of trees growing under various physical and physiological growth constraints. In the present study, we investigated the spatial distributions of β-(1–4)-D-galactan, xyloglucan and xylans (both less and highly substituted) in the opposite and tension wood fibres of bent Leucaena leucocephala by immunolabelling with monoclonal antibodies LM5, CCRCM1, LM10 and LM11 specific to these carbohydrate epitopes. The presence of non-lignified, tertiary wall layer is the typical tension wood characteristic associated with the reaction xylem fibres in Leucaena. LM5 labelling of opposite fibres showed weak labelling in the cell walls indicating less concentration of β-(1–4)-D-galactans while tension wood showed strong labelling in the tertiary wall layer suggesting the gelatinous layer (G-layer) has a strong cross linking with β-(1–4)-D-galactans. Xyloglucan distribution was more in the compound middle lamellae and the primary wall-S1 layer boundary of tension wood fibres as compared to that of opposite wood. A weak labelling was also evident near the boundary between the G-layer and the secondary wall of tension wood fibres. The secondary wall of opposite and tension wood fibres showed a strong distribution of both ls ACG Xs (LM10) and hs ACG Xs (LM11) while a weak labelling was noticed in the compound middle lamella. Tension wood fibres also showed strong xylan labelling mainly confined to the lignified secondary walls while the G-layer showed weak xylan labelling. In conclusion, our results suggest that β-(1–4)-D-galactans and xyloglucans could be implicated in the tensile stress generation within the G-layer of tension wood fibres of Leucaena leucocephala.

Characterization of MiFUT11 from Mangifera indica L.: A functional core α1,3-fucosyltransferase potentially involved in the biosynthesis of immunogenic carbohydrates in mango fruit

In higher plants, asparagine-linked oligosaccharides (N-glycans) in glycoproteins carry unique carbohydrate epitopes, namely, a core α1,3-fucose and/or a β1,2-xylose, which are common determinants responsible for the cross-reactivity of plant glycoproteins due to their strong immunogenicity. While these determinants and the relevant genes have been well characterized for herbaceous plants, information concerning whether many food plants cross-react with airborne pollens is not available. In this paper, we report on the characterization of a novel core α1,3-fucosyltransferase gene identified from Mangifera indica L., one of the major plants potentially related to food allergy. Based on sequence information of plant homologues, we amplified a candidate cDNA (MiFUT11) from pericarp tissue. An in vitro assay demonstrated that the recombinant MiFUT11 protein transfers a fucose unit onto both non-fucosylated and core α1,6-fucosylated oligosaccharides. A glycoform analysis using MALDI-TOF mass spectrometry showed that the introduction of the MiFUT11 cDNA increased the production of a core α1,3- and α1,6-fucosylated pauci-mannosidic oligosaccharide in Spodoptera Sf21 cells. Our findings suggest that MiFUT11 is a functional core α1,3-fucosyltransferase gene that is involved in the assembly of cross-reactive N-glycans in mango fruit.

False-Positive Results of Serological Tests for Allergy in Alcoholic Patients.

Alcohol consumption is associated with enhanced TH2 immune responses. Objective: To investigate the frequency of false-positive results in serological tests for allergy in alcoholic patients. A total of 138 alcoholic patients consecutively admitted to hospital underwent a panel of allergy tests that included serum total IgE, a multiallergen IgE test (UniCAP Phadiatop), and skin prick tests to relevant aeroallergens in the area, which were considered the standard reference for atopy. In selected cases with positive specific IgE (sIgE) to cross-reactive carbohydrate determinants (CCDs) on ImmunoCAP, we determined sIgE to hymenoptera venom components (ADVIA Centaur) and a microarray of 103 allergen components (ISAC). Increased serum total IgE (>170 IU/mL) was observed in 59/110 (54%) of nonatopic (skin prick test-negative) patients. The result of the multiallergen IgE test was positive in 46 nonatopic patients (42%). This finding was closely associated with high serum concentrations of total IgE and sIgE to CCDs. The vast majority of patients with positive CCD-sIgE showed positivity to glycosylated plant and hymenoptera allergen components on ISAC and ADVIA Centaur. Only 1 out of 26 patients with positive sIgE to CCD and hymenoptera venom developed honeybee venom allergy after a median follow-up of 166 months. Correlations between measurements of sIgE to CCD markers on ImmunoCAP, ADVIA Centaur, and ISAC were imperfect. Serological tests for allergy should be interpreted with caution in alcoholic patients, who frequently have increased levels of total IgE and CCD-sIgE and subsequent positivity of sIgE to glycosylated allergen components, irrespective of the method used.

Sialyl Glycan Expression on T Cell Subsets in Asthma: a correlation with disease severity and blood parameters

Memory T helper (Th) and regulatory T (Treg) cells play key roles in asthma. Certain sialyl carbohydrate determinants for selectins profoundly affect the migratory properties of memory Th cells, and the suppressive function of Treg cells. Previous studies have shown that the proportion of CCR4+ memory Th cells expressing sialyl 6-sulfo Lewis X (LeX) is elevated in asthma patients. We aim to investigate the roles of different sialyl glycans on T cell subsets in asthma. Using flow cytometry, we assessed the expression of three sialyl glycans, sialyl 6-sulfo LeX, cyclic sialyl 6-sulfo LeX, and sialyl LeX on memory Th and Treg cells, in the peripheral blood of asthmatic children. We also assessed the relationships between glycan-expressing cell percentages and asthma clinical parameters. Compared with controls, asthmatic children showed higher proportions of memory Th cells expressing sialyl LeX and sialyl 6-sulfo LeX. The proportions of memory Th cells with sialyl 6-sulfo LeX and cyclic sialyl 6-sulfo LeX expression in asthmatic children correlated with absolute eosinophil count and IgE level, respectively. Children with moderate-to-severe asthma had lower numbers of sialyl LeX positive Treg cells. Our study suggests that sialyl glycans on T cells may play important roles in the pathogenesis of asthma.

PEGylated immunoliposome-loaded endoglin single-chain antibody enhances anti-tumor capacity of porcine α1,3GT gene

Tumor could not be completely removed due to the absence of immune storm against tumor. The porcine α1,3 galactosyltransferase (α1,3 GT) induce the hyperacute rejection by synthesizing Galα1-3Galβ1-(3)4GlcNAc-R (αGal) on the surface of graft endothelial cells (ECs) during xeno-transplantation. This study aimed to develop anti-endoglin single-chain Fv fragments (ENG-scFv) conjugated PEGylated immunoliposomes (iLPs) to induce immune storm against tumor. Immune fluorescence was performed to detect the binding of ENG-scFv to human ENG, the endosomal/lysosomal escape of ENG-scFv-iLPs/α1,3 GT, and αGal expression in hENG-HEK293 cells. In vitro MTT assay was performed to measure ENG-scFv-iLPs/α1,3 GT cytotoxicity. NOD/SCID mouse born A549 tumor model was used to evaluate the therapeutic potency of ENG-scFv-iLPs/α1,3 GT. ENG-scFv-iLPs enabled efficient targeting delivery of α1,3 GT plasmid to ENG + tumors neovascular endothelial cells (TnECs), promoted endosomal/lysosomal escape due to the pH-sensitive ability, then synthesized carbohydrate epitope αGal on the surface of these cells to achieve the purpose of destroying the tumor. The mechanism of uptake for nanoparticles was energy driven, the clathrin-mediated endocytosis was the main endocytic pathway of the ENG-mAb-iLPs/α1,3 GT and lipid-raft-mediated of the ENG-scFv-iLPs/α1,3 GT, and macropinocytosis was also involved in intracellular entry. The inhibition of tumor angiogenesis and proliferation by ENG-scFv-iLPs/α1,3 GT was closely related to down-regulation of VEGF. Our findings establish an alternative therapeutic paradigm for scFv-conjugated nanoparticles to induce tumor cell apoptosis and inhibit tumor growth early. Such iLPs nanocarrier could efficiently release α1,3 GT to their distinct sites of action, where the endoglin + tumor neovascular endothelial cells (ENG + TnECs) exist, in a site-specific manner. Therefore, we believe that these scFv-targeted core-shell immunocomplexes are an important potential α1,3 GT delivery system for various solid tumor-targeted therapy.

Generation of cattle knockout for galactose-α1,3-galactose and N-glycolylneuraminic acid antigens.

Two well‐characterized carbohydrate epitopes are absent in humans but present in other mammals. These are galactose‐α1,3‐galactose (αGal) and N‐glycolylneuraminic acid (Neu5Gc) which are introduced by the activities of two enzymes including α(1,3) galactosyltransferase (encoded by the GGTA1 gene) and CMP‐Neu5Gc hydroxylase (encoded by the CMAH gene) that are inactive in humans but present in cattle. Hence, bovine‐derived products are antigenic in humans who receive bioprosthetic heart valves (BHVs) or those that suffer from red meat syndrome. Using programmable nucleases, we disrupted (knockout, KO) GGTA1 and CMAH genes encoding for the enzymes that catalyse the synthesis of αGal and Neu5Gc, respectively, in both male and female bovine fibroblasts. The KO in clonally selected fibroblasts was detected by polymerase chain reaction (PCR) and confirmed by Sanger sequencing. Selected fibroblasts colonies were used for somatic cell nuclear transfer (SCNT) to produce cloned embryos that were implanted in surrogate recipient heifers. Fifty‐three embryos were implanted in 33 recipients heifers; 3 pregnancies were carried to term and delivered 3 live calves. Primary cell cultures were established from the 3 calves and following molecular analyses confirmed the genetic deletions. FACS analysis showed the double‐KO phenotype for both antigens confirming the mutated genotypes. Availability of such cattle double‐KO model lacking both αGal and Neu5Gc offers a unique opportunity to study the functionality of BHV manufactured with tissues of potentially lower immunogenicity, as well as a possible new clinical approaches to help patients with red meat allergy syndrome due to the presence of these xenoantigens in the diet.

Fucosyltransferase 4 and 7 mediates adhesion of non-small cell lung cancer cells to brain-derived endothelial cells and results in modification of the blood\u2013brain-barrier: in vitro investigation of CD15 and CD15s in lung-to-brain metastasis

PurposeMetastatic non-small cell lung (NSCLC) cancer represents one of the most common types of brain metastasis. The mechanisms involved in how circulating cancer cells transmigrate into brain parenchyma are not fully understood. The aim of this work was to investigate the role of fucosylated carbohydrate epitopes CD15 and sialyated CD15s in cancer adhesion to brain-derived endothelial cells and determine their influence in blood–brain barrier (BBB) disruptionMethodsThree distinct, independent methods were used to measure brain endothelial integrity and include voltohmmeter (EVOM™), impedance spectroscopy (CellZscope®) and electric cell-substrate impedance sensing system (ECIS™). Two fucosyltransferases (FUT4 and 7) responsible for CD15 and CD15s synthesis were modulated in four human cancer cell lines (three lung cancer and one glioma).ResultsOverexpression of CD15 or CD15s epitopes led to increase in adhesion of cancer cells to cerebral endothelial cells compared with wild-type and cells with silenced CD15 or CD15s (p < 0.01). This overexpression led to the disruption of cerebral endothelial cell monolayers (p < 0.01). Knockdown of FUT4 and FUT7 in metastatic cancer cells prevented disruption of an in vitro BBB model. Surprisingly, although the cells characterised as ‘non-metastatic’, they became ‘metastatic’ -like when cells were forced to over-express either FUT4 or FUT7.ConclusionsResults from these studies suggest that overexpression of CD15 and CD15s could potentiate the transmigration of circulating NSCLC cells into the brain. The clinical significance of these studies includes the possible use of these epitopes as biomarkers for metastasis.