Ontogenic expression of histo-blood group antigens in the intestines of suckling pigs: lectin histochemical and immunohistochemical analysis

Summary The expression of intestinal histo-blood group AO and related antigens was investigated in piglets during an 8 week suckling period. Lectin histochemical and immunohistochemical analyses were undertaken on sections of resin-embedded intestinal tissue and semi-quantitative scoring systems were adopted for categories of lectins and monoclonal antibodies reactive with carbohydrate moieties present in core, backbone and terminal oligosaccharide sequences of histo-blood group antigens. Distinct age-related changes were observed in the terminal glycosylation of both secretory and membrane glycoconjugates. Histo-blood group A antigen was identified in intestinal mucin 5 weeks after birth and the precursor H antigen was found in goblet cells at week 1. H antigen was undetectable on intestinal membranes during the first 3 weeks of suckling but a conspicuous and sustained level of this form of fucosylation was apparent during the latter half of the suckling period. More complex membrane glycosylation involving further fucosylation and/or the expression of A antigen, was evident in the latter part of the suckling period. These temporal changes in membrane and secretory glycosylation may be physiologically important during intestinal adaptation and development in young pigs.Abbreviations GalNac N-acetylgalactosamine - Gal Galactose - GlcNac N-acetylglucosamine     

Stomach expression of histo-blood group antigens A and B in some vertebrates

Histo‐blood group antigens (HBGA) are genetically determined glycoproteins and glycolipids expressed not only on human erythrocytes but also in vertebrate tissues. Direct evidence for the immunobiological importance of their tissue localization in the evolutionary aspect is still lacking. The present study examines the expression of A and B HBGA in the stomach of free‐living vertebrates belonging to: Chondrichthyes, Actinopterygii, Amphibia, Reptilia, Aves, and Mammalia. HBGA were detected immunohistochemically on stomach paraffin sections from 11 species. In all classes from Actinopterygii to Mammalia HBGA expression was confined to stomach mucosa only. Antigenic heterogeneity in the pattern of expression and localization was observed. Smooth muscle tissue, endothelial and red blood cells were immunonegative, except for the reptile Emys orbicularis. Our results present the first comparative evidence for the expression of HBGA in the stomach of 11 free‐living vertebrate species from six classes, some of which have never been studied so far. It could be assumed that A and B antigens are constant and conservative structures with almost similar tissue localization. Their immunobiological role in the animal gastrointestinal tract might be possibly related to cell differentiation and homeostasis maintenance which would contribute to sustain the evolutionary stable ABH antigen cellular expression.     

Differential expression of ABH histo-blood group antigens and LAMPs in infantile hemangioma

Summary Although infantile hemangioma (IH) are the most common tumors of infancy, the mechanism of their proliferation and involution remains vague. Proliferation, differentiation and death of endothelial cells are the basic processes involved in their pathobiology. Here we hypothesize that the glycoconjugates ABH histo-blood group antigens (HBGA) and lysosome-associated membrane proteins (LAMPs) might be implied in both the differentiation and death of endothelial cells during vascular remodeling in IH. Proliferating and involuting IH were examined immunohistochemically for HGBA and LAMP expression together with vWF and CD31. Proliferative and apoptotic indices were determined. LAMPs were found in immature endothelium of proliferating IH. In involution an increased number of immunopositive cells stained with higher intensity was detected. The enhanced expression might be associated with augmented autophagy required for tissue remodeling during tumor involution. HBGA presented an opposite pattern of expression – they stained intensely the endothelium of mature capillaries, while the immature ones were positive for vWF. The presence of HBGA in endothelial cells of IH may be related to the differentiation process only, as well as to endothelial adhesion and angiogenesis. Novel evidence for differential expression of HBGA and LAMPs in proliferative and involutive phases of IH is presented.     

Aberrant expression of histo-blood group A type 3 antigens in vascular endothelial cells in inflammatory sites.

Histo-blood group ABH antigens are widely distributed in human tissues. The epitopes of ABH antigens are carried by at least four different peripheral core isotypes of internal carbohydrate backbones (type 1-4). Each type of ABH antigen is expressed tissue specifically, and aberrant expression of ABH antigens is often observed during oncogenesis. We immunohistochemically examined the expression of A type 3 antigens in wounded and diseased skin tissues (A and AB blood groups). In uninjured skin, the expression of A type 3 antigens was restricted to the eccrine sweat gland. In addition to the sweat glands, A type 3 antigens were found in vascular endothelial cells of the wound sites. The extent of A type 3 antigens expression related to postinfliction intervals. A significantly higher expression rate of A type 3 antigens in endothelial cells was also observed in diseased skin, suggesting that inflammation might induce A type 3 antigen expression in endothelial cells. Double-color immunofluorescence staining of the specimens showed that von Willebrand factor (vWF) was a core-protein of A type 3 determinants aberrantly expressed in endothelial cells in inflamed tissues, suggesting that aberrant expression of A type 3 antigens is involved in stabilization of vWF in inflammation.     

The distribution of type-2 chain histo-blood group antigens in normal cycling human endometrium

Summary The blood group ABO(H) determinants are major allogenic antigens in both erythrocytes and tissue of man. These antigens and related carbohydrates are markers of cellular maturation and differentiation in many epithelial tissues and have recently attracted great interest as tumor-associated antigens. Previous studies of endometrial tissues have indicated that glycosylation in this tissue may be related to hormonal stimulation. We have investigated the immunohistochemical distribution of type-2 chain histo-blood group-related carbohydrates in specimens of normal, cycling endometria obtained from hysterectomies on women with known ABO/Lewis erythrocyte type and saliva secretor status. N-acetyllactosamine and Le^x were demonstrated to be uninfluenced by the genetic background. A and Ale^y antigens were exclusively demonstrated in endometria from blood group A individuals, while Le^y was expressed in endometria from blood group 0 individuals mainly. The precursor N-acetyllactosamine as well as the terminal H, A, and ALe^y antigens were shown in only a few cells. In contrast, N-acetyllactosamine substituted by sialic acid and/or fucose residues (Le^x, sialosyl-Le^x, Le^y) were demonstrated in epithelial cells of normal, cycling endometrium, but with both quantitative and qualitative differences in staining relating to the menstrual cycle, indicating that type-2 chain antigens are expressed under both genetic and hormonal influence in human cycling endometrium.     

H blood group genotypes and expression of A and 0 antigens in pigs

Tests of over 2000 pigs, with the blood group reagents Ac, Ap and 0, and the reagents Ha, Hb, He, Hd, He and Hc/c, confirmed that the H locus affects the expression of A and 0 antigens. The 'exceptional' phenotypes Ap and — were found in Ha homozygotes of Miniature pigs and in Hcd homozygotes of a semi-inbred Landrace line. They were also found in unrelated pigs of other breeds, and moreover in some individuals with genotypes H ^a H ^cd, H ^ab H ^cd, H-H- and H ^a H ^ab or H ^ab H ^ab.
The mechanism of the effect of the H locus on the A phenotypes may consist in the interaction of certain, not yet completely defined, H genotypes, or in linkage of the H system with the hypothetical ' S ' locus.     

Synthesis and NMR studies on the ABO histo-blood group antigens: synthesis of type III and IV structures and NMR characterization of type I-VI antigens

The ABO histo-blood group antigens are best known for their important roles in solid organ and bone marrow transplantation as well as transfusion medicine. Here we report the synthesis of the ABO type III and IV antigens with a 7-octen-1-yl aglycone. Also described is an NMR study of the ABO type I to VI antigens, which were carried out to probe differences in overall conformation of the molecules. These NMR investigations showed very little difference in the ¹H chemical shifts, as well as ¹H–¹H coupling constants, across all compounds, suggesting that these ABO subtypes adopt nearly identical conformations in solution.     

The time of appearance of blood group antigens in miniature pigs

Erythrocytes of foetuses and piglets of miniature pigs of different age were tested with 50 to 55 blood group reagents of 15 genetic systems. Out of 49 blood factors found to be present in parent animals, 40 were present in 34–46-day-old foetuses. Factor Kb was detected in 66-day-old foetuses, and other factors of the K system (Ka, Kc, Kd and Ke) at 77 days of age. Factor A was demonstrated in one day old piglets, factor 0 not earlier than in piglets aged 13 to 24 days.     

The carbohydrate moiety and high molecular weight carrier of histo-blood group antigens are both required for norovirus-receptor recognition

Histo-blood group antigens (HBGAs) on human intestinal epithelium serve as receptors for noroviruses (NVs). These antigens also are expressed in milk and may act as decoy receptors to protect breast-fed infants and others against NV disease. In this study we demonstrated that human milk is highly variable in synthesis of HBGAs, which differs from that of saliva; a large quantity of small, soluble HBGAs are found in milk, but much less in saliva and are recognized by MAbs, but not by NVs. There is another group of HBGAs, of high MW, found in both milk and saliva, and recognized by both NVs and MAbs. These results suggest that the specificity of NVs and MAbs to HBGAs are different and the backbones in addition to the carbohydrate moiety are required for NV recognition. Further studies to define the structure and genetics of the high MW milk glycans are necessary.     

Comparative lectin histochemical analysis of the duodenal glands in various mammals

Composition and histotopography of lectin receptors have been studied in 12 species of mammals with various nutritional specialization: carnivorous, phytophagous and omnivorous. In cells of the duodenal glands of the carnivorous and omnivorous receptors to concanavalin A and lentil lectin (D-mannosoglycans ) are absent and they are present in the glands of the phytophagous animals. In cells of some parts of the glands presence of receptors to soya bean lectin (N-acetyl-D-galactosamine++) is the most characteristic sign of the duodenal glands in the carnivorous and phytophagous animals. Together with certain differences, depending on the nutritional way of the animals, specific peculiarities of lectins binding with glandulocytes of the duodenal glands are demonstrated. The data on rearrangement of the lectin receptors are obtained during the process of cellular differentiation. Presence of N-acetyl-D-galactosamine++ remnants-biding soya bean lectin in composition of oligosaccharide++ chains of glycoconjugates is a sign of low differential degree of the glandular cells. In more differentiated cells concealment in oligosaccharide chains of D-galactose remnants (peanut and castor-oil lectins receptors) by L-fucose, N-acetil-D-glucosamin remnants and sialic acid can have place; this is demonstrated as accumulation of receptors to wheat germ and Laburnum anagyroides lectins in the glandular cells.     

Cloning of a rat gene encoding the histo-blood group A enzyme. Tissue expression of the gene and of the A and B antigens.

The complete coding sequence of a BDIX rat gene homologous to the human ABO gene was determined. Identification of the exon-intron boundaries, obtained by comparison of the coding sequence with rat genomic sequences from data banks, revealed that the rat gene structure is identical to that of the human ABO gene. It localizes to rat chromosome 3 (q11-q12), a region homologous to human 9q34. Phylogenetic analysis of a set of sequences available for the various members of the same gene family confirmed that the rat sequence belongs to the ABO gene cluster. The cDNA was transfected in CHO cells already stably transfected with an alpha1,2fucosyltransferase in order to express H oligosaccharide acceptors. Analysis of the transfectants by flow cytometry indicated that A but not B epitopes were synthesized. Direct assay of the enzyme activity using 2' fucosyllactose as acceptor confirmed the strong UDP-GalNAc:Fucalpha1,2GalalphaGalNAc transferase (Atransferase) activity of the enzyme product and allowed detection of a small UDP-Gal:Fucalpha1,2GalalphaGal transferase (B transferase) activity. The presence of the mRNA and of the A and B antigens was searched in various BDIX rat tissues. There was a general good concordance between the presence of the mRNA and that of the A antigen. Tissue distributions of the A and B antigens in the homozygous BDIX rat strain were largely different, indicating that these antigens cannot be synthesized by alleles of the same gene in this rat inbred strain.     

Evolution of the interactions between GII.4 noroviruses and histo-blood group antigens: Insights from experimental and computational studies

Norovirus (NoV) is the major pathogen causing the outbreaks of the viral gastroenteritis across the world. Among the various genotypes of NoV, GII.4 is the most predominant over the past decades. GII.4 NoVs interact with the histo-blood group antigens (HBGAs) to invade the host cell, and it is believed that the receptor HBGAs may play important roles in selecting the predominate variants by the nature during the evolution of GII.4 NoVs. However, the evolution-induced changes in the HBGA-binding affinity for the GII.4 NoV variants and the mechanism behind the evolution of the NoV-HBGA interactions remain elusive. In the present work, the virus-like particles (VLPs) of the representative GII.4 NoV stains epidemic in the past decades were expressed by using the Hansenula polymorpha yeast expression platform constructed by our laboratory, and then the enzyme linked immunosorbent assay (ELISA)-based HBGA-binding assays as well as the molecular dynamics (MD) simulations combined with the molecular mechanics/generalized born surface area (MMGBSA) calculations were performed to investigate the interactions between various GII.4 strains and different types of HBGAs. The HBGA-binding assays show that for all the studied types of HBGAs, the evolution of GII.4 NoVs results in the increased NoV-HBGA binding affinities, where the early epidemic strains have the lower binding activity and the newly epidemic strains exhibit relative stronger binding intensity. Based on the MD simulation and MMGBSA calculation results, a physical mechanism that accounts for the increased HBGA-binding affinity was proposed. The evolution-involved residue mutations cause the conformational rearrangements of loop-2 (residues 390–396), which result in the narrowing of the receptor-binding pocket and thus tighten the binding of the receptor HBGAs. Our experimental and computational studies are helpful for better understanding the mechanism behind the evolution-induced increasing of HBGA-binding affinity, which may provide useful information for the drug and vaccine designs against GII.4 NoVs.     

Expression of histo-blood group antigens by lipopolysaccharides of Helicobacter pylori strains from asian hosts: the propensity to express type 1 blood-group antigens

Past studies have shown that the cell surface lipopolysaccharides (LPSs) of the ubiquitous human gastric pathogen Helicobacter pylori (a type 1 carcinogen) isolated from people residing in Europe and North America express predominantly type 2 Lewis x (Le(x)) and Le(y) epitopes and, infrequently, type 1 Le(a), Le(b), and Le(d) antigens. This production of Lewis blood-group structures by H. pylori LPSs, similar to those found in the surfaces of human gastric cells, allows the bacterium to mimic its human niche. In this study, LPSs of H.pylori strains extracted from patients living in China, Japan, and Singapore were chemically and serologically analyzed. When compared with Western H.pylori LPSs, these Asian strains showed a stronger tendency to produce type 1 blood groups. Of particular interest, and novel observations in H.pylori, the O-chain regions of strains F-58C and R-58A carried type 1 Le(a) without the presence of type 2 Le(x), strains R-7A and H607 were shown to have the capability of producing the type 1 blood group A antigen, and strains CA2, H507, and H428 expressed simultaneously the difucosyl isomeric antigens, type 1 Le(b) and type 2 Le(y). The apparent proclivity for the production of type 1 histo-blood group antigens in Asian H.pylori LPSs, as compared with Western strains, may be an adaptive evolutionary effect in that differences in the gastric cell surfaces of the respective hosts might be significantly dissimilar to select for the formation of different LPS structures on the resident H.pylori strain.     

Binding patterns of human norovirus-like particles to buccal and intestinal tissues of gnotobiotic pigs in relation to A/H histo-blood group antigen expression

Histo-blood group antigen (HBGA) phenotypes have been associated with susceptibility to human noroviruses (HuNoVs). Our aims were: (i) to determine the patterns of A/H HBGA expression in buccal and intestinal tissues of gnotobiotic (Gn) pigs; (ii) to determine if virus-like particles (VLPs) of HuNoV genogroup I (GI) and GII bind to A- or H-type tissues; (iii) to compare A/H expression and VLP binding patterns and confirm their binding specificities by blocking assays; (iv) to develop a hemagglutination inhibition test using buccal cells from live pigs to determine the Gn pig's A/H phenotype and to match viral strains with previously determined HuNoV VLP binding specificities; and (v) to determine the A/H phenotypes and compare these data to the infection outcomes of a previous study of 65 Gn pigs inoculated with HuNoV GII/4 strain HS66 and expressing A and/or H or neither antigen on their buccal and intestinal tissues (S. Cheetham, M. Souza, T. Meulia, S. Grimes, M. G. Han, and L. J. Saif, J. Virol. 80:10372-10381, 2006). We found that the HuNoV GI/GII VLPs of different clusters bound to tissues from four pigs tested (two A+ and two H+). The GI/1 and GII/4 VLPs bound extensively to duodenal and buccal tissues from either A+ or H+ pigs, but surprisingly, GII/1 and GII/3 VLPs bound minimally to the duodenum of an A+ pig. The VLP binding was partially inhibited by A-, H1-, or H2-specific monoclonal antibodies, but was completely blocked by porcine mucin. Comparing the A/H phenotypes of 65 HS66-inoculated Gn pigs from our previous study, we found that significantly more A+ and H(+) pigs (51%) than non-A+ and non-H+ pigs (12.5%) shed virus. From the 22 convalescent pigs, significantly more A+ or H+ pigs (66%) than non-A+ or H+ pigs (25%) seroconverted.     

Comparison of human saliva and synthetic histo-blood group antigens usage as ligands in norovirus-like particle binding and blocking assays

Blocking of norovirus-like particle binding to their cellular ligands, histo-blood group antigens with immune sera, is considered a surrogate norovirus neutralization assay. We compared human secretor positive saliva and synthetic biotinylated carbohydrates as a source of histo-blood group antigens in binding and blocking assays. Six norovirus capsid-derived virus-like particles belonging to genogroup I (GI-1-2001 and GI-3-2002) and genogroup II (GII-4-1999, GII-4-2010 New Orleans, GII-4-2012 Sydney and GII-12-1998) noroviruses were produced by a recombinant baculovirus expression system and binding profile to saliva type A, B and O and to synthetic antigens (A trimer, B trimer, H type 1, H type 3, Lewis^a and Lewis^b) was identified. Good correlation between virus-like particle binding to saliva type A and synthetic A trimer (r = 0.66, p < 0.05) and saliva type B and synthetic B trimer (r = 0.75, p < 0.05) was observed. Binding of each norovirus virus-like particle to the selected histo-blood group antigens was blocked by convalescent sera from NoV-infected subjects or type-specific mouse antisera. Our results support the use of either saliva or synthetic antigens in blocking assay to measure the ability of norovirus antisera to block virus-like particle binding to the carbohydrate ligands.     

Intermittent suckling enables estrus and pregnancy during lactation in sows: effects of stage of lactation and lactation during early pregnancy

Previously we demonstrated that pre-ovulatory LH and post-ovulatory progesterone (P4) concentrations in plasma were low and embryo development was retarded when sows were induced to ovulate during lactation by submitting them to intermittent suckling (IS). The present study investigated whether this was due to: (1) stage of lactation when IS was initiated, and (2) continuation of IS post-ovulation. Multiparous Topigs40 sows were studied under three conditions: conventional weaning at Day 21 of lactation (C21; n = 30), intermittent suckling from Day 14 of lactation (IS14; n = 32), and intermittent suckling from Day 21 of lactation (IS21; n = 33). Sows were separated from piglets for 12 h daily during IS. IS sows were either weaned at ovulation or 20 d following ovulation. One-third (21/63) of the IS21 and C21 sows had already ovulated or had large pre-ovulatory follicles at Day 21 and were excluded from further study. Initiation of IS at Day 14 instead of Day 21 of lactation tended to reduce P4 at 7 d post-ovulation (P = 0.07), did not affect pregnancy rate, and tended to reduce embryo survival (P = 0.06). Continuation of IS during pregnancy resulted in lower P4 at 7 and 12 d post-ovulation, tended to reduce embryo weight and pregnancy rate (P < 0.10), whereas embryo survival was not affected. This study presents data for a population of sows in which follicle growth and ovulation are easily triggered under suckling conditions. Further, when these sows are bred during lactation, initiation of IS at 21 rather than 14 d of lactation with weaning at ovulation yields the most desirable reproductive performance.