[Genetic susceptibility to norovirus infection]. / Sensibilité génétique aux infections à norovirus.

Norovirus (NoV) are the most common cause of acute gastroenteritidis in humans worldwide. They are transmitted through consumption of contaminated food, or mostly by direct person-to-person contact. However, susceptibility to NoV infection is variable. NoVs recognize carbohydrate ligand, including A, B, H and Lewis histoblood group antigen (HBGAs) for attachment to human epithelial cells. Synthesis of these HBGAs requires various glycosyltransferase encoded by the ABO, FUT2, FUT3 genes. The presence of distinct carbohydrates structures dependent upon the combined polymorphism at the FUT2, FUT3 and ABO loci influences susceptibility to NoV infection. NoV-glycan interactions studies show that different strains recognize specific HBGAs. Together with herd immunity, HBGAs play a major role in the epidemiology and evolution of NoVs.

Qualitative and quantitative analysis of the binding of GII.4 norovirus variants onto human blood group antigens.

Noroviruses (NoVs) are one of the leading causes of gastroenteritis in children and adults. For the last 2 decades, genogroup II genotype 4 (GII.4) NoVs have been circulating worldwide. GII.4 NoVs can be divided into variants, and since 2002 they have circulated in the population before being replaced every 2 or 3 years, which raises questions about the role of their histo-blood group antigen (HBGA) ligands in their evolution. To shed light on these questions, we performed an analysis of the interaction between representative GII.4 variants and HBGAs, and we determined the role of selected amino acids in the binding profiles. By mutagenesis, we showed that there was a strict structural requirement for the amino acids, directly implicated in interactions with HBGAs. However, the ablation of the threonine residue at position 395 (ΔT395), an epidemiological feature of the post-2002 variants, was not deleterious to the binding of the virus-like particle (VLP) to the H antigen, while binding to A and B antigens was severely hampered. Nevertheless, the ΔT395 VLPs gained the capacity to bind to the Lewis x and sialyl-Lewis x antigens in comparison with the wild-type VLP, demonstrating that amino acid residues outside the HBGA binding site can modify the binding properties of NoVs. We also analyzed the attachment of baculovirus-expressed VLPs from six variants (Bristol, US95/96, Hunter, Yerseke, Den Haag, and Osaka) that were isolated from 1987 to 2007 to phenotyped saliva samples and synthetic HBGAs. We showed that the six variants could all attach to saliva of secretors irrespective of the ABO phenotype and to oligosaccharides characteristic of the secretor phenotype. Interestingly, Den Haag and Osaka variants additionally bound to carbohydrates present in the saliva of Lewis-positive nonsecretors. The carbohydrate binding profile and the genetic and mutagenesis analysis suggested that GII.4 binding to Lewis x and sialyl-Lewis x antigens might be a by-product of the genetic variation of the amino acids located in the vicinity of the binding site. Analysis of the binding properties for the six variants by surface plasmon resonance showed that only post-2002 variants (i.e., Hunter, Yerseke, Den Haag, and Osaka) presented strong binding to A and B antigens, suggesting that the GII.4 evolution could be related to an increased affinity for HBGAs for the post-2002 variants. The combination of increased affinity for ABH antigens and of a newly acquired ability to recognize glycans from Lewis-positive nonsecretors could have contributed to the epidemiological importance of strains such as the Den Haag GII.4 subtype.

A new mucin-associated oncofetal antigen, a marker of early carcinogenesis in rat colon.

We report the characterization of an IgG2a monoclonal antibody, (MAb) 660, prepared against rat gastric high molecular weight glycoproteins. By immunoperoxidase staining, MAb 660 reacted only with the mucous cells of surface gastric epithelium and with a few duodenal goblet cells close to the pylorus in normal adult rats. In fetuses, it reacted with intestinal and colonic goblet cells. The adult colon was always negative. The MAb 660 stained 100% (30 of 30) of chemically induced colonic carcinomas and 100% (7 of 7) of duodenal carcinomas. Several weeks before the appearance of tumors, histologically normal glands, then hyperplasia and dysplasia were precociously stained with MAb 660. The tissue distribution was different from that of blood group related antigens and M1 fucomucins. The recognized antigen was not sensitive to neuraminidase treatment. After electrophoresis in polyacrylamide gel, staining with periodic acid-Schiff reagent and Western blotting showed that the MAb 660 recognized an epitope associated with high molecular weight glycoproteins. This epitope was unaffected by beta-mercaptoethanol reduction-periodate treatment and neuraminidase and trypsin digestion. However, trypsin digestion performed after beta-mercaptoethanol reduction destroyed the 660 epitope. These data suggest that the antibody could recognize the peptide moiety of the mucin rather than its carbohydrate moiety. Thus, the new antigen identified by MAb 660 is a mucin-type glycoprotein with an oncofetal behavior in the rat colon and is precociously expressed by precancerous colonic mucosa.

H blood group antigen carried by CD44V modulates tumorigenicity of rat colon carcinoma cells.

Expression of carbohydrate ABH blood group antigens is oncodevelopmentally regulated and their presence on tumor cells constitutes a prognostic factor. However, it is not clear whether they directly affect tumor behavior. Using a rat model of colon carcinoma, we previously observed an association between the presence of H blood group antigens and tumorigenicity in syngeneic animals. In the present study, we show by immunoprecipitation experiments that cell surface H blood group antigens of a highly tumorigenic clone (PROb) are essentially carried by splice variants of the CD44 molecule containing exon V6. PROb cells were then transfected with an antisense fragment of the gene coding for a rat alpha (1-2)fucosyltransferase. This enzyme allows synthesis of H antigens from various beta-galactoside precursors. Transfected subclones of PROb cells were obtained which had significantly decreased enzymatic activity and H antigenic cell surface levels. In contrast, no such changes were observed in control cells transfected with either the empty vector or with a sense fragment of the gene. Compared to controls, the antisense-transfected cells were far less tumorigenic in syngeneic animals. These results show that H blood group antigens at the surface of PROb colon carcinoma cells contribute to tumor progression. The presence of the fucosylated structures on CD44 could modulate the functions of this adhesion molecule.

Tk, a new colon tumor-associated antigen resulting from altered O-glycosylation.

Erythrocyte polyagglutination antigens T and Tn are truncated O-glycan chains that are also carcinoma-associated antigens. We investigated whether Tk polyagglutination antigen could similarly be a carcinoma-associated marker and a target of immunotherapy. Monoclonal antibody LM389 was raised against Tk erythrocytes and tested by immunohistochemistry. LM389 strongly reacted with 48% human colorectal carcinomas. Labeling of normal tissues was visible on epithelial cells, mainly digestive, but was confined at a supranuclear level. Expression of the antigen on cloned human carcinoma cells correlated with sialosyl-Tn expression. O-Sialoglycoprotein endopeptidase treatment revealed that on carcinomas and cell lines, the epitope was present on O-glycans. Antibody specificity was determined using synthetic carbohydrates. Direct binding and inhibition studies indicated that LM389 best ligands were terminated by two branched N-acetylglucosamine units. Screening of murine cellular cell lines with LM389 allowed development of an experimental model with Tk-positive and -negative cells in syngeneic BDIX rats. Vaccination of rats with Tk erythrocytes provided a protection against growth of rat Tk-positive, but not of Tk-negative, tumor cells in association with the development of antibodies. Taken together, the results indicate that Tk polyagglutination antigen is a new colorectal carcinoma-associated antigen, absent from the normal cell surface, resulting from alteration of O-glycans biosynthesis and with potential as a target of immunotherapy.

Cellular expression of H and B antigens in the rat olfactory system during development.

Developmental expression of H and B antigens in the rat olfactory system was studied from the embryonic day 14 up to the postnatal day 30. The H antigen was detected in the olfactory and vomeronasal epithelia as early as fetal day 14, whereas the B antigen first appeared 2 days later. The anti-H reagent reacted strongly with sensory receptors and weakly with supporting cells in both epithelia, whereas the anti-B reagent was specific for olfactory receptors. In the main olfactory epithelium, the H antigen was expressed from fetal day 19 by most of the receptor cells, whereas the B determinant was expressed from fetal day 16 to postnatal day 3 by only a few neuroreceptors mostly located near the epithelial surface. After the postnatal day 3, B positive neurons increased in number from the periphery toward the deeper mucosal layers and they were distributed over 3/4 of the epithelial thickness in 15- and 30-day-old rats. In the main olfactory bulb, a widespread glomerular B staining with variable binding intensity between adjacent glomeruli was already observed at birth. The vomeronasal receptor cells and their axon terminals in the accessory olfactory bulb exhibited a comparable developmental expression of the B antigen. Results suggest that the B antigen could be regarded as a marker of neuronal maturation of both the olfactory and vomeronasal receptor cells; moreover, its first appearance in the receptor cells might be temporally related to the formation of synapses between receptor axons and deutoneurons in the bulb.

Expression of ABH and X (Lex) antigens in various cells.

Using a panel of reagents specific to the various subtypes of ABH antigens, it could be demonstrated that platelets carry ABH type 2 monofucosylated determinants on intrinsic glycoproteins. The presence of these antigens is controlled by the H gene and correlates with the presence of alpha-2-L-fucosyltransferase and the absence of alpha-3-L-fucosyltransferase. In contrast, intrinsic ABH antigens were not found on mononuclear cells, correlating with the absence of alpha-2-L-fucosyltransferase on these cells. However, after transformation with the Epstein-Barr virus and stimulation with 12-O-tetradecanoylphorbol-13-O-acetate (TPA), B lymphocytes were found to express the H antigen under control of the H gene and not the Se gene. The lymphoblastoid cell lines also expressed the X and sialylated X antigens which are normally markers of the myeloid lineage. These antigens are also normally found in epithelial cells of the digestive tract, kidney proximal convoluted tubules and hepatocytes. The alpha-3-L-fucosyltransferase responsible for the synthesis of this antigen is present in the serum but we report the existence of two individuals, a mother and her daughter, who lack more than 90% of this serum enzyme. The young girl suffers from a congenital kidney anomaly: oligomeganephronic hypoplasia. Her kidney tubules are devoid of X antigen. However, she and her mother have the X antigen on their granulocytes and its sialylated form on their monocytes. It therefore appears that there are distinct genetic controls for the expression of antigen X in different body compartments. This would be quite similar to the H and Se gene controls in tissues of distinct embryological origins.

ABH and Lewis histo-blood group antigens, a model for the meaning of oligosaccharide diversity in the face of a changing world.

Antigens of the ABH and Lewis histo-blood group family have been known for a long time. Yet their biological meaning is still largely obscure. Based on the available knowledge about the genes involved in their biosynthesis and about their tissue distribution in humans and other mammals, we discuss here the selective forces that may maintain or propagate these oligosaccharide antigens. The ABO, alpha 1,2fucosyltransferase and alpha 1,3fucosyltransferase enzyme families have been generated by gene duplications. Members of these families contribute to biosynthesis of the antigens through epistatic interactions. We suggest that the highly polymorphic genes of each family provide intraspecies diversity that allows coping with diverse and rapidly evolving pathogens. In contrast, the genes of low frequency polymorphism are expected to play roles at the cellular level, although they may be dispensable at the individual level. In addition, some members of these three gene families are expected to be functionally redundant and may either provide a reservoir for additional diversity in the future or become inactivated. We also discuss the role of the ABH and Lewis histo-blood group antigens in pathologies such as cancer and cardiovascular diseases, but argue that it is merely incidental and devoid of evolutionary impact.

Influence of the original disease, race, and center on the outcome of kidney transplantation.

The low graft survival rate in black recipients (36 +/- 2% at 1 year) as compared with the graft survival rate in white recipients (48 +/- 1%) might be secondary to a higher incidence of vascular lesions, inducing hypertensive disease, in blacks than in whites. The relative frequency of malignant hypertension in black recipients was six times that of white recipients, and recipients with malignant hypertension had a significant lower graft survival rate (43 +/- 2%) than recipients with glomerulonephritis (54 +/- 1%). In addition, patients with vascular lesions (diabetes, malignant hypertension, and glomerulonephritis) showed significantly lower graft survival rates in black than in white recipients, in contrast to patients with primary tubular or interstitial lesions (polycystic kidneys and pyelonephritis), who showed similar graft survival rates in blacks and whites. Only a small fraction of this racial effect could be traced back to the higher incidence of Lewis-negative phenotypes in black recipients and a similar beneficial effect of transfusions, on graft survival, was observed in both black and white recipients. The effects of graft survival of age (6%), race (9%), and transfusions (18%) were significant in good (A) and poor (B) centers. No overlap between A and B centers was observed for any of these three parameters when analyzed separately. However, when the cumulative effects of these three risk parameters were analyzed together a partial overlap appeared, i.e., higher graft survival rates were observed in low-risk recipients that received transplants in B centers than in high-risk recipients that received transplants in A centers. Consequently, the selection of the recipient may play a role in the overall results of different transplantation units, leading to their classification into A or B centers, but cannot explain all of the differences between A and B centers.

Demonstration of human kidney differentiation antigens with monoclonal antibodies.

Six human differentiation antigens (EE24.6, EG9.11, EG14.1, EI16.1, EK8.1, EK17.1) have been defined using monoclonal antibodies obtained from mice immunized with embryonic kidney cells. Their histologic distribution was determined on frozen sections of embryonic, fetal, and adult human kidneys by immunofluorescence assay. EE24.6, an ureteral bud marker, was detected only on the germ layer of mature kidney urothelium. EG9.11 and EG14.1 were detected on the S-shaped bodies and also on the adult proximal convoluted tubule for the former and the glomerular basement membrane for the latter. EI16.1, a marker of condensed mesenchyme, was detected only on epithelial cells of adult proximal convoluted tubule. EK8.1 was found in the mesangium, connective tissue, and with particularly dense labeling in the basement membranes. This labeling pattern was present throughout renal organogenesis. EK17.1 recognized both cell and plasma human fibronectins. Staining for all antibodies was nearly identical in mesonephros and metanephros. These results demonstate that some antigens follow their embryonic destiny. They indicate an antigenic similarity between the mesonephros and the metanephros and, therefore, a very early appearance of these antigens. During differentiation, these antigens concentrate on more defined structures, and staining became increased with an increased degree of differentiation.

ABH and Lewis histo-blood group antigens in cancer.

Antigens of the ABH and Lewis histo-blood group family can be found on many normal cells, mainly of epithelial type. In carcinomas, altered expression of the various carbohydrate epitopes of this family occur, and are often strongly associated with either a good or bad prognosis. A review of the available data on these tumor-associated markers, their biosynthesis and their prognostic value is proposed here. For a long time it has been unclear whether their presence could affect the behavior of carcinoma cells. Recent data, however, indicate that they play biological roles in the course of tumor progression. The presence of sialyl-Le(a) or sialyl-Le(x), which are ligands for selectins, promotes the metastatic process by facilitating interaction with the endothelium of distant organs. The loss of A and B antigens increases cellular motility, while the presence of H epitopes increases resistance to apoptosis by mechanisms that remain to be defined. The Le(y) antigen has procoagulant and angiogenic activities. All these observations are used to present a model that may account for the described associations between the presence or loss of these markers and the outcome of disease. Finally, their potential clinical applications as tumor-associated markers or as targets of immunotherapy are reviewed.

Synthesis of type 2 human blood-group antigenic determinants. The H, X, and Y haptens and variations of the H type 2 determinant as probes for the combining site of the lectin I of Ulex europaeus.

Chemical syntheses of the human blood-group antigenic determinants derived from N-acetyllactosamine are described; namely, the 6-deoxy derivative, the 4'-epimer, and the 5 H type 2 [alpha LFuc(1 to 2)beta DGal-(1 to 4)beta DGlcNAc], X [beta DGal(1 to 4)[alpha LFuc(1 to 3)[beta DGlcNAc], and Y [alpha LFuc-(1 to 2)beta DGal(1 to 4)[alpha LFuc(1 to 3)]beta DGlcNAc] determinants as glycosides of 8-carboxymethyloctanol. In order to study the binding of the H type 2 determinant with the lectin I of Ulex europaeus, structures designed to specifically alter the hydrophilic and hydrophobic portions of the H type 2 determinant were also prepared; namely, the 6-deoxy derivative, the 4'-epimer, and the 5"-nor-homolog. The use of these structures, together with the H type 1 hapten and the N-deacetylated forms of both the H type 1 and H type 2 determinants, as inhibitors of the agglutination of O red cells by the lectin allowed the conclusion that the binding of the H type 2 determinant is hydrophobic; the binding involves a wedge-like portion of the determinant that is basically hydrophobic, except for the 5-hydroxymethyl group, which is at the tip of the wedge and forms an intramolecular hydrogen-bond with O-5 for acceptance by a hydrophobic cleft at the surface of the lectin. Blocking procedures involving alkoxymethyl groups and new experiences involving glycosylation reactions are described.

[Vaccination with genetically modified IL-2 secreting cells in a rat model of colonic carcinoma]. / Vaccination par des cellules génétiquement modifiées sécrétant de l'IL-2 dans un modèle de carcinome colique de rat.

Genetically engineered tumor cells secreting immunostimulatory molecules could facilitate the obtention of a vaccination against tumor antigens. To test this approach, we transfected genes encoding for rat and mouse IL-2 into PROb cells. These cells originate from a dimethylhydrazine induced colon carcinoma of BD IX rats. We observed an inhibition of the in vivo tumor growth directly proportional to the IL-2 secretion. An immunohistochemical analysis revealed that the tumors were infiltrated by leucocytes expressing the IL-2 receptor, suggesting their activation within the tumor. A strong delay of tumor growth was observed in rats challenged with PROb cells after a previous rejection of IL-2 secreting cells. Yet two rats out of six were completely protected. This protection is specific since rejection of PROb-IL-2 does not confer protection towards the syngeneic glioma A15A5. In addition, we could show by depletion experiments that NK/LAK, CD8, and CD4 lymphocytes were involved in the rejection of cells secreting large amounts of IL-2. Macrophages appear to be involved in the rejection process too, but also in the induction of an immune memory. Vaccination experiments using irradiated PROb IL-2 cells were performed. Only a partial protection towards a challenge with parental PROb cells could be obtained, also depending on the amount of secreted IL-2: the best protection being obtained after vaccination with cells synthesizing a small amount of IL-2. However, this protection was not superior to that obtained by coinjection of irradiated PROb cells and BCG.

Failure of monoclonal antibodies against tumor associated antigens to improve tumor targeting of LAK cells in a model of rat colon carcinoma.

The possibility of targeting LAK cells to the tumor by arming them with monoclonal antibodies directed against tumor associated antigens was tested in a rat model of colon carcinoma. Peritoneal carcinomatosis was generated by injection of cloned tumor cells and 111In-labeled LAK cells were injected in the tail vein after preincubation with the monoclonal antibodies themselves. It appeared that the antibodies did not significantly improve tumor targeting of LAK cells, most of the radioactivity being recovered in the spleen, the liver, the kidney or the lung, and only a small fraction in the tumor.

[Characterization of autoantigen (p105) in a model of colonic adenocarcinoma in the rat]. / Caractérisation d'un autoantigène (p105) dans un modèle d'adénocarcinome colique chez le rat.

Sera from BDIX rats bearing the syngeneic colon tumors PROb or REGb were analysed by Western blotting in order to detect a possible humoral response against the grafted tumor. The PROb clone grows progressively in syngeneic hosts and metastasizes, whereas the REGb clone grows slowly and then is rejected. This model was developed by F Martin and his group in Dijon, France. We observed that rats bearing PROb tumors only develop an antibody response against a water-soluble protein of 105 kDa (p105) which is expressed by both tumor clones. This antibody response has never been detected in rats bearing REGb tumors. The antigen p105 was also expressed by normal adult colon as well as some other foetal or adult tissues. It is also present in extracts from several tumor cell lines including human colorectal carcinoma cell lines. Moreover, the titer of detected antibody at day 30 was inversely correlated with the survival of rats after tumor inoculation, suggesting a possible facilitating role of this antibody response.

Immunohistological analysis of antibodies against ABH and other glycoconjugates in normal human pyloric and duodenal mucosae.

Eighty-eight monoclonal antibodies were tested by immunohistochemistry on human gastro-duodenal mucosae of known ABO, Lewis and Secretor phenotypes. Antibodies were classified among anti-A, anti-B, anti-AB, anti-H and other anti-glycoconjugates (I, i, T, Tn, Lewis, P1, Tk). Anti-A, B and AB antibodies were subdivided into subgroups with "broad" or "restricted" reactivity according to the extent of epithelial cell labeling. Anti-H antibodies were classified in accordance to their degree of sensitivity to the secretor phenotype. Among anti-T and anti-I antibodies, only one of each showed positive staining of epithelial cells. All anti-Lewis antibodies had distinct reactivities, although, they were clearly anti-Lewis reagents. Some anti-P1 antibodies labeled epithelial cells, irrespective of the ABO, Lewis and secretor phenotypes. One anti-Tk stained the Golgi apparatus of most epithelial cells, irrespective of the individual's phenotype. In conclusion, some of the antibodies tested were defined as very useful reagents for immunohistochemistry showing both specificity and sensitivity.

Fluorescent carbohydrate probes for cell lectins.

Fluorescein labeled carbohydrate (Glyc) probes were synthesized as analytical tools for the study of cellular lectins, i.e. SiaLe(x)-PAA-flu, Sia2-PAA-flu, GlcNAc2-PAA-flu, LacNAc-PAA-flu and a number of similar ones, with PAA a soluble polyacrylamide carrier. The binding of SiaLe(x)-PAA-flu was assessed using CHO cells transfected with E-selectin, and the binding of Sia2-PAA-flu was assessed by COS cells transfected with siglec-9. In flow cytometry assays, the fluorescein probes demonstrated a specific binding to the lectin-transfected cells that was inhibited by unlabeled carbohydrate ligands. The intense binding of SiaLe(x)-PAA-3H to the E-selectin transfected cells and the lack of binding to both native and permeabilized control cells lead to the conclusion that the polyacrylamide carrier itself and the spacer arm connecting the carbohydrate moiety with PAA did not contribute anymore to the binding. Tumors were obtained from nude mice by injection of CHO E-selectin or mock transfected cells. The fluorescent SiaLe(x)-PAA-flu probe could bind to the tumor sections from E-selectin positive CHO cells, but not from the control ones. Thus, these probes can be used to reveal specifically the carbohydrate binding sites on cells in culture as well as cells in tissue sections. The use of the confocal spectral imaging technique with Glyc-PAA-flu probes offered the unique possibility to detect lectins in different cells, even when the level of lectin expression was rather low. The confocal mode of spectrum recording provided an analysis of the probe localization with 3D submicron resolution. The spectral analysis (as a constituent part of the confocal spectral imaging technique) enabled interfering signals of the probe and intrinsic cellular fluorescence to be accurately separated, the distribution of the probe to be revealed and its local concentration to be measured.