Immunoelectron microscopic studies reveal differences in distribution of sialo-oligosaccharide receptors for Mycoplasma pneumoniae on the epithelium of human and hamster bronchi.

Long-chain sialo-oligosaccharides with poly-N-acetyllactosamine backbones (Ii antigen type) are major host cell receptors for the human pathogen Mycoplasma pneumoniae. Previous immunofluorescence studies of the human bronchial epithelium, using sequence-specific monoclonal antibodies to the branched I-type and linear i-type backbones, have indicated that sialylated and nonsialylated long-chain sequences of both types are richly expressed on the ciliated cells, where they are polarized at the apical aspects. These sequences are lacking in the goblet cells. In the present study, the display of these oligosaccharides has been investigated by electron microscopy (immunogold labelling) in the human bronchial epithelium and in that of the hamster, an animal model commonly used for M. pneumoniae infection. In the human bronchial epithelium, the long-chain branched sequences have been detected along the entire length of the cilia and on microvilli, whereas the linear sequences are confined to the microvilli and the basal aspects of the cilia. On the ciliated epithelial cells of the hamster, by contrast, the branched and linear sequences (sialo- and asialo-) have been detected exclusively on microvilli. A further striking difference is that in the hamster these structures are expressed in abundance on the goblet cells and in the intracellular globules. We suggest that the latter finding may partly explain the relatively large doses of M. pneumoniae required to establish experimental infection in the hamster, as the receptor-bearing secreted mucus may have a protective role in binding to the microorganisms, leading to their clearance by bronchociliary action.     

Expression of blood group antigens in urinary tract tumours: prospective fluorescence study using cryostat sections of fresh frozen tissues.

Cryostat sections of fresh frozen tissues were used in a prospective study of blood group H and A antigen fluorescence in 73 transitional cell carcinomas of the bladder. The aim was to evaluate antigen expression without subjecting the tumour tissues to organic solvents that extract blood group active glycolipids. Deletion of the genetically predicted antigen was twice as common in tumours of pT1 or greater stage than those of pTa stage and also twice as common in poorly differentiated than in moderately well differentiated tumours. The considerable heterogeneity and overlap, however, in patterns of reactivity in tumours of various histopathological stages and grades and the effect of secretor status on antigenicity meant that there was no obvious antigenic feature that correlated precisely with invasive stage or differentiation grade. It remains to be determined whether the antigen positive and antigen negative tumours represent different disease entities with differing clinical courses. Our results indicate, however, that studies of the blood group antigens in urinary tract tumours are more likely to be of value in research into biochemical disorders in the neoplastic process than in routine clinical assessment as a guide to treatment.     

Studies on the specificities of two IgM lambda cold agglutinins

The reactions of two monoclonal IgM lambda cold agglutinins, Sch and Sher, have been studied in detail with human and animal erythrocyte antigens. Although they were unusual in having lambda and not kappa polypeptide chains, they could be assigned to the anti I and anti-Pr₁ groups of cold agglutinins.

The findings with serum Sher indicated that the Pr₁ antigen may be more complex than previously thought. The occurrence of unexpectedly large numbers of specificities among monoclonal anti-I, anti-i and anti-Pr antibodies is discussed and it is suggested that each monoclonal antibody may recognize only a limited portion of a complex red cell antigen.

     

Cross idiotypic specificity among cold agglutinins in relation to combining activity for blood group-related antigens

The previous observations of cross idiotypic specificity of IgM cold agglutinins were further investigated in quantitative precipitin assays and attempts were made to relate shared idiotypic specificity to combining specificity for I, i and Pr antigens of red cells. Shared idiotypic determinants were observed among anti-I and anti-i antibodies which are now known to have combining specificity for determinants present on precursors of the ABH and Lewis blood group substances. There was evidence that one cold agglutinin, Ma, which had previously been shown to have an unusual kind of I specificity was deficient in the idiotypic determinants associated with the other anti-I proteins.     

Chemically synthesized solid phase oligosaccharide probes for carbohydrate-binding receptors. Interactions of the E-, L- and P-selectins with sialyl-Le(x) and O-sulphated forms linked to biotin or to polyacrylamide

There is a growing interest in chemically defined oligosaccharide reagents for identifying proteins that bind carbohydrates and determining the specificities of carbohydrate-binding proteins. Here, we compare three sets of chemically synthesized commercially available oligosaccharide conjugates as immobilized probes, for the binding signals that they elicit with known carbohydrate-binding receptors of the immune system, the E-, P- and L-selectins. The first set of conjugates is of oligosaccharides linked to biotin via a nine-carbon spacer. The second and third sets are multivalent derivatives in which the oligosaccharides are linked, via a three-carbon spacer to poly[N-(2-hydroxyethyl)acrylamide] (PAA) or to biotinylated PAA with an average of 20% substitution of the hydroxyethyl-amide groups by carbohydrate. The conjugates were immobilized on streptavidin-coated microwells if biotinylated, otherwise by drying in uncoated wells. The most robust binding curves, overall, were with the biotinylated PAA derivatives of the ligands immobilized on streptavidin wells. These reagents have permitted a reevaluation of selectin binding signals elicited by sialyl-Lewis(x) (SLe(x)) analogues having sulphate at position 6 of the galactose (6'SuSLe(x)) or of the N-acetylglucosamine (6SuSLe(x)). The results clarify the role of 6SuSLe(x), rather then 6'SuSLe(x), as a ligand for the selectins.     

Interaction of Mycoplasma pneumoniae with erythrocyte glycolipids of I and i antigen types.

The role of sialoglycolipids (gangliosides) as receptors for the human pathogen Mycoplasma pneumoniae was investigated by using purified gangliosides of known carbohydrate structures as inhibitors of the binding of 51Cr-labeled erythrocytes to sheet cultures of M. pneumoniae. We found that sialoglycolipids with long carbohydrate backbones of the poly-N-acetyllactosamine type were more potent inhibitors of M. pneumoniae binding than those with short carbohydrate chains. This is in accord with earlier inhibition data for glycoproteins and oligosaccharides. Thus, the inhibitory activity of a fraction of bovine erythrocyte gangliosides containing long backbone structures of I antigen type was approximately 200 times greater than that of the short chain gangliosides GM3 and GT1b. The binding of M. pneumoniae to erythrocytes of I and i antigen types was found to be comparable, indicating that M. pneumoniae in its adhesive specificity may not distinguish between the branched carbohydrate backbones of I type and the linear structures of i type. Thus, the production of autoantibodies to the backbone structures of I type rather than i type after infection with this agent may simply reflect a greater abundance of branched carbohydrate receptors of I type on the surface of host cells with which the mycoplasma forms immunogenic complexes.     

Mitochondrial and other tissue antibodies in relatives of patients with primary biliary cirrhosis

Clinical and immunological studies were made on 126 close relatives of twenty-seven patients with primary biliary cirrhosis and in healthy controls matched with them for age and sex. In each of two families there were two members with overt chronic liver disease, and an increased incidence of thyroid disorders was found in the patients and their families.Mitochondrial antibodies which were present in twenty-six of the twenty-seven probands, were also found in one sister with liver disease and in 7% of healthy relatives, while none of the controls reacted in the test (P<0·01). There was also a slight increase in nuclear and gastric parietal cell antibodies. Certain autoantibodies were strikingly clustered in some of the families.Serum immunoglobulins were measured in ninety-two relatives and showed a few instances of raised levels as well as some relatives with abnormally low values in the IgG, IgA and IgM classes.The findings suggest a familial or genetic abnormality of the immune responses as one of the predisposing factors in the development of primary biliary disease.     

VH gene analysis of Burkitt's lymphoma in children from north-western Iran

Cases of Burkitt's lymphoma (BL) from north-western Iran were investigated for the usage and somatic mutational pattern of their immunoglobulin variable region genes. Potentially functional V_H genes were amplified from 6/12 of the tumour masses and all of these were derived from the V_H3 family, with 4/6 being derived from the most commonly used V_H3 family member, V_3-23. All of the tumour sequences were mutated from their germline counterparts, to varying degrees, with a mean level of 5.8%, indicating that the cell of origin had encountered the germinal centre. Intraclonal sequence heterogeneity was also evident in 4/6 of the lymphomas, showing that the tumour cells had undergone further somatic mutation following neoplastic transformation. Analysis of the five potentially functional mutated V_H sequences showed a significant clustering of replacement mutations in the complementarity-determining region 2, consistent with a role for antigen in selection of tumour cell sequences. The pattern of extensive somatic mutation, and intraclonal variation, in these mainly EBV+ve tumours, was similar to that previously reported in V_H sequences of EBV+ve endemic BL (eBL) and EBV−ve sporadic BL (sBL), with the mean level of somatic mutation lying between those reported for eBL (7.7%) and sBL (4.0%). However, V_H gene bias and the distribution of mutations in the Iranian cases showed features which differed from those reported for endemic or sporadic BL.