Summary report on the ISOBM TD-6 workshop: analysis of 20 monoclonal antibodies against Sialyl Lewisa and related antigens. Montreux, Switzerland, September 19-24, 1997

The ISOBM TD-6 Workshop is the first international workshop on monoclonal antibodies against the Sialyl Lewisa (SLea) antigen. Eight research groups participated in a blind study to characterize the epitope binding, relative affinity and performance in immunoradiometric assays, of a panel of 20 monoclonal antibodies. The antibodies were tested against a diverse panel of neoglycoconjugates, purified antigens and human serum pools from gastrointestinal malignancies. Epitope specificities were determined for the majority of antibodies in the panel. Cross-reactivity with related saccharide structures was noted in several antibodies. Overall, the results of the TD-6 Workshop show further development of SLea immunoassays may yield yet more specific assays for the detection and management of gastrointestinal and other malignancies.     

Comparison of two enzyme-linked immunosorbent assays and one rapid immunoblot assay for detection of herpes simplex virus type 2-specific antibodies in serum

The sensitivities and specificities of three immunoassays for the detection of herpes simplex virus type 2 (HSV-2)-specific immunoglobulin G antibodies in serum, including the one-strip rapid immunoblot assay (RIBA; Chiron Corporation) and two indirect enzyme immunosorbent assays (EIA; Gull Laboratories and Centocor), were compared by testing a panel of 1,250 serum samples from individuals attending an outpatient clinic for sexually transmitted diseases. A qualitative agreement among the three assays was observed with 1,080 serum samples (86.4%); 291 of the serum samples (23.3%) were positive, 789 samples (63.1%) were negative, and 170 serum samples (13.6%) gave a discordant result. Results were considered conclusive when a concordant result was obtained with two of three assays. The sensitivities and specificities of the RIBA, the Gull EIA, and the Centocor EIA proved to be 99.2, 99.7, and 89.9% and 97.1, 96.7, and 99.3%, respectively. These results indicate that the Chiron RIBA and the Gull EIA are especially useful and reliable for the detection of HSV-2-specific antibodies in serum.     

Ganglioside structure dictates signal transduction by cholera toxin and association with caveolae-like membrane domains in polarized epithelia

In polarized cells, signal transduction by cholera toxin (CT) requires apical endocytosis and retrograde transport into Golgi cisternae and perhaps ER (Lencer, W.I., C. Constable, S. Moe, M. Jobling, H.M. Webb, S. Ruston, J.L. Madara, T. Hirst, and R. Holmes. 1995. J. Cell Biol. 131:951-962). In this study, we tested whether CT's apical membrane receptor ganglioside GM1 acts specifically in toxin action. To do so, we used CT and the related Escherichia coli heat-labile type II enterotoxin LTIIb. CT and LTIIb distinguish between gangliosides GM1 and GD1a at the cell surface by virtue of their dissimilar receptor-binding B subunits. The enzymatically active A subunits, however, are homologous. While both toxins bound specifically to human intestinal T84 cells (Kd approximately 5 nM), only CT elicited a cAMP-dependent Cl- secretory response. LTIIb, however, was more potent than CT in eliciting a cAMP-dependent response from mouse Y1 adrenal cells (toxic dose 10 vs. 300 pg/well). In T84 cells, CT fractionated with caveolae-like detergent-insoluble membranes, but LTIIb did not. To investigate further the relationship between the specificity of ganglioside binding and partitioning into detergent-insoluble membranes and signal transduction, CT and LTIIb chimeric toxins were prepared. Analysis of these chimeric toxins confirmed that toxin-induced signal transduction depended critically on the specificity of ganglioside structure. The mechanism(s) by which ganglioside GM1 functions in signal transduction likely depends on coupling CT with caveolae or caveolae-related membrane domains.     

Mechanism of N-acetylgalactosamine binding to a C-type animal lectin carbohydrate-recognition domain

The mammalian hepatic asialoglycoprotein receptor, a member of the C-type animal lectin family, displays preferential binding to N-acetylgalactosamine compared with galactose. The structural basis for selective binding to N-acetylgalactosamine has been investigated. Regions of the carbohydrate-recognition domain of the receptor believed to be important in preferential binding to N-acetylgalactosamine have been inserted into the homologous carbohydrate-recognition domain of a mannose-binding protein mutant that was previously altered to bind galactose. Introduction of a single histidine residue corresponding to residue 256 of the hepatic asialoglycoprotein receptor was found to cause a 14-fold increase in the relative affinity for N-acetylgalactosamine compared with galactose. The relative ability of various acyl derivatives of galactosamine to compete for binding to this modified carbohydrate-recognition domain suggest that it is a good model for the natural N-acetylgalactosamine binding site of the asialoglycoprotein receptor. Crystallographic analysis of this mutant carbohydrate-recognition domain in complex with N-acetylgalactosamine reveals a direct interaction between the inserted histidine residue and the methyl group of the N-acetyl substituent of the sugar. Evidence for the role of the side chain at position 208 of the receptor in positioning this key histidine residue was obtained from structural analysis and mutagenesis experiments. The corresponding serine residue in the modified carbohydrate-recognition domain of mannose-binding protein forms a hydrogen bond to the imidazole side chain. When this serine residue is changed to valine, loss in selectivity for N-acetylgalactosamine is observed. The structure of this mutant reveals that the beta-branched valine side chain interacts directly with the histidine side chain, resulting in an altered imidazole ring orientation.     

Field testing of a long-span arch steel culvert during backfilling and in service

The paper presents the first part of the in-situ measurements and data analyses for the tests conducted during backfilling and during service of a long-span corrugated steel culvert railway bridge over Skivarpsån, Rydsgård, Sweden. Static and dynamic tests were carried out measuring strains and displacements. Temperature readings were taken along with the measurements. Comparisons of moments during compaction showed that there is good agreement between test results and theoretical values. The theoretical calculation of the rise of the crown during compaction and the crown moments due to live load seem to be conservative, while the theoretical axial forces agree reasonably with the measured axial forces.     

Formation of white layers in steels by machining and their characteristics

White layers (WLs) produced in hard steels by machining have been characterized using nanoindentation, optical microscopy, transmission electron microscopy (TEM), and X-ray diffraction. The WL is found to have a hardness of 12.85±0.80 GPa, which is significantly greater than that of untempered martensite produced by various heat-treatment processes. The grain size in the WL is shown to be in the submicrometer range with values ranging, typically, between 30 and 500 nm. These two characteristics of the WL distinguish it from various structures formed in steels by heat treatment. The formation of WLs is promoted by conditions of moderate to high cutting speed in conjunction with tool flank wear. Based on a consideration of the strain, stress, and temperature states associated with the formation of WLs in machining, it is hypothesized that deformation of material to very large strains is the principal factor contributing to the formation of these layers with ultrafine grained or nanocrystalline structures. The large strain deformation and elevated temperatures prevailing in the machining zone could also trigger dynamic recrystallization or cause decomposition and partial dissolution of the cementite present in the steels.