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Item response theory (IRT) analyses have, over the past 3 decades, added much to our understanding of the relationships among and characteristics of test items, as revealed in examinees response patterns. Assessment instruments used outside the educational context have only infrequently been analyzed using IRT, however. This study demonstrates the relevance of IRT to personality data through analyses of Scale 2 (the Depression Scale) on the revised Minnesota Multiphasic Personality Inventory (MMPI-2). A rich set of hypotheses regarding the items on this scale, including contrasts among the Harris-Lingoes and Wiener-Harmon subscales and differences in the items measurement characteristics for men and women, are investigated through the IRT analyses.

Seventy-eight patients with haematological malignancies, received T-cell-depleted stem cell transplants and cyclosporin followed by delayed add-back of donor lymphocytes to prevent leukaemia relapse. The source of stem cells was bone marrow in 50 patients and granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood in 28 patients. In univariate analysis, only the CD34+ cell dose (but not the stem cell source or the T lymphocyte dose) and disease status were predictive for transplant-related mortality, relapse and survival. Patients receiving >/= 3 x 106 CD34+ cells/kg had an overall actuarial survival of 68% compared with 52%, 35% and 10%, respectively, for cell doses of 2-2.99, 1-1.99 and < 1 x 106/kg. Multivariate analysis of risk factors for relapse identified disease risk and CD34+ cell dose as the only factors. Relapse was 62.5% in 38 patients at high risk of relapse vs. 25% for 40 patients at intermediate or low risk. CD34+ cell doses of >/= 3 x 106/kg were associated with a 13.5% relapse vs. 48% for recipients of lower doses. This favourable effect of CD34+ cell dose on relapse was apparent in both high- and intermediate- plus low-risk groups. Our results support the potential benefit of a high stem cell dose in lowering transplant-related mortality (TRM) and in reducing relapse after allogeneic marrow or blood stem cell transplants.

We reevaluate here an earlier report of monosaccharide binding by the C-type lectin-like, leukocyte surface protein CD69 in the form of a recombinant soluble dimer, and we examine polysaccharide binding by the protein. We have expressed in Escherichia coli a new construct of the extracellular part (Q(65)-K(199)) of human CD69. We describe the folding in vitro to produce, in good yield, the protein in a soluble, disulphide-linked, dimeric form, and the results of binding experiments with monosaccharides: glucose, galactose, mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine, linked to bovine serum albumin. Monosaccharide-binding signals are not detectable. Among the polysaccharides, heparin, chondroitin sulphates A, B, and C, fucoidan, and dextran sulphate, CD69 dimer gives a weak binding signal with fucoidan., (Copyright 1999 Academic Press.)

Nonmyeloablative allogeneic stem cell transplantation has recently been explored as a safer alternative to conventional high-dose transplant regimens. Although a high incidence of mixed chimerism after nonmyeloablative procedures has been reported, the exact kinetics of engrafting donor cells in specific cellular lineages has yet to be defined. We investigated lineage-specific chimerism in 15 patients receiving an allogeneic peripheral blood stem cell (PBSC) transplant from an HLA-identical (n = 14) or a 5/6 antigen-matched sibling donor after a preparative regimen of cyclophosphamide and fludarabine. Donor chimerism was assessed weekly in T lymphocytes and myeloid cells by polymerase chain reaction (PCR) of minisatellite regions. Eight patients survived between 121 to 409 days after transplant. Ten of 14 patients surviving more than 30 days (71.4%) had delayed disease regression consistent with a graft-versus-malignancy (GVM) effect. One patient rejected the transplant with subsequent recovery of autologous hematopoiesis. Hematological recovery was rapid (median, 11 days to >/=500 neutrophils/microL) and was initially predominantly recipient in origin. Donor myeloid chimerism gradually supplanted recipient hematopoiesis and became fully donor in all survivors by 200 days after transplantation. In contrast, T-cell engraftment was more rapid, with full chimerism in 7 patients by day 30 and in 6 further patients by day 200 after cyclosporine withdrawal and donor lymphocyte infusion. Full donor T-cell engraftment preceded donor myeloid engraftment, acute graft-versus-host disease, and disease regression, consistent with a requirement for 100% donor T-cell chimerism for full expression of the alloresponse. These results emphasize the importance of lineage-specific chimerism analysis to successfully manipulate engraftment after nonmyeloablative allogeneic PBSC transplantation.

Two patients with chronic myeloid leukaemia (CML) received a non-myeloablative preparative regimen of cyclophosphamide and fludarabine, followed by an unmanipulated, G-CSF-mobilized, peripheral blood stem cell transplant from an HLA-identical sibling. Chimaerism, evaluated in myeloid and T-lymphoid lineages by PCR of minisatellite variable regions, showed day 14 post-transplant haemopoietic recovery to be 90% autologous in both patients. On day 30 the bone marrow showed only 1/20 and 2/18 donor metaphases. By day 100 post transplant both had 100% donor myeloid and lymphoid lineages as assessed by karyotype and minisatellite chimaerism analysis. They subsequently became RT-PCR negative for BCR-ABL. Both survive 7 and 14 months post transplant in molecular remission of CML. In one, donor T cells, stimulated with pre-transplant CML cells, induced 30-50% inhibition of pre-transplant leukaemic CFU-GM, but did not inhibit CFU-GM in the day 60 marrow (46% Ph-negative recipient, 54% donor). These results show that a non-myeloablative allotransplant can induce molecular remissions of CML through a graft-versus-leukaemia effect.

This study was aimed at investigating the efficacy of presentation of biotinylated oligosaccharides on streptavidin-coated microwells for interactions with (a) three monoclonal antibodies directed at sialyl-Lewisa (Le(a)) or sulfo-Le(a)-related sequences, and (b) the endothelium-leukocyte adhesion molecules, the E-, L- and P-selectins which recognize both the sulfo- and sialyl-Le(a) series. With the antibodies it was observed that if the biotinylated oligosaccharide incorporated the entire antigenic determinant, and additional saccharide length was not included, the biotinyl tag spacer length was a critical factor in the strength of the binding signal. If oligosaccharide chain beyond the determinant was included, the biotinyl tag spacer length was less important. The E-selectin binding data with the biotinylated sialyl- and sulfo-oligosaccharides were in overall accord with previous knowledge. With the L- and P-selectins, however, unexpectedly low binding signals were elicited by biotinyl sulfo-Le(a) sequences relative to those with the sialyl-analogs. This suppression was more pronounced with the rodent than the human L-selectin. Such differential availabilities of oligosaccharides displayed on streptavidin may relate to biological situations, such as the differential reactivities of the three selectins with a given oligosaccharide ligand presented on different carrier proteins, or on different O-glycan cores on mucin-type glycoproteins.

Antigen-specific immunotherapy of cancer depends on a consistent source of well-defined protein antigen. Production of recombinant protein offers the obvious solution to this problem but few comparisons of recombinant and native proteins in cellular immune assays have been reported. We report expression of a putative immunotherapy antigen, prostate-specific membrane antigen (PSMA), in insect cells using a baculovirus vector. T cells stimulated with recombinant PSMA or native PSMA derived from the LNCaP cell line recognized both native PSMA and recombinant, baculoviral PSMA. These data indicate that PSMA produced in Sf9 cells is immunologically cross-reactive with native PSMA and therefore suitable for immunotherapy as it is recognized by both cellular and humoral immune responses.

Purpose: A 50-year-old man developed progressive pulmonary metastasis resistant to interferon alfa-2b treatment 7 months after he underwent left nephrectomy for stage III renal cell carcinoma. We performed a nonmyeloablative allogeneic peripheral-blood stem-cell transplant in this patient to exploit a possible graft-versus-tumor effect from allogeneic lymphocytes., Materials and Methods: The conditioning regimen consisted of fludarabine and cyclophosphamide followed by a T-cell replete, granulocyte-colony stimulating-factor-mobilized peripheral-blood stem-cell transplant from his HLA-identical brother. Cyclosporine was administered from days -4 to +45 to prevent graft rejection and acute graft-versus-host disease (GVHD)., Results: Serial polymerase chain reaction analysis of hematopoietic lineage-specific minisatellites initiallyshowed mixed chimerism in CD14(+) and CD15(+) myeloid cells, CD3(+) T cells, and CD34(+) progenitor cells, with rapid conversion to 100% donor T-cell chimerism by day +60 and 100% donor myeloid cells by day +100. Serial computed tomography scans of the chest showed stable disease at day +30, slight regression of pulmonary lesions at day +63, and complete disappearance of all pulmonary metastatic disease by day +110. Mild transient acute GVHD disease of the skin occurred on day +60 and limited chronic GVHD of the skin occurred by day +200., Conclusion: The complete regression of metastatic disease, which has now been maintained for more than 1 year, is compatible with a graft-versus-tumor effect.

To investigate mechanisms of stem cell graft rejection we studied the allo-stimulatory potential of G-CSF mobilized peripheral blood progenitor cells (PBPC). CD34+ cells were purified (>95%) in a two-step procedure using immunoaffinity columns for CD34 selection and T-depletion. The capacity of CD34+ cells to stimulate allogeneic T-cell responses was compared with other cells from the same individual. CD34+ cells induced potent proliferative responses at stimulator:responder ratios of 1:20, but were approximately 50-fold less efficient compared to dendritic cells. Furthermore, CD34+ cells primed responses from partially matched allogeneic T cells in bulk cultures. Dual-colour flow cytometry showed that the co-stimulatory molecules B7.1, CD40 and ICAM-1 were absent on resting CD34-positive progenitor cells, but were induced during incubation with allogeneic lymphocytes due to a cytokine-mediated effect. Up-regulation of accessory molecules on CD34+ cells was reproduced by incubation with interferon-gamma or GM-CSF which enhanced the allo-stimulatory activity of CD34+ cells. Blocking studies with inhibitory antibodies suggested co-stimulatory functions for B7.2, ICAM-3, CD40 and LFA-3. CD34+ cells were more efficient in inducing allogeneic T-cell responses when compared to the unprocessed leukapheresis products. The reduced allo-stimulatory ability of G-CSF mobilized PBPC could be explained by the presence of CD3+ 4+ and CD3+ 8+ lymphocytes with suppressor activity. We conclude that current methods of stem cell selection for transplantation do not avoid allosensitization of the recipient and that further graft manipulation with add-back of lymphocytes or selection of subsets of CD34+ cells with reduced allo-stimulatory ability may reduce graft rejection.

New legislation may not always have its intended effect. Agencies targeted by the legislation must be aware of it, understand its demands, develop procedures to insure implementation, and monitor compliance. This paper reports a study of the extent to which several new state statutes impacted policy, procedures, and services for mentally ill offenders in a large urban Texas county. Key informant-interviews, document reviews and surveys of knowledge and opinions were used to assess implementation, understanding, and acceptance of these new statutes. Results were reported to local constituencies, and used to shape recommendations for local action. Following the study, the community formed a council that has made specific improvements in the system.

The interaction between glycosphingolipids and recombinant human GM2-activator was studied in a microwell binding assay. A-series gangliosides like GM3, GM2 and GM1 were strongly bound by the recombinant human GM2 activator. A weak binding was observed to GD1b and sulfatide, while neutral glycolipids were not bound. Optimal binding occurred at pH 4.2 and was inhibited by increasing concentrations of citrate buffer and NaCl. In contrast with these in vitro results the recombinant human GM2-activator is able to restore the degradation of GA2 in fibroblasts from patients with the AB variant of GM2 gangliosidosis in vivo.

Nine of 56 (20% actuarial) patients receiving a T cell-depleted, HLA-identical sibling BMT for hematological malignancy developed hemorrhagic cystitis (HC) 15-368 days post BMT. Hematuria was severe and prolonged (median duration 18 days). In eight patients (89%), a viral etiology was confirmed (four adenovirus, four polyomavirus). HC was associated with significant morbidity, with all patients requiring continuous bladder irrigation and transfusion support for blood loss and thrombocytopenia. HC occurring before day 100 was significantly associated with a reduction in long-term survival: 1/7 (14.3%) patients developing HC before day 100 became long-term survivors vs 21/49 (42.8%) without HC by day 100 (P = 0.034). In univariate analysis, HC was associated with a diagnosis of multiple myeloma (P = 0.02). There was a trend towards a higher incidence of HC in patients reactivating cytomegalovirus (CMV) compared with those remaining CMV negative (18.4 vs 5.5% respectively, P = 0.17). HC was not associated with graft-versus-host disease, or with the transplant dose of CD34+ progenitors or CD3+ cells, patient age or sex. Life-threatening, viral-induced HC and the unusually high incidence of adenovirus-induced HC may have been caused by immune deficiency associated with T cell depletion in this series.

Biotinyl-oligosaccharides are a relatively new generation of saccharide probes that enable immobilization of desired oligosaccharides on streptavidin matrices for studies of carbohydrate-protein interactions. Here we describe the facile preparation of biotinyl-l-3-(2-naphthyl)-alanine hydrazide (BNAH) derivatives of oligosaccharides, containing a strong UV absorbing and fluorescent group, in which the ring of the reducing-end monosaccharide is nonreduced. We evaluate reactivities of immobilized BNAH- N -glycans with plant lectins that recognize aspects of the oligosaccharide core or outer-arms. We make some comparisons with 2-amino-6-amidobiotinyl-pyridine (BAP) derivatives obtained by reductive amination, and 6-(biotinyl)-aminocaproyl-hydrazide (BACH) derivatives which have a longer spacer-arm. N -Glycan-BNAH and-BAP derivatives have, overall, comparable reactivities with lectins which recognize N -glycan outer-arms or the trimannosyl core, but only BNAH and BACH derivatives are bound by lectins which recognize the non-reduced core. Moreover, with Pisum sativum agglutinin (PSA) which additionally requires the fucosyl- N- glycan-asparaginyl core for high affinity binding, the immobilized BNAH derivative (which is an alanine hydrazide beta-glycoside) can substitute for the natural beta-glycosylasparaginyl core, whereas the BACH derivative (aminocaproyl-hydrazide-beta-glycoside) is less effective. BNAH is a derivatization reagent of choice, therefore, for solid phase carbohydrate-binding experiments with immobilized N -glycans.

The human L-selectin is known to bind to immobilized 3'-sialyl-Le(x) and -Le(a) oligosaccharides both under static and physiological flow conditions. Here the reactivities toward 3'-sulfated and 3'-sialyl-Le(a) and -Le(x) pentasaccharides are compared by in-vitro binding and inhibition assays using preparations of human L-selectin-IgG-Fc chimera in which the selectin is predominantly in di- and tetrameric form (paucivalent) or in the form of a complex with anti-IgG (multivalent). Affinity for the sulfated ligands is marginally greater than for the sialyl ligands, as judged by concentrations required to give 50% inhibition of the multivalent selectin binding to the immobilized sulfated and sialyl ligands. There is a striking difference, however, in the avidities of binding of the two L-selectin forms toward the sulfated and sialyl ligands when these are immobilized in the clustered state: the paucivalent selectin gives detectable binding only to the sulfated ligands when these are immobilized as neoglycolipids on plastic microwells (up to 100 pmol immobilized per well) whereas the multivalent L-selectin binds well to both classes of ligand. Moreover, binding of the paucivalent selectin form is effectively inhibited only by the sulfated ligand, although binding of the multivalent selectin is inhibitable by both the sulfated and sialyl ligands. Such striking valency-dependent differences in ligand binding avidity and inhibitability may be manifest in vivo with the membrane-bound L-selectin, as marked variations occur in its density of expression on leukocytes. Thus, for the purpose of selecting inhibitors for development of therapeutic anti-inflammatory compounds, experimental designs based on the paucivalent L-selectin would more clearly single out compounds with broad spectrum anti-adhesive activities toward the both the high- and low-avidity interactions of the cell adhesion protein.

This communication is concerned with the binding specificity of the leukocyte-adhesion molecule L-selectin (leukocyte homing receptor) towards structurally defined sulphated oligosaccharides of the blood group Le(a) and Le(x) series, and of the glycosaminoglycan series heparin, chondroitin sulphate and keratan sulphate. The recombinant soluble form of the rat L-selectin (L-selectin-IgG Fc chimera) investigated here was shown previously to bind to lipid-linked oligosaccharides 3-O, 4-O and 6-O sulphated at galactose, such as sulphatides and a mixture of 3-sulphated Le(a)/Le(x) type tetrasaccharides isolated from ovarian cystadenoma, as well as to the HNK-1 glycolipid with 3-O sulphated glucuronic acid. In the present study, the L-selectin investigated in both chromatogram binding and plastic microwell binding experiments using neoglycolipids was found to bind to the individual 3-sulphated Le(a) and Le(x) sequences (penta-, tetra- and trisaccharides), and with somewhat lower intensities to their non-fucosylated analogues. Glycosaminoglycan disaccharides of keratan sulphate, heparin and chondroitin sulphate types were also bound by L-selectin in one or both assay systems, leading to the conclusion that clustered glycosaminoglycan oligosaccharides with 6-O sulphation of N-acetylgalactosamine, N-acetylglucosamine or glucosamine, 4-O sulphation of N-acetylgalactosamine, 2-O sulphation of uronic acid, N-sulphation of glucosamine and, to a lesser extent, the non-sulphated uronic acid-containing disaccharides, can support L-selectin adhesion. As inflammatory chemokines (short-range stimulators of lymphocyte migration which trigger integrin activation) are known to bind to endothelial glycosaminoglycans, we propose that the binding of the lymphocyte membrane L-selectin to endothelial glycosaminoglycans may provide a link between the selectin-mediated and integrin-mediated adhesion systems in leukocyte extravasation cascades. The possibility is also raised that lymphocyte L-selectin interactions with glycosaminoglycans may contribute to pathologies of glycosaminoglycan-rich tissues, e.g. cartilage loss in rheumatoid arthritis and inflammatory lesions of the cornea.

A diversity of high-affinity oligosaccharide ligands are identified for NKR-P1, a membrane protein on natural killer (NK) cells which contains an extracellular Ca(2+)-dependent lectin domain. Interactions of such oligosaccharides on the target cell surface with NKR-P1 on the killer cell surface are crucial both for target cell recognition and for delivery of stimulatory or inhibitory signals linked to the NK cytolytic machinery. NK-resistant tumour cells are rendered susceptible by preincubation with liposomes expressing NKR-P1 ligands, suggesting that purging of tumour or virally infected cells in vivo may be a therapeutic possibility.

In this study, the radiation doses to occupants of two reinforced concrete buildings at Nagasaki, who survived the immediate effects of the nuclear weapon detonation, are determined using state-of-the-art radiation transport techniques. The radiation doses at all locations in the buildings are calculated using the Three-Dimensional Oak Ridge Discrete Ordinates Radiation Transport Code which was constructed especially for this task. This code represents a new and unique capability that has been previously reported. This study resulted in case-by-case lists of doses to occupants and an uncertainty analysis. These data have been used in a companion study as the basis for determining a new value of the dose producing a 50% risk of fatality.

Carbohydrate recognition by the human endothelial-leukocyte adhesion molecule, E-selectin, has been investigated by binding studies using 3H-labeled Chinese hamster ovary cells expressing different levels of the transfected full-length adhesion molecule and a series of structurally defined oligosaccharides linked to the lipid phosphatidylethanolamine dipalmitoate (neoglycolipids) and synthetic glycolipids chromatographed on silica gel plates or immobilized on plastic wells. Evidence is presented for density-dependent binding of the membrane-associated E-selectin not only to 3'-sialyl-lacto-N-fucopentaose II (3'-S-LNFP-II) and 3'-sialyl-lacto-N-fucopentaose III (3'-S-LNFP-III) which express the sialyl Le(a) and sialyl Le(x) antigens, respectively, but also to the nonsialylated analogue LNFP-II; there is a threshold density of E-selectin required for binding to these sialylated sequences, and binding to the nonsialylated analogue is a property only of cells with the highest density of E-selectin expression. The presence of fucose linked to subterminal rather than to an internal N-acetylglucosamine is shown to be a requirement for E-selectin binding, and although the presence of sialic acid 3-linked to the terminal galactose of the LNFP-II or LNFP-III sequences substantially enhances E-selectin binding, the presence of 6-linked sialic acid abolishes binding. E-selectin binding is unaffected in the presence of the blood group H fucose (alpha 1-2 linked to galactose to form the Le(b) antigen). However, the binding is abolished when in addition alpha 1-3-linked N-acetylgalactosamine to the galactose (blood group A antigen) is present. These results indicate that some E-selectin-mediated adhesive events may be influenced by blood group status.

Binding specificity of the major surfactant protein SP-A from human and dog lung has been investigated. Radiobinding experiments have shown that both proteins bind in a Ca(2+)-dependent manner to galactose, mannose, fucose, and glucose linked to bovine serum albumin. These results are in accord with a previous study in which monosaccharides were linked to agarose (Haagsman, H. P., Hawgood, S., Sargeant, T., Buckley, D., White, R. T., Drickamer, K., and Benson, B. J. (1987) J. Biol. Chem. 262, 13877-13880). Chromatogram overlays in conjunction with in situ liquid secondary ion mass spectrometry (TLC-LSIMS) of several purified glycosphingolipids and neoglycolipids as well as binding assays with glycolipids immobilized on plastic wells, demonstrate recognition of galactose (human and dog SP-A), glucose, and lactose (human SP-A) in association with specific lipids. In addition, the occurrence of several neutral and acidic glycosphingolipids in human and rat extracellular surfactants and rat alveolar type II cells is described. Selected components among the neutral glycolipids are bound by radiolabeled human SP-A; these are identified by TLC-LSIMS as predominantly ceramide mono- and disaccharides (human surfactant) and ceramide tri- and tetrasaccharides (rat surfactant and type II cells). A recombinant carbohydrate recognition domain (CRD) of human SP-A inhibits the binding of human SP-A to galactosyl ceramide and to galactose- and mannose-bovine serum albumin, indicating that the CRD is directly involved in the binding of SP-A to these ligands. These results provide evidence for a novel type of binding specificity for proteins that have Ca(2+)-dependent CRDs and raise the possibility that glycosphingolipids are endogenous ligands for SP-A.

The aim of the present study has been to investigate the binding specificity of the soluble 14,000-dalton lectin of bovine heart muscle towards immobilised oligosaccharides in clustered form. To this end, chromatogram overlay assays and quantitative plastic-microwell-binding assays have been performed using several natural glycolipids and neoglycolipids containing one or more of the disaccharide units, beta-D-Galp-(1----4 or 3)-D-GlcNAc or beta-D-Galp-(1----4)-D-Glc and related structures. The microwell assay gave the most consistent results. It was observed that for binding by the soluble lectin the optimal sequence, which is beta-D-Galp-(1----4 or 3)-D-GlcNAc, must occur at the nonreducing end of longer oligosaccharides when linked to lipid. These oligosaccharides may be of poly(N-acetyllactosamine) type or they may be mono- or multi-antennary, complex-type chains in which the disaccharide is joined directly to a trimannosyl core. The lectin bound to such immobilised lipid-linked oligosaccharides on which the terminal D-galactosyl groups are substituted with alpha-L-Fucp-(1----2), alpha-D-Galp-(1----3), or alpha-NeuAc-(2----3) groups. However, no binding was detected if the terminal D-galactosyl groups were substituted with an alpha-NeuAc-(2----6) group or the subterminal N-acetylglucosamine units with an alpha-L-Fucp-(1----3 or -4) group. Internally located N-acetyllactosamine units where the D-galactose units are disubstituted by beta-D-GlcNacp-(1----3) and -(1----6) units, as in branched poly(N-acetyllactosamine) backbones were not bound by the bovine lectin. These results are in accord with previous observations on the bovine lectin and the corresponding human and rat lectins, using structurally defined oligosaccharides as inhibitors of binding. The results of comparative binding experiments using paragloboside and ceramide hexasaccharide which contain one and two N-acetyllactosamine units, respectively, joined in linear sequence to the lactosylceramide core, were equivocal with respect to the availability of the internal N-acetyllactosamine units for binding by the bovine lectin.

Two different mannose-binding proteins (MBP-A and MBP-C), which show 56% sequence identity, are present in rat serum and liver. It has previously been shown that MBP-A binds to a range of monosaccharide-bovine serum albumin conjugates, and that, among oligosaccharide ligands tested, preferential binding is to terminal nonreducing N-acetylglucosamine residues of complex type N-linked oligosaccharides. In order to compare the binding specificity of MBP-C, an expression system has been developed for production of a fragment of this protein which contains the COOH-terminal carbohydrate-recognition domain. After radioiodination, the domain has been used to probe natural glycoproteins, neoglycoproteins, and neoglycolipids. Like MBP-A, MBP-C binds several different monosaccharides conjugated to bovine serum albumin, including mannose, fucose, and N-acetylglucosamine, although binding to the last of these is relatively weaker than observed for MBP-A. The results of binding to natural glycoproteins and to neoglycolipids containing oligosaccharides derived from these proteins are most compatible with the interpretation that MBP-C interacts primarily with the trimannosyl core of complex N-linked oligosaccharides, with additional ligands being terminal fucose and perhaps also peripheral mannose residues of high mannose type oligosaccharides. This binding specificity is thus quite distinct from that of MBP-A. The presence of multiple MBPs with distinct binding specificities in preparations derived from serum and liver explains conflicting conclusions which have been reached about carbohydrate recognition by these proteins.

A monoclonal antibody has been raised against a cross-reactive idiotypic determinant expressed on human autoantibodies with anti-I and -i specificities. The McAb is directed against a conformational epitope requiring interaction of H- and L-chains for maximal expression. This epitope was strongly expressed on the monoclonal protein of one out of 100 patients with paraproteinaemia and the peripheral blood lymphocytes of one out of 50 cases of B-cell leukaemia. A small amount of the epitope is detectable among immunoglobulins in normal serum.

The aim of this study was to investigate whether I antigen occurs in association with Mycoplasma pneumoniae in a form that may be immunogenic during natural infection or experimental immunization. I antigen activity was detected by radioimmunoassay in suspensions of M. pneumoniae MY11965 and in the soluble phase of mycoplasma lysates prepared with Triton X-100. There was evidence for the occurrence of I antigen in at least two macromolecular forms. The first form partitioned in the lipid phase following chloroform-methanol extraction and chromatographed on thin-layer chromatograms as a ceramide decasaccharide. The second form was associated with the residue after lipid extraction and was solubilized by treatment with sodium dodecyl sulfate or pepsin; this component was tentatively designated a glycoprotein or polysaccharide and was not investigated further. In a lipid extract from mycoplasmas that had been surface labeled by the galactose oxidase-NaB3H4 method, two 3H-labeled glycolipids were detected as minor components which chromatographed on thin-layer chromatograms in the region of an authentic I-active ceramide decasaccharide. However, no significant radioactivity was incorporated into glycolipids after metabolic labeling with [3H]glucosamine. These observations suggested that the mycoplasmas contained surface-associated glycolipids with I antigen activity that were of exogenous origin. This was supported by the observations that horse, rabbit, and fetal calf sera contained I antigen activity and that the I antigen activity in M. pneumoniae cultures reflected the levels found in the sera included in the culture media. From rabbit serum, which expressed the highest antigen activity, an I-active glycolipid was isolated that chromatographed as a ceramide decasaccharide. I-active substances passively adsorbed onto M. pneumoniae are potentially immunogenic. However, we consider these unlikely to be the main stimulus for autoantibody production in natural infection, since the autoantibodies elicited are restricted to the I carbohydrate antigen and there is a lack of antibodies to other glycolipids that may be adsorbed from serous and cellular components of the host tissues. In our view, the more likely stimulus is the specific complex formed between the mycoplasma and the sialo-oligosaccharide receptors of the Ii antigen type, as suggested previously.

A peroxidase has been isolated from pooled specimens of human cervical mucus. Investigation of substrate and inhibitor specificity, pH optimum, chromatography on Sephadex G-200, disc electrophoresis, ultraviolet absorption spectrum and the effects of temperature and ionic strength variation are described. A study of the steady-state kinetics is reported using hydrogen peroxide and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) as substrates and a catalytic mechanism is proposed.

The VH specificities of two rabbit antisera raised against the H chains of two human IgM cold agglutinins were studied with the aid of proteins on which the VH sequence data are available. They were found to recognize a new VH antigen (VHMar), which may represent a subgroup of VHI. This antigen is found on anti-Ii antibodies which have cross-reactive idiotypes. It is moderately well-expressed in normal gamma globulin and strongly expressed on 8% of unselected myeloma and macroglobulinaemia proteins. There is evidence to suggest that a proportion of the cross-reactive idiotypes among the anti-Ii antibodies involve this VH antigen.

In this report the carbohydrate antigens expressed on the three oligosaccharide domains, core, backbone and peripheral, of mucin-type glycoproteins are briefly reviewed in the light of recent observations with monoclonal antibodies. These have revealed that a number of cell-surface antigens which behave as tumour-associated and differentiation antigens of man or mouse are abundantly expressed on the carbohydrate chains of a variety of secreted mucins of human and animal origins and they belong to an antigen system which also includes the major blood group antigens. Examples are given of the use of well-characterized anti-carbohydrate antibodies to derive structural information on (a) mucin-type glycoproteins of human B lymphocyte membranes, (b) the high molecular weight glycoproteins of the normal human gastric and distal-colon mucosae and (c) tumour-derived glycoproteins from these two organs. Major differences between the antigenicities of the normal stomach and distal-colon, and between their tumour-derived glycoproteins, and the important effect of the secretor status in the expression of these antigens are described. These observations have enabled a better understanding of the individual and tissue differences in the expression of tumour-associated antigens. The possibility is raised that these carbohydrate structures (many of which also occur on certain N-linked oligosaccharides and glycolipids) are components of receptor systems for endogenous ligands. More tangible evidence is cited for the role of certain structures in this family of saccharides as receptors for infective agents.

A beta-galactoside-binding endogenous lectin extracted from bovine heart binds to the surface of baby hamster kidney (BHK) cells. The binding to and agglutination of cells is reduced in certain ricin-resistant mutants (Ric cells) in parallel with the decreased number of binding sites for the selective agent, ricin, a galactose-specific plant lectin. However, clear differences in the binding specificities of bovine lectin and ricin are shown by the effect of neuraminidase. BHK cells and Ric mutant cells treated with neuraminidase bind similar amounts of the bovine lectin compared with untreated cells, and ricin binding is greatly increased. The mammalian lectin immobilised on inert glass mediates the attachment and spreading of normal BHK cells and agglutinates these cells in solution. Ricin-resistant mutant cells respond poorly. These results are consistent with a role of endogenous lectins in cellular adhesiveness and show that cell adhesion may be regulated by the density of specific surface receptors for lectins.

Human amniotic fluid has been shown to contain blood group i as well as I antigens. Crude extracts of amniotic fluids at 16-23 weeks of gestation were in general more active than those obtained at term. A pool of amniotic fluids which had A, B, H as well as I and i activity was fractionated with an insolubilized anti-I (Group 3 type) immunoadsorbent column. There was evidence for the occurrence of I and i determinants on macromolecules carrying A, B and H determinants, for the fraction specifically retained by the column was enriched by 50-60-fold in I and i activity and by at least 10-fold in A, B and H activity. In the fraction not retained by the column there remained the bulk of the A, B, H activity in addition to I activity of Group I type which is known to be distinct from the Group 3 determinant. With the aid of specific immunochemical fractionation procedures, human amniotic fluid should prove useful in structural studies of the several I and i determinants and of their relationship to other blood group determinants.

Carbohydrate recognition by bovine serum conglutinin has been investigated by inhibition and direct binding assays using glycoproteins and polysaccharides from Saccharomyces cerevisiae (baker's yeast), and neoglycolipids derived from N-acetylglucosamine oligomers, mannobiose and human milk oligosaccharides. The results clearly show that conglutinin is a lectin which binds terminal N-acetylglucosamine, mannose and fucose residues as found in chitobiose (GlcNAc beta 1-4GlcNAc), mannobiose (Man alpha 1-3Man) and lacto-N-fucopentaose II [Fuc alpha 1-4(Gal beta 1-3)GlcNAc beta 1-3Gal beta 1-4Glc] respectively.

With the aim of obtaining monospecific antibodies against the beta-galactoside-binding lectin of bovine heart muscle, spleen cells from Lou rats immunized with lectin were fused with the rat myeloma line Y3.Ag1.2.3. Two immunoglobulin M (IgM)-producing clones, designated NIBy 142-36/8 and NIBy 143-9/5, derived from separate fusions, were used to generate ascites containing high-titre binding activity against the 13kDa component in preparations of lectin. Direct evidence that haemagglutinating activity is associated with the 13kDa protein was obtained by the specific elution of 13kDa polypeptides with haemagglutinating activity from an immobilized antibody adsorbent. Solid-phase radiobinding assays and immunoblotting of isolated lectins and/or muscle homogenates confirmed the earlier indications with conventional antisera that the beta-galactoside-binding lectins of bovine, human and monkey muscle tissue are antigenically related.

1. The theory of plane curves was applied to the graphical methods used in enzyme kinetics and a mathematical analysis of the possible graph shapes is given. 2. The belief that allosterism can be inferred from steady-state data alone is subjected to criticism and the mathematical significance of sigmoid curves and non-linear double-reciprocal plots is explored. 3. It is suggested that the usual methods of interpreting steady-state kinetic data are often based on over-restrictive assumptions which prevent maximum utilization of the available data. 4. Methods for obtaining the degree of the rate equation from graph shapes obtained directly from initial-rate measurements and from replots of asymptotic behaviour as chi approach the level 0 and chi approach the level infinity are discussed. 5. Detailed proofs of the theorems given in the text have been deposited as Supplementary Publication SUP 50049 (10 pages) at the British Library (Lending Division), Boston Spa, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1975), 145, 5.

A human serum containing a monoclonal anti-(blood-group I) antibody was used to investigate the distribution of blood-group-I antigen on erythrocyte membrane components. Sodium dodecyl sulphate/polyacrylamide-gel-electrophoresis profiles of immuneprecipitates by using 3H-labelled (by the galactose oxidase/NaB3H4 method) and 125I-labelled solubilized stroma were compared. Different radioactive profiles were revealed by the two radiolabelling methods. In the immunoprecipitates the predominant 125I radioactivity within the gel had the electrophoretic mobility of Band-3 protein (apparent mol.wt. 90 000--100 000), whereas the 3H radioactivity revealed a diffusely migrating component(s) (apparent mol.wt. range 40 000--70 000) in addition to radioactivity compatible with glycolipids at the dye front. The diffusely migrating 3H-labelled component was shown to have a similar electrophoretic mobility to a subpopulation of erythrocyte poly(glycosyl)ceramides with blood-group-I activity.

We have evaluated the pharmacokinetics in patients of a monoclonal antibody (19-9) F(ab')2 fragment coupled with DTPA and labeled with 111In. In addition to imaging and organ uptake determinations, serum and urine samples were analyzed to help determine the in vivo behavior of the label. Using a competitive binding assay, the immunoreactivity of the coupled fragment was found to be indistinguishable from that of the unmodified fragment. The absence of radiocolloids in the injectate was confirmed as was the in vivo stability of the attached DTPA groups. By a variety of techniques, we show that the only significant source of label instability was transcomplexation to circulating transferrin. About 9% per day of label exposed to transferrin (about 1-2% of the injected dose) dissociated with slight bone marrow accumulation. Following i.v. administration, serum activity levels fell rapidly (T 1/2 alpha 2 hr, T 1/2 beta 19 hr). Whole-body clearance of the label was slow (T 1/2 160 hr) and may be attributed entirely to urinary excretion (0.26% of the injected dose per hour). Organ accumulation was greatest in the liver and persisted after rapidly attaining high values (20% of the injected dose). A total of 14 cancer patients were studied, nine with identifiable sites of metastatic disease from colorectal [8], pancreatic [2], ovarian [3], or small cell lung [1] primaries. Eight of the 12 sites of documented tumor were visualized by external imaging (67%) most distinctly at 48-72 hr postadministration.

A long-term study was conducted to determine the possible chronic toxicity and oncogenicity of methylene chloride. Rats and hamsters were exposed by inhalation to 0, 500, 1500, or 3500 ppm of methylene chloride for 6 hr per day, 5 days a week, for 2 years. No exposure-related cytogenetic effects were present in male or female rats exposed to 500, 1500, or 3500 ppm. Females rats exposed to 3500 ppm had an increased mortality rate while female hamsters exposed to 1500 or 3500 ppm had decreased mortality rates. Carboxyhemoglobin values were elevated in rats and hamsters exposed to 500, 1500, or 3500 ppm with the percentage increase in hamsters greater than in rats. Minimal histopathologic effects were present in the livers of rats exposed to 500, 1500, or 3500 ppm. Decreased amyloidosis was observed in the liver and other organs in hamsters exposed to 500, 1500 or 3500 ppm. While the number of female rats with a benign tumor was not increased, the total number of benign mammary tumors was increased in female rats in an exposure-related manner. This effect was also evident in male rats in the 1500- and 3500-ppm exposure groups. Finally, male rats exposed to 1500 or 3500 ppm had an increased number of sarcomas in the ventral neck region located in or around the salivary glands. Therefore, in this 2-year study, some effects were observed in male and female rats exposed to 500, 1500, or 3500 ppm of methylene chloride. In contrast, hamsters exposed to the same exposure concentrations had less extensive spontaneous geriatric changes, decreased mortality (females), and lacked evidence of definite target organ toxicity.

1. The mechanism of inhibition of enzymes by metal ion-chelating reagents is discussed and equations derived. 2. Two distinct mechanisms are postulated and graphical methods are given for differentiating between them. 3. Where the metal ion is actually removed from the enzyme to form a co-ordination complex in solution, a procedure is described for obtaining the stability constant for metal-enzyme interaction, the number of metal ions involved and the stoicheiometry of metal ion-ligand interaction.

In this study carbohydrate-mediated interactions of the envelope glycoprotein, gp120, of HIV-1 were investigated. Oligosaccharide probes (neoglycolipids), prepared from the N-glycosidically-linked chains of the natural and recombinant forms of gp120, were used in conjunction with the intact glycoprotein to investigate reactivities with a soluble carbohydrate-binding protein (lectin) known as mannose-binding protein in human serum. Evidence is presented that the high-mannose-type oligosaccharides with seven, eight and nine mannose residues from both forms of gp120 are recognized by the serum lectin, and that these reactivities are unrelated to CD4 recognition. Reactivities of the two forms of envelope glycoprotein with macrophages derived from human blood monocytes and with the mannose-specific macrophage endocytosis receptor isolated from human placental membranes were also investigated. Evidence is presented that both forms of gp120 bind to the macrophage surface by multiple interactions in addition to CD4 binding, and that among these interactions is a carbohydrate-mediated binding to the endocytosis receptor. We propose that such carbohydrate-mediated interactions could form the basis of viral attachment to a variety of healthy and diseased tissues.

The chemical nature of the important new chromogen ABTS [2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulphonic acid)] is described together with an account of the redox and spectroscopic properties of the system ABTS--H2O2--peroxidase. Keq. is calculated and a study of the steady-state kinetics over a whole range of substrate concentrations is reported. By using novel methods of kinetic analysis, an interpretation of the results is given which requires some extension of the classical peroxidase mechanism.

This paper reports two studies of the expressive and instrumental characteristics of communication associated with the Type A coronary-prone behavior pattern. Using Norton's communicators' style instrument to measure expressive behavior of college students, Study 1 with 50 men and 79 women showed that Type A persons are more dominant, contentious, precise, animated, and dramatic communicators than Type B persons. Study 2 with 48 men and 80 women yielded no differences in the use of persuasive strategies by Type A and Type B college undergraduates.