P- and E-selectin use common sites for carbohydrate ligand recognition and cell adhesion.

The selectins are a family of three calcium-dependent lectins that mediate adhesive interactions between leukocytes and the endothelium during normal and abnormal inflammatory episodes. Previous work has implicated the carbohydrate sialyl Lewis(x) (sLe(x); sialic acid alpha 2-3 galactose beta 1-4 [Fucose alpha 1-3] N-acetyl glucosamine) as a component of the ligand recognized by E- and P-selectin. In the case of P-selectin, other components of the cell surface, including 2'6-linked sialic acid and sulfatide (galactose-4-sulfate ceramide), have also been proposed for adhesion mediated by this selectin. We have recently defined a region of the E-selectin lectin domain that appears to be directly involved with carbohydrate recognition and cell adhesion (Erbe, D. V., B. A. Wolitzky, L. G. Presta, C. R. Norton, R. J. Ramos, D. K. Burns, R. M. Rumberger, B. N. N. Rao, C. Foxall, B. K. Brandley, and L. A. Lasky. 1992. J. Cell Biol. 119:215-227). Here we describe a similar analysis of the P-selectin lectin domain which demonstrates that a homologous region of this glycoprotein's lectin motif is involved with carbohydrate recognition and cell binding. In addition, we present evidence that is inconsistent with a biological role for either 2'6-linked sialic acid or sulfatide in P-selectin-mediated adhesion. These results suggest that a common region of the E- and P-selectin lectin domains appears to mediate carbohydrate recognition and cell adhesion.

The three members of the selectin receptor family recognize a common carbohydrate epitope, the sialyl Lewis(x) oligosaccharide.

The selectins (lectin-EGF-complement binding-cell adhesion molecules [LEC-CAMs]) are a family of mammalian receptors implicated in the initial interactions between leukocytes and vascular endothelia, leading to lymphocyte homing, platelet binding, and neutrophil extravasation. The three known selectins, L-selectin (leukocyte adhesion molecule-1 [LECAM-1]), E-selectin (endothelial-leukocyte adhesion molecule-1 [ELAM-1]), and P-selectin (GMP-140) share structural features that include a calcium-dependent lectin domain. The sialyl Lewis(x) carbohydrate epitope has been reported as a ligand for both E- and P-selectins. Although L-selectin has been demonstrated to bind to carbohydrates, structural features of potential mammalian carbohydrate ligand(s) have not been well defined. Using an ELISA developed with a sialyl Lewis(x)-containing glycolipid and an E-selectin-IgG chimera, we have demonstrated the direct binding of the L-selectin-IgG chimera to sialyl Lewis(x). This recognition was calcium dependent, and could be blocked by Mel-14 antibody but not by other antibodies. Recognition was confirmed by the ability of cells expressing the native L-selectin to adhere to immobilized sialyl Lewis(x). These data suggest that the sialyl Lewis(x) oligosaccharide may form the basis of a recognition domain common to all three selectins.

Identification of an E-selectin region critical for carbohydrate recognition and cell adhesion.

E-selectin elicits cell adhesion by binding to the cell surface carbohydrate, sialyl Lewis X (sLe(x)). We evaluated the effects of mutations in the E-selectin lectin domain on the binding of a panel of anti-E-selectin mAbs and on the recognition of immobilized sLe(x) glycolipid. Functional residues were then superimposed onto a three-dimensional model of the E-selectin lectin domain. This analysis demonstrated that the epitopes recognized by blocking mAbs map to a patch near the antiparallel beta sheet derived from the NH2 and COOH termini of the lectin domain and two adjacent loops. Mutations that affect sLe(x) binding map to this same region. These results thus define a small region of the E-selectin lectin domain that is critical for carbohydrate recognition.

Changes in creatine kinase and its isoenzymes in human fetal muscle during development.

The total creatine kinase activity and its isoenzyme patterns were investigated in the skeletal muscle of 87 fetuses, of which 80 were presumed normal, 5 were anencephalic and 2 were "at risk" for Duchenne muscular dystrophy (DMD). No differences, either in total enzyme activity or in the isoenzyme distributions, were found between the anencephalic fetuses or those at risk for DMD, when compared to normal fetuses of similar gestation. Creatine kinase activity was found to rise steadily throughout embryonic life. During fetal development, the isoenzyme pattern in skeletal muscle was observed to change from the initial prevalence of the brain (BB) type, to the predominance of the muscle (MM) form. The most pronounced change occurred between the 6th and the 16th week of gestation, a period characterized by the rapid fusion of myoblasts to form myotubes and the concomitant production of myofibrils. It is proposed that there is a close association between the creatine kinase isoenzymes spectrum and the stage of muscle development.

Validation of a congener specific method for ortho and non-ortho substituted polychlorinated biphenyls in fruit and vegetable samples.

An on-line procedure has been developed and validated for the clean-up and fractionation of ortho and non-ortho-PCBs in fruit and vegetable samples. The procedure combines silica/acid and carbon/glass fibre columns with gas chromatography-mass spectrometry. Chromatography on carbon/glass fibre allowed collection of mono-ortho/di-ortho and non-ortho-PCB fractions, which were determined separately by GC-MS. The method was validated by replicate analyses and by inter-laboratory comparison of data for PCB congeners determined in fruit and vegetable samples collected in South Wales. The concurrent determination of ortho and non-ortho substituted PCBs is reported with recoveries ranging from 55-95% and a mean intra-laboratory precision (%COV) of 9.5% for apple extracts.

PCB and PCDD/DF concentrations in egg and poultry meat samples from known urban and rural locations in Wales and England.

A survey was undertaken of PCB and PCDD/DF congeners in eggs and poultry meat from a smallholding close to a chemical waste incinerator, other sites in the surrounding district, and three rural locations. The concentrations from the site close to the incinerator were appreciably greater than those found elsewhere, although the contrast was less marked for poultry meat than eggs. All types of poultry produce displayed noticeable variations in congener composition when the samples were grouped according to geographical origin. These results support the view that the environment in which poultry live does influence the PCB and PCDD/DF characteristics of their products. Exposure calculations indicated that consumption of eggs from the site close to the incinerator would constitute a substantial proportion of recommended daily intakes for such contaminants and at the present time these products are not being eaten.

PCB and PCDD/DF congeners in locally grown fruit and vegetable samples in Wales and England.

A survey was undertaken of PCB and PCDD/DF congeners in fruit and vegetables grown in an urban areas close to a chemical waste incinerator and three rural locations. All of the concentrations detected were low and there was considerable overlap between those found in urban and rural samples. Some similarities with the congener composition of air samples were identified and concentrations in apple skin were noticeably higher than those in the flesh of the fruit. These results suggest that atmospheric deposition was an important contamination pathway. Assessments using the highest concentrations found indicated that consumption of such fruit and vegetables would represent an additional 3% of the normal dietary intake for PCBs and 8% for PCDD/DFs.

Transfer and uptake of polychlorinated dibenzo-p-dioxins and furans (PCDD/Fs) and polychlorinated biphenyls (PCBs) into meat and organs of indoor and outdoor reared pigs.

This study was designed to investigate how and to what extent PCDD/Fs and PCBs are transmitted from exposure sources to porcine muscle and other tissues derived from pigs. The experimental approach involved two longitudinal studies in which indoor and outdoor pigs were reared to market readiness using typical animal husbandry practices; closely matched samples of soil, feed, bedding, meat, etc. were collected and analysed for PCDD/Fs and PCBs. The total PCDD/F + PCB WHO-TEQs in pig liver were much higher than in meat and kidney samples from the same animals and exceeded the current relevant European Union maximum limits (6 ng PCDD/F-TEQ kg⁻¹ fat). Liver samples were also characterised by much lower PCB contributions to the total TEQ than for the corresponding meat and kidney samples, and by a predominance of many of the hepta- and octa-substituted PCDD/Fs. At ages approaching market readiness, TEQ values in meat samples from outdoor pigs tended to be slightly higher than those from comparable ages in the indoor programme, possibly due to additional intake from soil. Biotransfer factors (BTFs) were derived for each of the 39 PCDD/F and PCB congeners measured. Interpretation of the findings focused particularly on trends in four selected congeners, namely: 2,3,7,8-TCDD, 2,3,4,7,8-PeCDF, PCB 153 and PCB 169. Increases in the BTF for PCB 169 in the pig-rearing programmes were noticed when the diet changed from being dominated by sow's milk to feed. Much higher transfer factors for many of the more heavily chlorinated PCDD/Fs (e.g. 2,3,4,7,8-PeCDF) were found in liver compared with meat or kidney samples from the same animals. Soil consistently accounted for at least 30% of input for many hexa- or higher chlorinated PCDD/Fs, while it rarely representing more than 10% of the total intake.

The assimilation of dioxins and PCBs in conventionally reared farm animals: occurrence and biotransfer factors.

The assimilation of PCDD/Fs and PCBs in chickens, pigs and sheep was investigated in studies using conventional animal husbandry practices. Closely matched samples of muscle (meat), liver, kidneys, eggs, milk, feed, soil and grass were collected of which 105 were analysed. The data obtained were consistent with the PCB and PCDD/F TEQ concentrations to be expected in rural background locations. A slight decline in TEQ values in meat with increasing age was evident in pigs, sheep and broiler chickens. Higher TEQ values in meat from outdoor pigs compared to those raised indoors, and an increase in TEQs in eggs as a result of free-range conditions might be attributable to additional contaminant intakes from soil. TEQ values in samples of sheep meat were slightly higher than those for pigs and chickens and market ready lowland sheep showed higher meat TEQs than the highland species. PCDD/F TEQs were considerably higher in the liver than meat. Contaminant transfer from dietary sources was investigated using biotransfer factors (BTFs) which tended to be higher in chickens than in sheep or pigs. BTFs for all animals declined in magnitude with age, but on average, BTFs for pigs and chickens showed a sharper initial decline than for sheep. The relative magnitude of the BTFs usually followed the order: (highest first) PCB 153, PCB 169, PCB 126, 1,2,3,7,8-PeCDD/2,3,4,7,8-PeCDF and 2,3,7,8-TCDD. This may suggest that higher chlorinated congeners accumulate more readily in meat tissues. Congener-specific BTF variations were found to be associated with variables such as dietary composition during rearing, differences between feed and animal species.

An enzyme-linked immunosorbent assay using biotinylated heparan sulfate to evaluate the interactions of heparin-like molecules and basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) is a member of the heparin-binding growth factor family that interacts with cell surface heparan sulfate (HS) proteoglycans and extracellular matrix heparin. Here we report the development of a simple and sensitive assay that used biotinylated HS or heparin to bind to bFGF coated onto 96-well microtiter plates. Bound labeled HS or heparin was reacted with enzyme-linked streptavidin and results were recorded as optical density. Increased molar excess of biotin resulted in increased incorporation of biotin and higher signal without compromising binding. Glycosaminoglycans and modified heparins were assayed for their ability to compete with biotinylated HS for binding to bFGF. Inhibition of that binding by heparin and HS but not by chondroitin sulfate A or C, dermatan sulfate, or keratan sulfate demonstrated the specificity of the glycosaminoglycan binding. Structural modifications of heparin produced various degrees of inhibition with high structural specificity. Although removal of N-sulfates or 2,3-O-sulfate groups resulted in significant loss of inhibition, removal of 6-O-sulfates had little affect on binding. Carboxyl reduction or N-acetylation following N-desulfation produced heparinoids with moderate changes in binding capacity. Results from this assay are in agreement with previous data from our laboratory and reports from other researchers with respect to the specificity of glycosaminoglycan binding to bFGF and the role of 2,3-O- and 6-O-sulfate groups of heparin. The flexibility of this assay, in both the amount of label incorporated and the variability of solid substrate, makes this an excellent tool to study other heparin binding proteins.

Phase I study of a murine monoclonal anti-lipid A antibody in bacteremic and nonbacteremic patients.

Nine patients with suspected gram-negative bacterial sepsis were studied to determine the safety, pharmacokinetics, and immunogenicity of XMMEN-0E5, a murine immunoglobulin M monoclonal antibody directed against the core lipid A region of bacterial endotoxin. Antibody was administered by single intravenous infusion of 1 to 4 h duration at doses ranging from 0.1 to 15 mg/kg. Five patients had positive blood cultures for gram-negative bacteria, one patient had Torulopsis septicemia, one patient had gram-negative bacterial meningitis, and two patients were culture negative. No evidence of antibody-mediated toxicity was observed at any dose level. The serum half-life of the antibody was approximately 10 h at doses of 0.1 to 7.5 mg/kg and approximately 18 h at a dose of 15 mg/kg. No apparent difference in clearance of antibody was observed between bacteremic and nonbacteremic patients. Human anti-mouse antibodies were detected in the sera of three evaluable patients that received doses equal to or greater than 2.0 mg/kg but not in patients that received lower doses of antibody. This study demonstrates that XMMEN-0E5 is well tolerated at doses from 0.1 to 15 mg/kg and may be immunogenic at doses of 2.0 mg/kg and above. Controlled trials to establish the efficacy of this antibody in the treatment of gram-negative bacteremia are indicated.

Structure-function studies on selectin carbohydrate ligands. Modifications to fucose, sialic acid and sulphate as a sialic acid replacement.

The selectins are a family of carbohydrate-binding proteins that have been implicated in the initial interaction between leukocytes and the vascular endothelium. The three members of this family will bind to the sialyl-Lewisx epitope [Sia alpha 2-3 Gal beta 1-4 (Fuc alpha 1-3) GlcNAc] and related oligosaccharides. In this report, we examine the molecular details of that recognition using synthesized carbohydrates with specific modifications on the sialyl-Lewisx epitope. E- and L-Selectin require hydroxyl groups at the 2, 3 and 4 positions of the fucose residue. P-Selectin, however, requires only the 3-position hydroxyl group, while tolerating removal of the oxygen at positions 2 or 4 of fucose residue. Modifications of the glycerol side chain or the N-acetyl group of the sialic acid have little effect on the binding of any of the selectins. All three selectins bind efficiently to an oligosaccharide with a sulphate replacement for the sialic acid [sulpho-Lewisx, or SO4-3Gal beta 1-4 (Fuc alpha 1-3) Glc-ceramide]. For E-Selectin, binding to sulpho-Lewisx appears to be equivalent to binding to sialyl-Lewisx, while for L- and P-Selectin binding to the sulphated structure shows characteristics distinct from sialyl-Lewisx recognition. Taken together, these data indicate that, while all three selectins can recognize sialyl-Lewisx, E-, L- and P-Selectin each display distinct carbohydrate ligand preferences.

Human antibody responses to components of the monoclonal antimelanoma antibody ricin A chain immunotoxin XomaZyme-MEL.

Human antibody responses to immunotoxin components were evaluated in 21 melanoma patients who were treated with XomaZyme-MEL, a murine monoclonal antimelanoma antibody-ricin A chain conjugate. Twenty of the 21 melanoma patients produced antibodies against ricin A chain, while 15 of 21 produced antibodies reactive with the murine monoclonal antibody component. Both IgM and IgG antibody responses were produced. Immunoglobulin responses were usually detected 1 to 2 weeks following initiation of therapy, with peak levels generally attained 2 to 4 weeks posttherapy. Titers of the anti-ricin A chain antibodies were generally higher than those of the antimurine monoclonal antibodies for the dose range tested. There was no clear correlation between the dose of immunotoxin administered and the antibody titer. By use of a competitive flow cytometry assay, antiidiotype responses were demonstrated in eight of 10 melanoma patients who had antimurine antibodies. Both the kinetics of appearance and the relative titers of the antiidiotype responses generally corresponded to the antimurine responses. The development of antimmunotoxin antibodies can reduce the therapeutic potential of immunotoxins through several mechanisms. The development of antibodies in a significant number of patients suggests that optimally effective, repeated courses of therapy will require some procedure for suppressing or abrogating the response against the immunotoxin.

The effects of cyclophosphamide on the toxicity and immunogenicity of ricin A chain immunotoxin in rats.

We conducted a study to determine if treatment with cyclophosphamide (CY) could suppress the formation of anti-murine and anti-ricin A chain antibodies in rats treated with a murine monoclonal antibody-ricin A chain immunotoxin (IT). Female Sprague-Dawley rats received intravenous doses of IT at a dose of 5 mg/kg body weight alone or in combination with CY at a dose level of either 10 or 20 mg/kg body weight. The IT was given as one or two courses consisting of five consecutive daily intravenous injections (days 0 to 4, or days 0 to 4 and days 21 to 25 of the study). Cyclophosphamide was given on days 2, 4, 6, 13, and 17 of the study to the group receiving a single course of IT; additional doses of CY were administered on days 23, 25, and 27 to the group receiving two courses of IT. On days 4, 14, 21, 28, and 35, animals from each group were evaluated for antibodies to murine IgG and ricin A chain, and for clinical laboratory parameters and histopathology. Animals receiving IT alone developed significant titers of both anti-murine and anti-ricin A chain antibodies. Compared with the response in the animals receiving single-course IT, the response to both of the components of the IT was significantly increased on days 28 and 35 in the animals receiving a second course of IT. The groups receiving a combination of either one or two courses of CY and IT demonstrated a significantly decreased antibody response to both the murine IgG and the ricin A chain compared with the group receiving IT alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural requirements for the carbohydrate ligand of E-selectin.

The acute inflammatory response requires that circulating leukocytes adhere to, and then migrate through, the vascular wall at the site of injury or infection. Several receptors have been implicated in this adhesion and migration process, including the selectins, a family of carbohydrate-binding proteins. The ligand for one of these proteins, E-selectin (LECAM-2, ELAM-1) has been described by several groups to contain a polylactosamine structure bearing a terminal sialic acid residue and at least one fucose residue. We report here a more detailed investigation into the minimum structural requirements for carbohydrate recognition by E-selectin. Using both direct binding and inhibition studies we demonstrate that the sialyl Lewisx tetrasaccharides Sia(alpha 2-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc, and Sia(alpha 2-3)Gal(beta 1-4)[Fuc(alpha 1-3)]Glc are the smallest oligosaccharides recognized by the lectin. In addition, an oligosaccharide containing the sialyl Lewisa epitope is also recognized, but less avidly. We propose a structural model of functional groups necessary for recognition by E-selectin, based on these data and additional experiments on modifications of sialic acid and the reducing terminal saccharide.

Sulfated malto-oligosaccharides bind to basic FGF, inhibit endothelial cell proliferation, and disrupt endothelial cell tube formation.

The interaction of basic FGF (bFGF) with heparin, heparan sulfate and related sugars can potentiate or antagonize bFGF activity, depending on the size of the saccharide used. Oligosaccharides based on heparin structures, as small as six sugar residues, have been demonstrated to bind to bFGF and block its activity, while larger structures (> 10 sugar residues) tend to potentiate bFGF. In this study we have synthesized a series of compounds designed to test the requirements of size and sulfation for binding of oligosaccharides to bFGF. These oligosaccharides are not derived from heparin, but rather, are linear chains of glucose linked alpha 1-4 (malto-oligosaccharides) that have been chemically sulfated. In addition to bFGF binding, these compounds were tested for their ability to block basic functions of endothelial cells that are known to be mediated, at least in part, by bFGF. We report that the ability of sulfated malto-oligosaccharides to block binding of bFGF to heparan sulfate was dependent on the size (at least a tetrasaccharide is required), and the degree of sulfation. The activity profile in the bFGF ELISA closely correlated with the ability of these compounds to block REEC or HMVEC tube formation on Matrigel. There was a similar relationship of size and sulfation to the ability of the sulfated malto-oligosaccharides to inhibit endothelial cell growth for most human and rat EC types tested. The single exception was REEC cell growth. One isolate of these cells was stimulated by sulfated malto-oligosaccharides rather than inhibited by them, while a second isolate was neither stimulated nor inhibited. This stimulation showed no correlation with inhibition of bFGF binding in the ELISA assay, suggesting that growth of this cell type was probably not dependent on bFGF. Compounds derived from this series of sulfated, malto-oligosaccharides have the potential to function as bFGF antagonists, are relatively easy to produce, and possess relatively low anticoagulant properties.

Sialyl Lewis X mimics derived from a pharmacophore search are selectin inhibitors with anti-inflammatory activity.

The selectins, a family of adhesion receptors involved in leukocyte extravasation, recognize sialyl Lewis X (sLe(x); NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc) and related oligosaccharides. We used conformational energy computations, high field NMR, and structure-function studies to define distance parameters of critical functional groups of sLe(x). This sLe(x) pharmacophore was used to search a three-dimensional data base of chemical structures. Compounds that had a similar spatial relationship of functional groups were tested as inhibitors of selectin binding. Glycyrrhizin, a triterpene glycoside, was identified and found to block selectin binding to sLe(x) in vitro. We substituted different sugars for the glucuronic acids of glycyrrhizin and found the L-fucose derivative to be the most active in vitro and in vivo. A C-fucoside derivative, synthesized on a linker designed for stability and to more closely approximate the original sLe(x) pharmacophore, resulted in an easily synthesized, effective selectin blocker with anti-inflammatory activity.