Mouse monoclonal antibodies with specificity for the melanoma-associated ganglioside disialyllactosylceramide (GD3) also react with the structural analogue disialylparagloboside.

A mouse monoclonal IgM antibody, 4.2, has previously been shown to bind preferentially to the surface of human malignant melanoma cells and to have specificity for the GD3 ganglioside (NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----4GlcCer). Using overlay of antibodies on thin-layer chromatograms with glycolipids of various sources, it was shown that antibody 4.2, a further IgM and two IgG3 mouse monoclonal antibodies, selected on the basis of reactivity with GD3, also bound with similar strength to the structural analogue NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----4GlcNac beta 1----3Gal beta 1----4GlcCer or disialylparagloboside. The SK-MEL 28 melanoma cell line used for immunization was shown to contain a large amount of GD3 but to lack disialylparagloboside. The demonstrated cross-reactivity may be of importance when considering the use of these antibodies for biochemical and medical purposes.

The mono- and difucosyl blood group B glycosphingolipids of rat large intestine differ in type of core saccharide.

Two blood group B-active glycosphingolipids were isolated from rat large intestine and characterized by mass spectrometry, proton NMR spectroscopy and methylation analysis. The following structures were concluded: Gal alpha 1----3(Fuc alpha 1----2)Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc beta 1----1Cer and Gal alpha 1----3(Fuc alpha 1----2)Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----3Gal beta 1----4Glc beta 1----1Cer. The two glycolipids thus differ in their core saccharides (type 1 and type 2 chain, respectively) and therefore must have different pathways for biosynthesis.

An antigen present in rat adenocarcinoma and normal colon non-epithelial stroma is a novel Forssman-like glycolipid based on isoglobotetraosylceramide.

A glycolipid with blood group A activity detected in the non-epithelial stroma of normal rat colon but not in epithelial cells (Hansson, G.C., Karlsson, K.-A., and Thurin, J. (1984) Biochim. Biophys. Acta 792, 281-292), was purified to homogeneity from normal rat colon and rat colon adenocarcinoma. Mass spectrometry and 1H-NMR spectroscopy of the intact permethylated derivative and gas chromatography after degradation revealed the structure GalNAc alpha 1----3GAINAc beta 1----3Gal alpha 1----3Gal beta 1----4Glc beta 1----1Cer, with the predominant ceramide containing sphingosine and non-hydroxylated 24:0 fatty acid. This identifies this glycolipid as a novel Forssman-like glycolipid, which is a tumor-associated antigen by definition, since it is not present in the normal rat large intestinal epithelium cells but in rat adenocarcinoma derived from these cells.

Novel isoglobo-neolacto-series hybrid glycolipid detected by a monoclonal antibody is a rat colon tumor-associated antigen.

Isoglobotetraosylceramide (GalNAc(beta 1-3)Gal(alpha 1-3)Gal(beta 1-4)Glc (beta 1-1)Cer), the major glycolipid species in dimethylhydrazine-induced rat tumors of colorectal origin, was not detected in epithelial cells of normal colon but was present in the non-epithelial stroma and could be extracted from each of nine tumors studied. Monoclonal antibodies produced against isoglobotetraosylceramide detected this and another novel rat tumor-associated glycolipid not present in epithelial cells nor in non-epithelial stroma of normal rat colon (Brodin, T., Thurin, J., Strömberg, N., Karlsson, K.-A. and Sjögren, H.O. (1985) Eur. J. Immunol. 16, 951-956). This novel glycolipid was present in 8/9 of the studied tumors and was also present in two in vitro cell clones. These were originally obtained from a W49/T4 colon tumor isograft. The novel glycolipid was characterized by mass spectrometry, 1H-NMR, and methylation analysis as a hybrid between the isoglobo- and neolacto-series, with the structure GalNAc(beta 1-3)Gal(alpha 1-3)Gal(beta 1-4)GlcNA(beta 1-3)Gal (beta 1-4)Glc(beta 1-1)Cer.

Studies on the binding of bacteria to glycolipids. Two species of Propionibacterium apparently recognize separate epitopes on lactose of lactosylceramide.

Two species of Propionibacterium were analysed regarding their binding to glycosphingolipids. Bacteria were labeled with 125I and selective interaction with glycolipids on thin-layer chromatograms was revealed by autoradiography. The carbohydrate site in common for active molecular species appeared to be lactose. The two bacteria differed, however, in the overall binding pattern on the chromatogram, probably due to recognition of separate epitopes on lactose. P. freudenreichii bound only to lactosylceramide while P. granulosum also recognized substituted lactosylceramide: Gal alpha 1----3Gal beta 1----4Glc beta Cer, GlcNAc beta 1----3Gal beta 1----4Glc beta Cer and Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc beta Cer were active, but Gal-alpha 1----4Gal beta 1----4Glc beta Cer was inactive. Also, there was an interesting dependence on ceramide structure in the case of lactosylceramide. P. freudenreichii bound to lactosylceramide with sphingosine and non-hydroxy fatty acids but not to species with sphingosine and 2-hydroxy fatty acids, phytosphingosine and non-hydroxy fatty acids or phytosphingosine and 2-hydroxy fatty acids. For P. granulosum the situation was reversed. This may be explained by an influence of ceramide structure on the presentation of the two lactose epitopes at the assay surface. These results were supported by curves from the binding of labeled bacteria to glycolipids coated in microtiter wells and in part by binding to glycolipid-coated chicken erythrocytes.

A novel approach to the study of glycolipid receptors for viruses. Binding of Sendai virus to thin-layer chromatograms.

A method for the binding of virus to a silica gel thin-layer chromatogram is presented. After development the chromatogram is overlayed with the 125I-labelled virus and the bound virus is autoradiographed. Alternatively, the unlabelled virus may be detected after exposure to monoclonal antibody and labelled anti-antibody. The Sendai virus strain used did not bind to brain gangliosides earlier proposed to be receptors, but bound to human erythrocyte gangliosides. This finding may be explained by the existence of Sendai virus variants with different receptor specificities.

A novel Ser O-glucuronidation in acidic proline-rich proteins identified by tandem mass spectrometry.

Human acidic proline-rich salivary protein PRP-1 and its C-terminally truncated form PRP-3 were analyzed by electrospray tandem mass spectrometry. Post-translational modifications were detected and characterized. A pyroglutamic acid residue was demonstrated at the N-terminus, Ser-8 and Ser-22 were shown to be phosphorylated and an O-linked glucuronic acid conjugation was identified. The latter modification was located to Ser-17 and found to be present in approximately 40% of the polypeptides.

Glycolipids of human large intestine: difference in glycolipid expression related to anatomical localization, epithelial/non-epithelial tissue and the ABO, Le and Se phenotypes of the donors.

Human large intestine specimens were obtained during elective surgery from donors of known blood group ABO, Lewis and secretor phenotypes. The intestinal epithelial cells were isolated from the non-epithelial tissue in one case and in another case mucosa tissue was obtained by scraping. Total non-acid glycolipid and ganglioside fractions were isolated from the tissue specimens, analyzed by thin-layer chromatography and detected by chemical reagents and autoradiography after staining the plate with various blood group monoclonal antibodies and bacterial toxins. The amount of non-acid glycolipids present in the large intestine epithelial cells was 3.9 micrograms/mg of cell protein and in the non-epithelial tissue 0.39 mg/g dry tissue weight. The epithelial cells contained monoglycosylceramides and blood group Lea pentaglycosylceramides as major compounds together with small amounts of diglycosylceramides. In addition, trace amounts of tri- and tetra-glycosylceramides together with more complex glycolipids were present. The non-epithelial tissue contained mono-, di-, tri- and tetra-glycosylceramides as major non-acid components. Blood group ABH glycolipids were present in trace amounts in the non-epithelial part of the large intestine. Lea pentaglycosylceramide was the major blood group glycolipid present in all Le-positive individuals independent of the secretor status. Leb glycolipids were present in trace amounts in secretor individuals but completely lacking in non-secretors. Trace amounts of X antigens were found in all individuals, while Y antigens were only present in secretor individuals. The Lea, Leb, X and Y glycolipids were located in the epithelial cells. The gangliosides were present mainly in the non-epithelial tissue (65-350 nmol of sialic acid/g dry weight) and only trace amounts (less than 0.014 nmol/mg of cell protein) were found in the epithelial cells. The major gangliosides of the non-epithelial tissue were identified as GM3, GM1, GD3, GD1b, GT1b and GQ1b. In addition, several minor gangliosides were also present. Binding of cholera toxin to the thin-layer plate revealed trace amounts of the GM1 ganglioside in the epithelial cell ganglioside fraction.

Gln-Gly cleavage: correlation between collision-induced dissociation and biological degradation.

Tryptic digestion of the 150-residue human acidic salivary proline-rich protein 1 (PRP-1) generated eight peptides, two of which corresponded to the N-terminal 30-residue segment. In each of the other six tryptic peptides, a consensus repeat with the structure PQGPPQQGG was present. A facile Gln-Gly cleavage between the second and the third residues of the repeat was observed during collision-induced dissociation experiments. We postulate possible mechanisms to account for this reactivity, involving attack on the peptidyl carbonyl group by the Gln sidechain. Significantly, the Gln-Gly cleavage has been shown to be biologically important in the bacterial degradation of PRPs in saliva, generating bacteria-binding Pro-Gln C-termini. We suggest a link between the gas-phase chemistry and the biochemical degradation of these molecules.

Evaluation of various models for respiratory inductance plethysmography calibration.

We evaluated one nonlinear and two linear models of the ventilatory system while calibrating the respiratory inductance plethysmograph (RIP) against a pneumotachometer. A calibration method involving voluntary varying rib cage and abdominal contributions to tidal volume in a single body position was utilized. The influence on accuracy of the choice of respiratory phase during calibration was assessed. Both tidal and intratidal volumes were evaluated. Ten adults with no history of respiratory disorders went through RIP calibration and validation in the sitting and supine positions. A linear calibration model, relating lung volume changes from the start of inspiration or expiration to rib cage and abdominal excursions from initiation of respiratory motion, had the best accuracy. The choice of respiratory phase for calibration did not affect accuracy. RIP generally underestimated lung volume at the start of inspiration and overestimated lung volume at the end of inspiration. RIP was more accurate in the supine than the sitting position, probably because of limited spine flexion in the supine position.

Agglutinin and acidic proline-rich protein receptor patterns may modulate bacterial adherence and colonization on tooth surfaces.

Bacterial binding to salivary proteins may in part account for individual differences in the colonization of tooth surfaces. High-molecular-weight glycoproteins, agglutinins, mediate S. mutans adherence, whereas acidic proline-rich proteins mediate adherence of other early-colonizing streptococci and Actinomyces. The aim of the present study was to examine the composition of adherence-related salivary proteins and dental plaque micro-organisms in three individuals with a low, moderate, and high capacity to mediate S. mutans adherence. The S. mutans (strain Ingbritt) binding activity resided with a 300-kDa agglutinin which was six-fold more prevalent in the high S. mutans binding saliva compared with the low one. Binding to all three salivas was completely blocked by a monoclonal anti-agglutinin antibody. The moderate S. mutans binding saliva was found to contain adherence-inhibiting components. Furthermore, the low and moderate S. mutans binding salivas mediated binding of A. naeslundii strain LY7 to a greater extent than the saliva with high S. mutans binding. The A. naeslundii binding activity resided with the acidic proline-rich proteins (APRPs) and paralleled the relative content of 106- and 150-residue APRPs. Low A. naeslundii binding coincided with an almost two-fold higher ratio of 106/150 APRPs compared with the high A. naeslundii binding saliva. During conventional gel filtration, a degradation of the acidic, basic, and glycosylated proline-rich proteins was evident in the saliva with high S. mutans and low A. naeslundii binding. This saliva donor had a comparably high rate of dental plaque formation, high counts of S. mutans, and low counts of other streptococci and Actinomyces.

The association of bacterial adhesion with dental caries.

Saliva adhesion of bacteria is a key event in oral biofilm formation. Here, we used partial least-squares (PLS) analysis to correlate adhesion of cariogenic (Streptococcus mutans Ingbritt) and commensal (Actinomyces naeslundii LY7) model bacteria, and their agglutinin and acidic proline-rich protein ligands, respectively, with high and low caries experiences in 38 children reflecting today's skewed caries distribution. Adhesion of S. mutans was among the factors correlating strongest with high caries experience when PLS modeled together with traditional factors (e.g., sugar intake, lactobacilli counts). Saliva phenotypes with high agglutinin levels and Db-s (an acidic PRP variant) coincided with both high caries experience and S. mutans adhesion. A. naeslundii adhesion correlated with low caries experience. Non-Db phenotypes (i.e., acidic PRP-1 and PRP-2 variants) coincided with both low caries experience and S. mutans, but high A. naeslundii, adhesion. Thus, bacterial adhesion may modulate susceptibility and resistance to dental caries.

Ventilation inhomogeneity assessed by nitrogen washout and ventilation-perfusion mismatch by capnography in stable and induced airway obstruction.

Few studies have been published on gas distribution in the lung during acute and stable airway obstruction in children. Multiple breath nitrogen (N(2)) washout is an established method for assessing ventilation inhomogeneity, while the tidal breathing capnogram may be used as an indicator of ventilation-perfusion (V(')(A)/Q) mismatch. We hypothesized that significant V(')(A)/Q mismatch is not seen in stable airway obstruction unless obstruction is severe, and that stable and induced airway obstruction of similar severity would result in different degrees of V(')(A)/Q mismatch. To test this hypothesis, we performed spirometry measurements of forced expiratory volume in 1 sec (FEV(1)), multiple breath N(2) washout, and tidal breathing capnography in 11 young patients (9-30 years) with cystic fibrosis, 37 asthmatic patients (8-18 years), and 34 healthy subjects (7-20 years). Lung function was measured at rest, after airway obstruction induced by cold dry air hyperventilation or methacholine challenge, and after beta(2)-agonist treatment. V(')(A)/Q mismatch was assessed from the slopes of the phases II and III of the capnogram. We observed a normal capnogram during stable obstruction of moderate severity despite significant ventilation inhomogeneity. In patients with severe stable obstruction and in those with induced airway obstruction significant ventilation inhomogeneity and pathological capnograms were seen. Induced airway obstruction, resulted in a more pathological capnogram than stable obstruction of similar severity. beta(2)-agonist treatment reduced ventilation inhomogeneity, but did not improve the capnogram. Our findings are compatible with the presence of an efficient pulmonary blood flow regulatory mechanism that adequately compensates for chronic ventilation inhomogeneity of moderate severity, but not for severe or sudden airway obstruction.

Breathing pattern variability during bronchial histamine and methacholine challenges in asthmatics.

Breathing pattern variability was determined in 10 asthmatic adolescents during repeated bronchial histamine and methacholine challenges (HiCh/MeCh). The purpose was to provide information on ventilatory control in asthmatics by comparing the variability of the various breathing pattern parameters at rest and during induced bronchial obstruction. Changes in variability during bronchial obstruction might be explained by either anxiety effects causing increased variability or by the minimization of the work of breathing causing decreased variability. Ventilation was monitored by respiratory inductive plethysmography in order to minimize the effects on the spontaneous pattern of breathing. Breath-to-breath and day-to-day variability were determined concerning respiratory frequency (fR), inspiratory tidal volume (VTI), inspiratory ventilation (V'I), inspiratory time to total cycle time ratio (TI/TTOT), mean inspiratory flow (VTI/TI, an index of ventilatory drive), rib cage fraction of VTI (VRC/VTI), and maximum compartmental amplitude to VTI ratio (MCA/VTI; an index of rib cage and abdominal phasing). No difference in any parameter was found regarding breath-to-breath coefficient of variation (CV = SD/mean) between recordings at baseline, after saline inhalation and after threshold dose of the provocative agents, i.e. > 20% fall in FEV1. Variability was less for MCA/VTI and VRC/VTI (mean CV 1.3 and 7.7%, respectively) than for TI/TTOT, fR, VTI/TI, VTI, and V'I (14.2, 15.8, 20.9, 22.2 and 21.1%, respectively) (P < 0.01). Likewise, the day-to-day variability did not differ in any parameter between recordings at baseline, after saline inhalation and after threshold dose. The variability was less for MCA/VTI (0.7%) than for TI/TTOT, VRC/VTI, V'I, VTI/TI, fR and VTI (7.1, 12.1, 12.8, 14.2, 13.0 and 15.4%) (P < 0.05). Furthermore, TI/TTOT was less variable than VTI (P < 0.05). Thus, the ventilatory pattern was quite reproducible on a day-to-day basis, despite considerable breath-to-breath variability. Ventilatory drive and tidal volumes were more variable than the rib cage and abdominal phasing, the respiratory timing and the rib cage fraction of tidal volume. The lack of difference in variability between rest and induced bronchial obstruction indicates that other factors than anxiety or minimization of the work of breathing are important for the control of respiration in asthmatics during bronchial challenge.

Hyperventilation during bronchial challenges in asthmatics: reproducibility and assessment of contributing factors.

Among asthmatics, the ventilatory response is heterogeneous during bronchial challenge. This study aimed to investigate the reproducibility of the response and to assess possible causes for hyperventilation. Repeated bronchial histamine and methacholine challenges (HiCh/MeCh) were performed in 10 asthmatic adolescents. Ventilation was monitored by respiratory inductive plethysmography (RIP), in order to minimally affect the spontaneous breathing pattern. FEV1 and the volume of trapped gas (measured as the volume of air mobilized by five maximal breaths after a multiple breath nitrogen washout to 2% N2), were used to assess mainly central and peripheral airways obstruction, respectively. When FEV1 had decreased by at least 20%, mean inspiratory flow (VTI/TI) increased by 21% and minute ventilation (V'I) by 21% and 23% during HiCh and MeCh, respectively (both P < 0.05). No correlation was found between the magnitude of the ventilatory response and either: the degree of FEV1 decline, the increase in gas trapping, SaO2 decline or the increase in dyspnoea score. Histamine challenge after beta 2-agonist pre-treatment was associated with increased ventilatory drive in one patient despite the absence of bronchial obstruction, indicating that histamine might directly stimulate afferent airway nerves which cause hyperventilation. The intra-individual variability of the ventilatory response (increase in V'I and VTI/TI) was more than 100% of the mean ventilatory response, while the variability of the bronchomotor response was about 25% of the mean bronchomotor response. Thus, during induced bronchial obstruction in asthmatics, the occurrence of hyperventilation and its intensity are not related to either the degree of central or peripheral airways obstruction, or to the degree of dyspnoea. The reproducibility of the ventilatory response is poor. The ventilatory response appears to be the result of a complex interaction between several afferent stimuli and central ventilatory control.

Structures of the eight- to nine-sugar glycolipids of human blood group A erythrocytes.

Two glycolipid fractions, isolated in 1975 from blood group A1 erythrocytes and shown on the basis of direct-inlet mass spectrometry to contain eight- and nine-sugar A-type sequences, have been reinvestigated by fast-atom-bombardment mass spectrometry and overlay analysis with selected monoclonal anti-A antibodies. The presence of three separate glycolipids was concluded, consistent with a common paragloboside backbone [beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-D-Glc] and a typical erythrocyte ceramide component (sphingosine, and 22-, 23-, 24-, and 25-carbon nonhydroxy fatty acids). It is proposed that they carry A determinants based on Type 1 [beta-D-Galp-(1----3)-beta-D-GlcpNAc], Type 2 [beta-D-Galp-(1----4)-beta-D-GlcpNAc], and Type 3 [beta-D-Galp-(1----3)-alpha-D-GalpNAc] chains, respectively. The Type 1 (eight sugars) and Type 3 (nine sugars) glycolipids appeared in mixtures of both the native and the acetylated form. The existence of Type 1 glycolipid, which appears to be a genuine erythrocyte glycolipid as concluded from the ceramide composition, had been predicted earlier by other workers.

Carbohydrates as recognition molecules for bacterial adhesins: methodology and characteristics.

Future attempts at developing inhibitors of dental plaque formation necessitate characterization of the bacterial-host, as well as the inter-bacterial recognition processes. Bacterial binding to a panel of solid-phase reference glycolipids was used to reveal the recognition of internal receptor sequences, low-affinity cooperative interactions, and adhesin variants with slightly shifted receptor epitopes. This epitopic variation may be a mechanism for shifting the host and tissue tropism of the bacteria, and may have evolved in response to the topography and expression of receptor epitopes at the host tissue surfaces.

Studies on human minor salivary gland secretions using the Periotron method.

Secretions from minor salivary glands were estimated in 127 individuals by the Periotron method of measuring fluid output from different mucosal sites, and outputs were related to different variables. Large intra- and interindividual variations in secretions (expressed as microliter/cm2 per min) were observed, with means of 0.9 for the palatal, 4.8 for the labial and 16.0 for the buccal mucosal sites. Age had no influence on the secretion rate, but women had 10-20% lower values from all three sites than men (p < 0.05). Individuals wearing upper dentures or using tobacco had 300 and 27% increased palatal secretion rates, respectively (p < 0.001, p < 0.05). In addition, those being treated with diuretics had 15% lower rates of secretion from buccal mucosal glands (p < 0.05), and those complaining of oral dryness had 21% lower fluid output from the labial mucosa (p < 0.05). These results support the use of minor salivary glands in combination with the Periotron method to study mucosal secretions and functions.

Interaction of viruses, bacteria and bacterial toxins with host cell surface glycolipids. Aspects on receptor identification and dissection of binding epitopes.

An overview and perspective is presented on animal cell surface carbohydrate (primarily lipid-linked oligosaccharides) as specific receptors for viruses, bacteria and bacterial toxins. Although carbohydrate has been known for many years to be specific attachment sites for these ligands, it is only in very recent time that carbohydrate technology and receptor assays in combination afford a rational approach. One generalization from present experience is the property of microbiological ligands to recognize sequences placed internally in an oligosaccharide chain which differs from antibody recognition of short sequences which most often involves terminally placed determinants. This is of both biological and technical importance. Biologically it may assure attachment by avoiding differences between host individuals often residing in terminal parts (e.g. blood group determinants), and may also make a shift of target cells by mutations more efficient. Technically this property is an important help when dissecting narrow binding epitopes, and for disclosing receptor-binding variants with only slight differences in binding epitopes (e.g. different epitopes on the same disaccharide). Such variants representing a kind of "epitope drift" are probably a consequence of point mutations in the binding site of the lectin-like proteins to select a proper host environment. Current technology allows an efficient screening for carbohydrate receptors with interesting consequences for applications within medicine (diagnosis and therapy) and biotechnology.

Error analysis of a natural breathing calibration method for respiratory inductive plethysmography.

Respiratory volumes are measured non-invasively by recording rib cage and abdominal motions using respiratory inductive plethysmography (RIP). Qualitative diagnostic calibration (QDC) of RIP is based on the natural variability in the relative rib-cage-to-abdomen contribution during tidal breathing. ODC does not require subject cooperation but it has previously been shown that accuracy may deteriorate when breathing pattern changes. The aim of this study was to investigate the causes and situations where QDC accuracy deteriorates. The QDC method was compared to PRA (calibration during voluntarily preferential rib cage or abdomen breathing) in ten adults. A reference RIP calibration was obtained from all validation data (REF). The PRA method had better accuracy than the ODC method (p<0.01). The volumetric error ranged between 10% and 136% with QDC and between 5% and 33% with PRA. The PRA calibration factors were within 6% of those from REF, while the QDC rib-cage factor was underestimated by 15% and the abdominal factor was overestimated by 38%. Small natural variability in the relative rib-cage-to-abdomen contribution was related to poor accuracy. Each compartment's variability depended on its magnitude, which is a violation of the QDC assumptions.