[New directions for psychotherapy research: an evaluation of a research protocol and a methodological and technical framework proposal]. / Nouvelles perspectives pour la recherche en psychothérapie: évaluation d'un protocole de recherche et proposition d'un dispositif méthodologique et technique.

INTRODUCTION: A recent report of the French research institute INSERM, based on a comprehensive review of the work done on the evaluation of psychotherapies Psychothérapies: trois approches évaluées, has shown the lack of research in France on this topic, notably in psychodynamic psychotherapy. The development of such research is needed. The first part of the paper deals with the limits of the third generation of studies on psychotherapy (medical model, use of RCT, DSM...) on which the INSERM report is based and reviews the existing propositions for a fourth generation of research in the field. METHODOLOGICAL FINDINGS: In the second part, a process-outcome research protocol developed by the authors, according to these new proposals as well as several on-going researches [J Clin Psychol, 27 2 (1998) 217-26, J Pragm Case Stud 3 (2000)(2), Arch Gen Psychiatry 59 (2002) 505-10, Psychother Res 12 3 (2002) 251-72 and Br J Psychiatry 165 (1994) 4-8] is presented. The proposed methodology is based on controlled single case studies. Quantitative and qualitative data are associated for the definition of the diagnosis, as well as initial, intermediate and final measures. Process analysis is used to describe the main characteristics of the on-going psychotherapy at different moments in time. It is thus possible to gain access to what is really done during the therapy and not only to what is supposed to be done, based on a manual or even on the name of the theory used by the therapist. DESIGN OF THE STUDY: This methodology was tested during a one-year pilot study, in true conditions of psychotherapy with outpatients. The different phases of the analysis are presented: several tools dedicated to the observation, formalisation and data analysis are integrated in a coherent iterative process during the whole therapy. The interests and limits of each tool (ESM, DSM, PPQS, Hoglend, CORE...) are described together with the first results of the pilot study. DISCUSSION: The overall architecture of a database designed to collect, search and analyse data is provided in the last part of the paper. CONCLUSION: This framework offers two possibilities at the same time: it provides therapists with the ability to follow the evolution of their cases and to compare them with similar cases. It provides researchers with the ability to drive true comparative analysis, based on psychotherapies conducted in real situations and on detailed-enough descriptions to obtain significant outcomes.

[Distinction, limits and complementarity between efficacy and effectiveness studies: new perspectives for psychotherapy research]. / Distinction, limites et complémentarité des recherches d'efficacité potentielle et d'efficacité réelle: nouvelles perspectives pour la recherche en psychothérapie.

Perron et al. (2004) criticize the collective expertise conducted by the INSERM on the efficacy of different psychotherapy methods for different mental disorders. They say the work is biased in favour of Cognitive and Behavioral Therapies (CBT), while there is a negative bias regarding other methods, in particular psychodynamic therapies. Philippe Cialdella, a specialist of methodology and quantitative analysis in psychiatry, exposes an counter-argument in 10 points and brings welcome clarifications on the methodology and statistics. There is neither statistical bias in the INSERM report (which anyway is not a meta-analysis, but a literature analysis), nor in the first or second level studies on which it is based, mainly Randomised Controlled Trials (RCT). Though some arguments of P. Cialdella could be discussed in detail, his work comes to the conclusion that we can thus trust the results of the INSERM on one point: positive results can be obtained by psychotherapy with the experimental patients involved in the RCTs, and these findings are reliable. This is an important result in favour of psychotherapy, but it does not answer a very fundamental question: do we have reliable evidence that there is no systematic difference between the population constituted of patient-treatment-therapist (as a whole) found in RCTs, and the population constituted of patient-treatment-therapist (as a whole) in real contexts? A detailed analysis of what are efficacy studies and their methodology shows that this is not the case, and that it is hence not possible to generalize the results obtained by RCTs to clinical practice in real situations. Comorbidities and complex pathologies, choice of the therapist by the patient, interpersonal factors, conditions of use of manuals, contextual and social parameters, amongst other parameters make the real situation radically different from the RCT one, and the results impossible to generalize. Effectiveness studies in real situations do not solve the problem either. They have low internal validity, and though the population studied is close to a real one, too many uncontrolled parameters make the results unreliable. Moreover, outcome studies, whether they are efficacy or effectiveness studies, generally test a therapy "as a whole" versus another one, as defined in a manual, or by psychotherapy "trademarks". This design totally fails to offer a detailed view of what really happens during the therapy between the patient and the therapist, and it gives no possible insights into the change process elements. Only clinically highly representative quasi-experimental prospective studies can help us to understand change processes in real situations and incrementally improve treatment procedures. Daniel B. Fishman in the year 2000 made an ambitious and really interesting proposal about using pragmatic case studies in a systematic and scientific manner. Numerous reliable methodological tools, such as the Psychotherapy Process Q-Sort for example, as well as the tremendous innovations made in information and communication technologies make it possible today to implement such a proposal. It will enable us to really compare the resulting differences between the different approaches used in clinical settings, in relations with the type of intervention carried or in the real therapies, during clinically significant periods of time, and with various and ecologically valid samples. This is a possible solution to the difficult question of the use of outcome research to improve psychotherapy practice.

Porcine submaxillary gland GDP-L-fucose: beta-D-galactoside alpha-2-L-fucosyltransferase is likely a counterpart of the human Secretor gene-encoded blood group transferase.

Partial amino acid sequence of GDP-L-fucose:beta-D-galactoside alpha-2-L-fucosyltransferase purified from porcine submaxillary glands was determined. Amino acid sequence analysis yielded 100, 93.3, and 84.2%, and 75, 46.6, and 84.2% sequence identity between 12-, 15-, and 19- amino acid tryptic peptides generated from porcine enzyme and amino acid residues 61-72, 111-125, and 308-326 and 89-100, 139-153, and 338-356 of the human Secretor and H type alpha-2-fucosyltransferases, respectively. Higher amino acid sequence homology of the porcine enzyme with the predicted sequence for the human Secretor locus as compared with H gene-encoded blood group beta-D-galactoside alpha-2-L-fucosyltransferase suggests that porcine alpha-2-fucosyltransferase highly corresponds to the human Secretor gene-encoded enzyme.

Elevated expression of H type GDP-L-fucose:beta-D-galactoside alpha-2-L-fucosyltransferase is associated with human colon adenocarcinoma progression.

GDP-L-fucose:beta-D-galactoside alpha-2-L-fucosyltransferase (EC 2.4.1.69) is a key enzyme in the biosynthesis of fucosylated type 1 and 2 lactoseries structures, such as Lewis b and the H type 2 and Lewis Y, respectively, that are accumulated in colon adenocarcinoma. Analysis of the mRNA transcript level for the human H gene-encoded beta-D-galactoside alpha-2-L-fucosyltransferase revealed 40- and 340-fold increases in the mRNA levels in all adenocarcinomas and tumor cell lines, respectively, compared to normal colon mucosa where a low level of mRNA transcript was detected. A variable increase in mRNA transcript levels was observed in 50% of adenomatous polyps. Nucleotide sequence analysis of the protein coding region of the cDNAs derived from normal colon, adenoma, and colon adenocarcinoma revealed 100% homology, suggesting that there are no tumor-associated allelic variations within the H beta-D-galactoside alpha-2-L-fucosyltransferase cDNA. These results suggest that beta-D-galactoside alpha-2-L-fucosyltransferase expression highly correlates with malignant progression of colon adenocarcinoma.

Influence of different N- and O-linked carbohydrates on the retention times of synthetic peptides in reversed-phase high-performance liquid chromatography.

Glycopeptides consisting of 6-19 amino acid residues and different mono- and disaccharides attached to single asparagine and serine residues were synthesized on solid-phase and were characterized by reversed-phase high-performance liquid chromatography and circular dichroism. It was shown that the decreased retention times due to glycosylation could be correlated with the increasing length of the sugar moiety. Phosphorylation of the same sequences reduced the retention times 1.6 times more than glycosylation with monosaccharides did. The binding to the column was dependent on the structure of the disaccharide when derivatized and glycosylated asparagine, the building block of N-glycopeptide syntheses was studied. However, this structural dependence of the elution times disappeared in the final glycopeptides. Although both glycosylation and phosphorylation resulted in altered secondary structure of the peptide backbone, it appears that the retention times reflect the increased hydrophilicity more strongly than induced conformational orientation on the surface of the bonded phase.

Synthesis and conformational studies of N-glycosylated analogues of the HIV-1 principal neutralizing determinant.

The principal neutralizing determinant (PND) of HIV-1 is found in the V3 loop of the envelope glycoprotein. Antibodies elicited by peptides from this region, containing the GlyProGlyArgAlaPhe (GPGRAF) sequence, were able to neutralize diverse HIV-1 isolates [Javaherian et al. (1990) Science 250, 1590-1593]. The GPGR tetrapeptide was predicted to adopt a type II beta-turn conformation. Earlier, we showed that glycosylation of synthetic T cell epitopic peptides at natural glycosylation sites stabilized beta-turns [Otvös et al. (1991) Int. J. Pept. Protein Res. 38, 467-482]. To evaluate the secondary structure modifying effect of the introduction of an N-glycosylated asparagine residue and to find a correlation between conformation and a possible PND potential, a series of glycopeptide derivatives, N(sugar) GPGRAFY-NH2 (4a-f), have been prepared, together with the parent peptides GPGRAFY-NH2 (2) and NGPGRAFY-NH2 (3), by solid-phase peptide synthesis [sugars: (a) beta-D-glucopyranosyl (Glc); (b) beta-D-galactopyranosyl (Gal); (c) Glc-beta(1----4)-Glc; (d) 2-acetamido-2-deoxy-beta-D-glucopyranosyl (GlcNAc); (e) 2-acetamido-2-deoxy-beta-D-galactopyranosyl (GalNAc); (f) GlcNAc-beta(1----4)-GlcNAc; sugars are attached through a beta (1----N beta) linkage to asparagine (N).] Peptides 2-4 were characterized by amino acid analysis, reversed-phase HPLC, and fast atom bombardment mass spectrometry. Circular dichroism (CD) and Fourier-transform infrared (FT-IR) spectroscopic studies were performed in trifluoroethanol (TFE) and water (D2O was used in FT-IR experiments). Nonglycosylated peptides showed significantly different CD spectra in aqueous and TFE solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of the secretor-type beta-galactoside alpha 1----2-fucosyltransferase from human serum.

The secretor-type beta-galactoside alpha 1----2-fucosyltransferase from human serum was purified by hydrophobic chromatography on phenyl-Sepharose, ion-exchange chromatography on sulfopropyl-Sepharose, and affinity chromatography on GDP-hexanolamine-Sepharose. Final purification of the enzyme was achieved by high pressure liquid chromatography gel filtration and resulted in a homogeneous protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the radiolabeled protein. The native enzyme appears as a molecule of apparent Mr 150,000 as determined by gel filtration high pressure liquid chromatography. The apparent Mr of the enzyme resolved in the presence of beta-mercaptoethanol by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was determined to be 50,000, indicating a multisubunit structure of the enzyme. Secretor-type alpha 1----2-fucosyltransferase is a glycoprotein as determined by WGA binding properties. A comparison of the Mr of the native blood group H gene encoded with the secretor-type beta-galactoside alpha 1----2-fucosyltransferases as well as comparison of subunit Mr for both enzymes suggests structural similarity. The alpha 1----2 linkage formed between alpha-L-fucose and terminal beta-D-galactose by the purified H- and secretor-type alpha 1----2-fucosyltransferases was determined by 1H NMR homonuclear cross-irradiation analysis of the oligosaccharide products. The substrate specificity and Km values calculated from the initial rate using various oligosaccharide acceptors showed that purified enzymes differ primarily in affinity for phenyl-beta-D-galactopyranoside and GDP-fucose as well as type 1 (Gal beta 1----3GlcNAc), 2 (Gal beta 1----4GlcNAc), and 3 (Gal beta 1----3GalNAc) oligosaccharide acceptors. The secretor-type alpha 1----2-fucosyltransferase shows significantly lower affinity than the H enzyme for phenyl-beta-D-galactopyranoside and GDP-fucose as well as for type 2 oligosaccharide acceptors. On the contrary, type 1 and 3 oligosaccharide acceptors are preferentially utilized by the secretor-type enzyme as compared with the H enzyme. The enzymes also differ in several physicochemical properties, implying nonidentity of the two enzymes (Sarnesto, A., Köhlin, T., Thurin, J., and Blaszczyk-Thurin, M. (1990) J. Biol. Chem. 265, 15067-15075).

Purification of the beta-N-acetylglucosaminide alpha 1----3-fucosyltransferase from human serum.

A beta-N-Acetylglucosaminide alpha 1----3-fucosyltransferase was purified from human serum by ammonium sulfate precipitation, hydrophobic chromatography on phenyl-Sepharose, ion-exchange chromatography on sulfopropyl-Sepharose, affinity chromatography on GDP-hexanolamine-Sepharose, and finally high pressure liquid chromatography gel filtration. Gel filtration chromatography of the native enzyme revealed a Mr of 45,000. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified protein also appeared as a single molecular species of Mr 45,000. In contrast to the multisubunit beta-galactoside alpha 1----2-fucosyltransferases with an apparent Mr of 150,000, present in human serum, the native beta-N-acetylglucosaminide alpha 1----3-fucosyltransferase is a monomer with a Mr of 45,000. The enzyme is glycosylated, as revealed by wheat germ agglutinin binding properties. The alpha 1----3 linkage formed by the enzyme between alpha-L-fucose and the penultimate beta-N-acetylglucosamine by the purified enzyme was confirmed by 1H NMR homonuclear cross-irradiation analysis of the oligosaccharide product. The specificity of the purified enzyme is restricted to type 2 structures, as revealed by its reactivity with different substrates and from the Km values calculated from the initial rate data using various oligosaccharide acceptors. The enzyme has the ability to utilize the N-acetyl-beta-lactosamine determinant (Gal beta 1----4GlcNAc) and the sialylated (NeuAc alpha 2----3Gal beta 1----4GlcNAc) and fucosylated (Fuc alpha 1----2Gal beta 1----4GlcNAc) derivatives of N-acetyl-beta-lactosamine and thus is distinct from both the human Lewis gene-encoded enzyme and the alpha 1----3-fucosyltransferase of the myeloid cell type.

Glycosylation of synthetic peptides breaks helices. Phosphorylation results in distorted structure.

Two proposed glycosylation sites are located within T cell epitopes of rabies virus glycoprotein, namely VVEDEGCTNLSGF (VF13; amino acids 29-41) and GKAYTIFNKTLM (GM12; amino acids 312-323). To explore the effects on peptide conformation due to post-translational modifications, we synthesized glycosylated and phosphorylated versions of the two peptides and compared their structures with the native peptide using CD and FT-IR spectroscopy. After the modifications, i.e., glycosylation on Asn with one or two N-acetyl-glucosamine or glucose residues or phosphorylation on Ser, the low to medium degree of helicity of the unmodified peptides disappears as indicated by CD measurements in water-trifluoroethanol mixtures. Incorporation of one sugar moiety into either peptide resulted with a high probability in a type I (III) beta-turn formation with almost identical spectra for the different peptides. Elongation of the carbohydrate in GM12 only slightly enhanced this effect. In contrast, phosphorylation of VF13 caused distorted conformation of the peptide backbone. This novel and direct demonstration of a change in secondary structure by glycosylation (or phosphorylation) might be an important element in determining peptide antigen structure and function.

Purification of H gene-encoded beta-galactoside alpha 1----2 fucosyltransferase from human serum.

The human serum enzyme, beta-galactoside alpha 1----2 fucosyltransferase, presumably blood group H gene-encoded, was purified to homogeneity from serum of AB and mixed secretor phenotype individuals. The purification procedure involved chromatography on phenyl-Sepharose, S-Sepharose, GDP-hexanolamine-Sepharose, and high pressure liquid chromatography gel filtration. The enzyme was purified 10 x 10(6)-fold, with a final specific activity of 23.6 units/mg for the phenyl-beta-O-galactoside acceptor. The apparent Mr of the H gene-encoded beta-galactoside alpha 1----2 fucosyltransferase was determined as 200,000 and 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in nonreducing and reducing conditions, respectively. The Mr of native enzyme was found by gel filtration chromatography to be 148,000. The subunit structure as well as the sensitivity of the enzymatic activity to beta-mercaptoethanol suggest that the native enzyme exists in polymeric form of covalently bound subunits. Lectin binding properties of the purified molecule indicate that the enzyme is glycosylated. Another human serum beta-galactoside alpha 1----2 fucosyltransferase, presumably Se gene-encoded, was separated from the H enzyme by adsorption on S-Sepharose cation exchange matrix. A comparison of the kinetic parameters of the initial rate data of both alpha 1----2 fucosyltransferases revealed differences between Km values for various oligosaccharide acceptors. Higher Km values for the phenyl-beta-O-galactoside acceptor and a lower Km for the lacto-N-tetraose-beta-O-PA8 type 1 acceptor for the enzyme that adsorbed to S-Sepharose compared with nonadsorbed enzyme were observed. The two enzymes also were differentiated by binding properties to S-Sepharose and electrophoretic mobilities on native gel electrophoresis. We, therefore, postulate that the enzyme which does not adsorb to S-Sepharose and adsorbed enzyme are structurally different molecules and they represent the H and Se gene-encoded beta-galactoside alpha 1----2 fucosyltransferases, respectively.

Conformational analysis of the tumor-associated carbohydrate antigen 19-9 and its Lea blood group antigen component as related to the specificity of monoclonal antibody CO19-9.

A structural comparison between the synthetic, tumor-associated 19-9 tetrasaccharide, NeuAc alpha 2----3Gal beta 1----3GlcNAc(4----1 alpha Fuc)-O(CH2)8CO2CH3 and its Lea blood group antigen component, Gal beta 1----3GlcNAc(4----1 alpha Fuc)-O(CH2)8CO2CH3 was carried out by two-dimensional 1H NMR spectroscopy and hard-sphere energy calculations. Significant chemical shift differences between the two molecules were detected only for protons at or near the linkage site of NeuAc to the Lea trisaccharide core. Coupling constants for the ring protons of both molecules did not suggest major deviation from the 4C1 chair conformation for Gal and GlcNAc, the 1C4 conformation for Fuc, or the 2C5 conformation for NeuAc. Two-dimensional nuclear Overhauser enhancement experiments revealed through-space, inter-proton interactions that corresponded to some extent with those predicted by diffraction data and hard-sphere energy minimization programs for both saccharides. However, a significant number of interactions did not obey the distance dependence predicted from a rigid structure model. These data suggest that, while the average conformation of the 19-9 antigen's Lea core may be invariant to NeuAc alpha 2----3Gal linkage, the dynamics of the Lea trisaccharide are altered upon sialylation. Data also indicate that the terminal NeuAc linkage is more flexible than the inter-residue bonds of the core trisacharide. This analysis, in combination with the fact that the monoclonal anti-19-9 antibody CO 19-9 does not cross-react with the Lea antigen, provides evidence in favor of NeuAc as an epitope-creating unit involved directly at the antibody binding site. However, given the possible role of variable dynamics in epitope formation, these results do not preclude crucial roles in antibody recognition for regions on the 19-9 antigen that are distanced from NeuAc.

Coupling strategies in solid-phase synthesis of glycopeptides.

N-beta-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-asparaginyl peptides [Asn(GlcNAc)] corresponding to T-helper cell determinants were synthesized on solid-phase. Various amino-terminal- and carbohydrate-protecting groups were used on the glycosylated asparagine residue which was coupled to the peptide chain. We found that coupling rates decreased with increased size of the protected carbohydrate part of the acylating agent. Double couplings with an O-unprotected saccharide, as in Fmoc-Asn(GlcNAc)-OH resulted in acceptable coupling rates even with a synthetically difficult sequence corresponding to the T-cell epitopic peptide from the C-terminus of pigeon cytochrome c. The observed coupling rates on this peptide as well as on a T-cell epitopic pentapeptide, derived from the rabies virus N-protein, were comparable to those of conventional solid-phase peptide syntheses. The Fmoc-Asn(GlcNAc)-OH used can be prepared easily from commercially available components. The described glycopeptides will be used to probe effects of N-glycosylation on the immune recognition of viral glycoproteins.

Antigenic profile of tumor progression stages in human melanocytic nevi and melanomas.

Sixteen monoclonal antibodies that were obtained after immunization of BALB/c mice with intact melanoma cells or extracts of melanoma cells were tested for reactivity with normal and malignant melanocytic cells in situ, using an immunoperoxidase technique on frozen tissue sections. Sections representing six histopathologically defined stages of tumor progression, ranging from normal melanocytes to highly malignant metastatic lesions, were used. Thirteen monoclonal antibodies (MAbs) did not stain normal melanocytes in situ, whereas three MAbs weakly stained between 1 and 12.5% of melanocytes in 6-22% of the skin sections examined. MAb B 73.1, which was produced by immunization of mice with human natural killer cells and which binds to the Fc receptor of natural killer cells and granulocytes, reacted exclusively with malignant cells that represent the last two stages of tumor progression, vertical growth phase (VGP) primary melanoma and metastatic melanoma. All other antibodies showed variable reactivity with benign proliferative lesions or radial growth phase (RGP), an early stage of primary melanoma. Staining by MAbs that were reactive with gangliosides, unknown antigens, receptors, and two proteins (120/94 kDa protein and 250 kDa glycoprotein) showed a gradual increase in subsequent stages of tumor progression. Two steps in tumor progression were characterized by significant quantitative changes in the expression of antigens detected by the MAbs used in this study. First, mature nevus cells showed significantly higher reactivity with a panel of six MAbs, when compared to normal melanocytes. Second, a separate panel of six MAbs discriminated between RGP and VGP primary melanoma cells. No significant differences in antigen expression were found between dysplastic nevus cells and RGP melanoma, except that some antigens (nerve growth factor receptor and GD2/GD3 gangliosides) appear to be expressed at lower levels in RGP lesions, nor did VGP primary and metastatic melanomas show significant differences in antigen expression. These results suggest that (a) tumor progression of melanocytic cells in vivo is accompanied by significant quantitative differences in the expression of antigens, (b) some of the antigens examined here are associated with biologically aggressive malignant lesions but not normal or premalignant melanocytic cells, and (c) RGP primary melanoma cells are antigenically more similar to nevus cells than to VGP primary melanoma 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.

Monoclonal antibodies to glycolipids of the isoglobo-series detect tumor-associated antigens in rats.

Monoclonal antibodies (MAbs) directed to 2 neutral glycolipids, isoglobotetraosylceramide and a 6-sugar isogloboneolactoseries hybrid glycolipid, previously shown to be enriched in rat colorectal tumor tissue, were produced by immunization with purified glycolipids. Four MAbs were selected which demonstrate 3 different specificity patterns when tested for binding to purified and crude preparations of neutral glycolipids, cultured tumor cells and fibroblasts and frozen tissue sections. MAbs 14.2 and 14.10, but not 14.3, stained most epithelial colorectal carcinomas, rat testis and a subpopulation of cells in the rat gastric mucosa. However, all 3 MAbs showed strong staining and binding to sections and cultured clones of the cytokeratin-negative tumor of colorectal origin, which was originally used for preparation of the glycolipid immunogens. The observed difference between MAbs 14.2/10 and MAb 14.3 could not be explained by differences in binding to the 2 original glycolipids used for screening. However, MAbs 14.2/10 were demonstrated to bind to high-molecular-weight glycoprotein(s) (HMW-gp's) previously shown to carry determinants for syngeneic antibodies and extracted from epithelial colorectal tumor tissue after extensive lipid extraction. This suggests that a protein-bound carbohydrate determinant, with similarities to the oligosaccharide part of the isoglobo-series glycolipids, is responsible for this cross-reactivity. The staining of rat testis could be explained by the strong expression in this tissue of glycolipids with 8-10 sugar residues bound by the 14.2/10 but not 14.3 MAbs. The cell-surface expression of the 6-sugar hybrid glycolipid was demonstrated by complement-dependent cytotoxicity and immunofluorescent staining of viable cells.

Blood group antigens in the intrahepatic biliary tree. I. Distribution in the normal liver.

The distribution of six blood group-related antigens, A, B, H, Lea, Leb and sialylated Lea antigens, in the intrahepatic biliary tree was studied. These carbohydrate antigens in fixed normal liver tissues were immunostained with the use of highly specific monoclonal antibodies in an avidin-biotin-peroxidase complex method. Bile ducts expressed both ABH and Lewis blood group antigens. However, only Lewis blood group antigens could be detected in the bile ductules. Canaliculo-ductular junctions could be clearly delineated with the anti-Lewis blood group antibodies, especially with anti-Lea antibody. Small Lewis antigen-positive cells were scattered intralobularly. They were adjacent to parenchymal liver cells, mainly in Rappaport zone 1, and apparently in continuity with the portal biliary tree. Sialylated Lea antigen was found in some septal bile ducts. No blood group antigen could be detected in the bile canaliculi. These results indicate that (1) biliary tract of a given size has its own pattern of blood group antigen expression, and (2) biliary epithelial cells are not identical with regard to the phenotypic expression of their structural carbohydrates. In future, it will be possible to classify biliary epithelial cells by their blood group antigen expression.

Glycosphingolipids of cultured human colon carcinoma cells and their drug-resistant sublines.

Human colon carcinoma cells were analyzed for lipid phosphorus, cholesterol and glycosphingolipids. Ceramide mono-, di- and trihexosides and sulfatides were isolated by column and thin-layer chromatography and determined quantitatively on the basis of their hexose content. The complex lipid fractions so isolated were only partially resolved with the material available. Gangliosides GM2 and GM3 and globoside were major components of the fraction and were determined on the basis of their hexose, hexosamine and neuraminic acid content. The HCT 116, 116a and 116b cells contained no fucolipids. Cell lines resistant to mitomycin C, teniposide and etoposide were developed and analyzed. Over the 5 year period of the study sulfatides declined to about one-fourth of their original amounts in both parent and drug-adapted cells. HCT 116 cells adapted to mitomycin C and teniposide had 30% less ceramide monohexoside and a 45% greater cholesterol to lipid phosphorus ratio than the parent cells. Reductions in ceramide dihexoside in the drug-adapted cells were greater than those of the ceramide monohexoside. Galabiosyl ceramide was the major ceramide dihexoside in all the cells and accumulated in HCT 116a to levels 4-6-fold greater than that of the other lines as the only dihexoside.

Biosynthetic pathways for the Leb and Y glycolipids in the gastric carcinoma cell line KATO III as analyzed by a novel assay.

The biosynthetic pathways for the difucosylated type 1 and 2 glycolipids, Leb and Y, respectively, were investigated in the gastric carcinoma cell line KATO III, using a novel chromatogram binding assay. The type of fucosylation obtained was deduced from the binding pattern of monoclonal antibodies specific for the biosynthesized glycolipid products using microsomal fractions as the source of enzyme, pure glycolipids and non-radioactive GDP-fucose as acceptor and donor substrates, respectively. The Leb glycolipid (Fuc alpha 1----2Gal beta 1----3GlcNAc(4----1 alpha Fuc) beta 1----3LacCer) was synthesized mainly via the blood group H, type 1, precursor (Fuc alpha 1----2Gal beta 1----3GlcNAc beta 1----3LacCer). However, the Lea glycolipid (Gal beta 1----3GlcNAc(4----1 alpha Fuc)beta 1----3LacCer) also served as a precursor for the alpha 1----2 fucosyltransferase, thus allowing conversion of Lea to Leb. This biosynthetic route represents either an "aberrant" specificity of the Fuc alpha 1----2 transferase associated with these gastric carcinoma cells and/or a new member of the alpha 1----2 fucosyltransferase family. The Y glycolipid (Fuc alpha 1----2Gal beta 1----4GlcNAc(3----1 alpha Fuc)beta 1----3LacCer) was synthesized exclusively via the classical pathway using the blood group H type 2 glycolipid (Fuc alpha 1----2Gal beta 1----4GlcNAc beta 1----3LacCer) as precursor. The X glycolipid (Gal beta 1----4GlcNAc(3----1 alpha Fuc)beta 1----3LacCer) did not serve as an acceptor substrate for the alpha 1----2 fucosyltransferase(s) present. The use of non-radioactive sugar-nucleotides as donor substrate, defined glycolipid precursors as acceptor substrates and of specific monoclonal anti-glycolipid antibodies for detection provides a rapid and highly specific assay for analyzing biosynthetic pathways of glycosyltransferases.