Alginate oligosaccharides modify hyphal infiltration of Candida albicans in an in vitro model of invasive human candidosis.

AIMS: A novel alginate oligomer (OligoG CF-5/20) has been shown to potentiate antifungal therapy against a range of fungal pathogens. The current study assessed the effect of this oligomer on in vitro virulence factor expression and epithelial invasion by Candida species. METHODS AND RESULTS: Plate substrate assays and epithelial models were used to assess Candida albicans (CCUG 39343 and ATCC 90028) invasion, in conjunction with confocal laser scanning microscopy and histochemistry. Expression of candidal virulence factors was determined biochemically and by quantitative PCR (qPCR). Changes in surface charge of C. albicans following OligoG treatment were analysed using electrophoretic light scattering. OligoG induced marked alterations in hyphal formation in the substrate assays and reduced invasion in the epithelial model (P < 0·001). Significant dose-dependent inhibition of phospholipase activity in C. albicans was evident following OligoG treatment (P < 0·05). While OligoG binding failed to affect alterations in surface charge (P > 0·05), qPCR demonstrated a reduction in phospholipase B (PLB2) and SAPs (SAP4 and SAP6) expression. CONCLUSION: OligoG CF-5/20 reduced in vitro virulence factor expression and invasion by C. albicans. SIGNIFICANCE AND IMPACT OF THE STUDY: These results, and the previously described potentiation of antifungal activity, define a potential therapeutic opportunity in the treatment of invasive candidal infections.

Differential susceptibility of transferrin glycoforms to chymotrypsin: a proteomics approach to the detection of carbohydrate-deficient transferrin.

Transferrin exhibits heterogeneity in glycosylation characteristic of pathological changes in alcohol abuse and congenital disorders in glycosylation. This study investigated an alternative approach in the detection of carbohydrate-deficient transferrin based on the premise that glycosylation may afford some degree of protection to proteolytic action. Differential susceptibility to proteolysis by chymotrypsin was demonstrated for normal glycosylated and nonglycosylated recombinant human transferrin, using reverse-phase (RP) HPLC, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, and LC-tandem mass spectrometry (MS/MS). Peptide fragmentation profiles were consistent with a predominantly high-specificity cleavage pattern of chymotrypsin. The observed peptide fragmentation profile showed that the C-lobe of recombinant full-length nonglycosylated transferrin (rhTf-NG) appeared to be preferentially cleaved, while cleavage of the N-lobe was restricted to the N-terminal and link sequence regions. Although chymotryptic cleavage sites abound in the N-lobe, their resistance to cleavage was independent of glycosylation. Compared to previous studies of lactoferrin, our data suggest disparity in the role by which glycosylation exerts a protective effect in the siderophilin family. It was clear from the transferrin digestions analyzed by HPLC that N-linked glycosylation did confer protection from proteolysis by chymotrypsin. After fragmentation, a range of peptides representing previously cryptic epitopes were identified as potential candidates for an immunological approach to differentiate between the different transferrin glycoforms. Based on its proximity to the Asn413 glycosylation site, a 15-mer peptide, m/z 1690.472 (NKSDNCEDTPEAGYF), was identified as a suitable candidate for raising anti-peptide antibodies for subsequent immunological detection. This novel approach could form the basis for an alternative assay or reference method for the detection of carbohydrate-deficient transferrin.

Interfering with cancer: a brief outline of advances in RNA interference in oncology.

RNA interference (RNAi) is a potent and ubiquitous gene-silencing mechanism that is generating considerable excitement in the fields of molecular biology and gene therapy. It is now in widespread use for loss-of-function analysis in many diseases including cancer. Nevertheless, RNAi is still in its infancy, with new discoveries appearing on a monthly basis. This article presents a brief outline of the history and recent advances in RNAi with a specific focus on its potential in oncology.

Tumor marker workshops.

Since 1996, the nine ISOBM Workshops have so far characterized more than 300 monoclonal antibodies to a variety of tumor markers that include CA125, AFP, PSA, MUC1, Cytokeratins, Sialyl Le(a), hCG, CEA, ALP, and more recently SCC, and S100. Besides the basic characterization of antibodies and their epitope configurations, several workshops have also addressed specific problems associated with multiple antigen variants. These workshops have been able to make significant advances well beyond those possible through any normal collaboration study. The data and impact of these workshops with their summary reports are reviewed.

New immunoassays for MUC1 in breast cancer.

Eleven experimental immunofluorometric assays (IFMAs) were made using antibodies previously tested for epitope specificities. These assays were compared with six commercially available immunoassays. The clinical performance of these experimental assays was evaluated by analysing sera from 138 breast cancer patients and 105 female blood donors. The clinical performance of these assays was evaluated at a set specificity of 0.94. The highest overall sensitivity (0.56) was observed in the experimental assay with the antibody BC2 as solid phase and GP1.4 as the tracer antibody. This combination also showed the highest sensitivity in stage I/II breast cancer. The Truquant assay (Biomira) had an overall sensitivity of 0.51, and the highest sensitivity in stages III and IV at 0.65 and 0.94, respectively. The remaining commercial assays, with sensitivity ranging from 0.67 to 0.79, were below the top five experimental assays that showed sensitivity values between 0.79 and 0.85. The findings from our current study suggest that further development in MUC1 immunoassays could improve the detection of relapse in breast cancer patients.

MUC1: antibodies and immunoassays.

High molecular weight mucins represent a unique challenge as tumor markers by virtue of their complex array of epitopes. The list is dominated by the high molecular weight mucins MUC1, CEA and CA125. While the currently accepted role for these tumor markers is in the prediction and detection of relapse, it is possible that their sensitivity and specificity can be improved. Although immunoassays detecting the tumor marker MUC1 are both sensitive and specific for predicting relapse in breast cancer, so far they are not in widespread use in the follow-up of this disease. Are there new combinations of conventional reagents that could improve assay sensitivity, or should we be looking for more radical changes in assay design incorporating combinatorial technology?

Immunomagnetic DNA aptamer assay.

DNA aptamers, oligonucleotides with antibody-like binding properties, are easy to manufacture and modify. As a class of molecules, they represent the biggest revolution to immunodiagnostics since the discovery of monoclonal antibodies. To demonstrate that DNA aptamers are versatile reagents for use as in vitro diagnostic tools, we developed a hybrid immunobead assay based on a 5'-biotinylated DNA thrombin aptamer (5'-GGTTGGTGTGGTTGG-3') and an anti-thrombin antibody (EST-7). Our results show that the thrombin DNA aptamer is capable of binding to its target molecule under stringent in vitro assay conditions and at physiological concentrations. These findings also support the view that DNA aptamers have potential value as complementary reagents in diagnostic assays.

CA 125: the end of the beginning.

CA 125, a high-molecular-weight mucin, was first defined in 1981 by the monoclonal antibody OC125. Until recently, it has defied many attempts to purify it from a variety of sources, although many research groups have successfully raised antibodies that bind to CA 125. Nevertheless, CA 125 has demonstrated its considerable value as a marker in monitoring patients with ovarian cancer. This year, two research groups have succeeded in cloning the high-molecular-weight mucin CA 125. Their findings are summarized and the significance discussed in light of existing data from the human genome.

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

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

Carbohydrate affinity PAGE for the study of carbohydrate-binding proteins.

Immobilized neoglycoconjugates covalently cross-linked into a polyacrylamide gel can be used to detect and characterize carbohydrate-binding proteins. The neoglycoconjugates comprise two active groups, saccharide and allyl, located on a poly(2-hydroxyethylacrylamide) backbone. The allyl group cross-links with the polyacrylamide gel matrix, while the saccharide groups are available for specific protein interactions. This neoglycoconjugate gel is prepared as a thin layer within the stacking region of a polyacrylamide gel, and electrophoresis is performed according to native, non-denaturing conditions. Carbohydrate-binding proteins, specific for the immobilized neoglycoconjugates, are thus retarded during electrophoresis, while simultaneously permitting the separation of nonbinding proteins according to size and charge. This new approach can be used to study carbohydrate-binding proteins in the pathology of disease or infection.

Invasion potential and N-acetylgalactosamine expression in a human melanoma model.

Reactivity of the N-acetylgalactosamine-binding Helix pomatia agglutinin (HPA) in tumours has been associated with poor prognosis and metastasis development. In our LOX/FEMX-I human melanoma model, the binding of HPA correlates with experimental lung metastasis formation in athymic nude mice. In the present study, the metastatic potential of 2 human melanoma cell lines (LOX and FEMX-I) was assessed in relation to carbohydrate and invasive phenotype. Immunocytological and invasion assays highlighted significant differences between these 2 cell lines. Immuno-cytochemical analysis confirmed the widespread expression of HPA-binding glycoconjugates on LOX but not FEMX-I cells. One of these HPA-binding glycoconjugates, the Tn antigen, was expressed highly on the surface of LOX cells but only weakly in the cytoplasm of FEMX-I cells. The sialyl Tn antigen was expressed in FEMX-I but not in LOX cells. There was no difference between the cell lines in adhesion/rate of trapping in athymic nude mouse lung tissues. In Matrigel invasion assays, LOX cells demonstrated an invasion potential more than 6 times greater than that observed with FEMX-I cells. Matrigel invasion of LOX cells was inhibited after incubation with HPA (89%) compared to controls with HPA and GalNAc blocking sugar or without HPA (p < 0.0005 at 5 df). In contrast, there was no inhibitory effect with the anti-Tn antibody IE3. Invasion of FEMX-I cells was not affected by the lectin and the IE3 antibody. Immuno-cytochemical analysis revealed expression of the terminal galactose- and polylactosamine-binding lectin galectin 3 (Mac-2) in these melanoma cell lines. Expression of both the lectin and its receptor may be a contributory feature in the pulmonary invasion of LOX melanoma cells. Overall, our findings suggest that HPA-binding glycoconjugates other than the alphaGalNAc-O-Ser/Thr of the Tn antigen may be important in the extracellular matrix invasion of LOX melanoma cells.

Summary report on the ISOBM TD-4 Workshop: analysis of 56 monoclonal antibodies against the MUC1 mucin. San Diego, Calif., November 17-23, 1996.

Sixteen research groups participated in the ISOBM TD-4 Workshop in which the reactivity and specificity of 56 monoclonal antibodies against the MUC1 mucin was investigated using a diverse panel of target antigens and MUC1 mucin-related synthetic peptides and glycopeptides. The majority of antibodies (34/56) defined epitopes located within the 20-amino acid tandem repeat sequence of the MUC1 mucin protein core. Of the remaining 22 antibodies, there was evidence for the involvement of carbohydrate residues in the epitopes for 16 antibodies. There was no obvious relationship between the type of immunogen and the specificity of each antibody. Synthetic peptides and glycopeptides were analyzed for their reactivity with each antibody either by assay of direct binding (e.g. by ELISA or BiaCore) or by determining the capacity of synthetic ligands to inhibit antibody binding interactions. There was good concordance between the research groups in identifying antibodies reactive with peptide epitopes within the MUC1 protein core. Epitope mapping tests were performed using the Pepscan analysis for antibody reactivity against overlapping synthetic peptides, and results were largely consistent between research groups. The dominant feature of epitopes within the MUC1 protein core was the presence, in full or part, of the hydrophilic sequence of PDTRAPAP. Carbohydrate epitopes were less easily characterized and the most useful reagents in this respect were defined oligosaccharides, rather than purified mucin preparations enriched in particular carbohydrate moieties. It was evident that carbohydrate residues were involved in many epitopes, by regulating epitope accessibility or masking determinants, or by stabilizing preferred conformations of peptide epitopes within the MUC1 protein core. Overall, the studies, highlight concordance between groups rather than exposing inconsistencies which gives added confidence to the results of analyses of the specificity of antimucin monoclonal antibodies.

Selection of carbohydrate-binding cell phenotypes using oligosaccharide-coated magnetic particles.

Neoglycoconjugate coated magnetic beads were assessed for their ability to selectively isolate human cells with known anti-carbohydrate reactivity. Four lung cancer cell lines, NCI-H146, NCI-N417D, SKMES-1, EKVX; two acute lymphoblastic leukemia lines, MOLT-4 and CCRF-CEM; and the anti- Le(c) (isolactosamine) hybridoma, LU-BCRU-G7, were tested. The neoglycoconjugates (biotinylated pseudopolysaccharides) bound uniformly to streptavidin coated magnetic beads as demonstrated by FITC labeled lectin. Streptavidin beads alone did not bind to any of the cell types. The anti- Le(c) hybridoma cell line, LU BCRU-G7, demonstrated binding only to Le(c) pseudopolysaccharide coated magnetic beads. Subsequent incubation in the presence of unlabeled pseudopolysaccharide resulted in the release of the beads from the cell surface. Although there was some heterogeneity within the individual lung and leukemic cell lines, positive cells showed strong rosette formation with the coated beads. The Adi disaccharide coated beads showed binding in all four lung cancer cell lines, with the Le(c) and the H (type1) pseudopolysaccharide-bead conjugates only reactive in the N417 and H146 SCLC lines. The range of L-selectin ligand-coated beads were all successful in binding to the acute lymphoblastic leukemia cell lines MOLT4 and CCRF-CEM. This approach provides a versatile model for the study of cell-surface carbohydrate interactions that should find application in many areas of cell biology.

Immunobead filtration: a novel approach for the isolation and propagation of tumor cells.

We have developed a method to facilitate the isolation and expansion of tumor cells from body fluids and tissue biopsies. Antibody-conjugated magnetic beads (immunobeads) were used to isolate tumor cells from blood, bone marrow, ascitic/pleural fluids, and enzyme-digested tissue biopsies. Filtration of the resulting cell suspension through a 20-micron nylon monofilament filter secured to the base of polystyrene 96-well strips purged the bead-rosetting cell fraction of contaminating normal cells and unbound beads. Tumor cells that bound the magnetic beads were retained on the membrane due to their increased size and concentrated into a small area (0.332 cm2), thus maintaining a high cell density. The filters provided a stable and uniform three-dimensional matrix for cell growth, with a total surface area of 1.42 cm2 available for cell attachment. The filters could be easily removed from the base of the 96-well strips to facilitate handling and transfer between culture vessels. Tumor cells grown on the filters could subsequently be harvested using trypsin/EDTA or left in situ for immunostaining with conventional immunohistochemical procedures. Filter-grown cells have shown extended passage in conventional cell culture in six cases. In two of five cases, the orthotopic implantation of confluent filters that contained approximately 10(4) cells/8 x 8 mm filter successfully produced tumors in nude mice after only 4 weeks. Our new approach may be of value in improving the success rate of generating long-term cultures from previously unproductive sources of tumor cells and thus may yield a greater variety of cell lines/strains for the study of malignant disease.

Brain metastasis model in athymic nude mice using a novel MUC1-secreting human breast-cancer cell line, MA11.

The MA11 human breast-cancer cell line was established with cells isolated from a bone-marrow sample using immunomagnetic beads conjugated to the anti-MUC1 antibody BM-2. The cell line showed a selective preference for metastasising to the brain in athymic nude mice. Following injection of MA11 cells into the left ventricle of the heart, brain metastases developed in 87% (20/23) animals, with a mean latency until development of neurological symptoms of 65 days. Necropsy and histological examination revealed tumour nodules of varying sizes throughout the brain, invading both grey and white matter of both hemispheres, and with extensive involvement of the cerebellum. MRI spin-echo images indicated brain lesions in some animals that were subsequently confirmed by histology. Three mice showed small tumour nodules (1-2 mm) in the lung, and 2 had solitary lesions (< 1 mm) within the spinal cord. Metastases were not detected in bone, liver, adrenal gland, kidney, spleen or heart. The human MUC1 mucin, as determined by a europium-based immunoradiometric assay, was detected in the serum of 9/11 animals that showed histological evidence of brain metastases. The mucin could not be found in mouse serum samples taken before day 46. The concentration range of MUC1 observed was from <1 to >50 U/ml, and did not appear to correlate with the size or number of tumours as determined from histological sections. This new model provides an opportunity to study the mechanisms of clinically relevant organ-selective metastases and may be of use in evaluating novel treatment for brain metastases in breast cancer.

Monoclonal antibody LU-BCRU-G7 against a breast tumour-associated glycoprotein recognizes the disaccharide Gal beta 1-3GlcNAc.

The monoclonal antibody LU-BCRU-G7, that was generated by in vitro immunization, shows clinical value as a prognostic marker in early stage breast carcinoma. It has now been characterized with regard to its binding epitope. Using a recently described method based on the construction of N-substituted polyacrylamide (PAA) derivatives of carbohydrates (pseudopolysaccharides), the structure of the epitope for the monoclonal antibody LU-BCRU-G7 has been determined. Competitive binding assays and inhibitory enzyme-linked immunosorbent assays (ELISAs) using these pseudopolysaccharides have shown the LU-BCRU-G7 epitope to be a disaccharide Gal beta 1-3GlcNAc (Lec; where Gal is D-galactose, Glc is D-glucose and GlcNAc is N-acetyl-D-glucosamine). Both galactose and N-acetyl glucosamine moieties are essential for binding. Substitution on C-2 or C-3 of the terminal galactose abolished binding, as did galactose-alpha terminated oligosaccharides. The galactose moiety alone, as expressed by the Gal beta-PAA conjugate, appeared to be a more important feature of the epitope than the GlcNAc-PAA conjugate, which failed to bind or inhibit the LU-BCRU-G7 antibody. In the N-acetyl glucosamine moiety, binding was decreased but not eliminated by fucose substitution, as in Lea, or change in configuration of C-4, as in Gal beta 1-3GlcNAc. Omission of the NAc group resulted in complete loss of activity. The tetrasaccharide lacto-N-tetraose, although containing the terminal Lec disaccharide, does not react with the antibody, suggesting conformational interference of the binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Helix pomatia agglutinin binding in human tumour cell lines: correlation with pulmonary metastases in nude mice.

The extent of lectin binding by three human melanoma (LOX, FEMX-1 and SESX) and two sarcoma lines (MHMX and OHSX) was related to their potential for experimental metastasis formation in athymic nude mice. The Helix pomatia agglutinin (HPA), which recognises the N-acetyl-D-galactosamine ligand, showed differential binding to the cell lines in a manner that correlated with their ability to give lung colonies after i.v. injection in the mice (P < 0.005). The degree of HPA binding and lung colony formation of the cell lines studied was ranked in the following order, LOX > MHMX > OHSX > SESX > FEMX-I. Similar patterns were not observed with the other lectins used in this study (WGA, Con A, PNA and UEA-I). The high HPA reacting LOX melanoma line shows extensive pulmonary metastatic formation with no extrapulmonary colonies, whereas the low HPA reacting FEMX-I cells give only extrapulmonary metastases with no detectable colonies in the lungs. Precoating of tumour cells with HPA prior to injection did not reduce the ability of cells to give pulmonary metastases, suggesting that the HPA epitope was not functionally associated with the pulmonary metastatic potential observed in nude mice. These findings support recent human studies of a correlation between HPA binding and incidence of metastasis, however, our data indicate that there is no causal relationship. Further analyses are required to identify the specific HPA-binding glycoconjugates that may be involved.

Prognostic value of a breast cancer-associated glycoprotein detected by monoclonal antibody LU-BCRU-G7.

The immunohistochemical reactivity of a second generation murine monoclonal antibody (LU-BCRU-G7), raised against a novel fucosylated glycoprotein of M(r) 2300,000, has shown a significant association with prognosis of early stage carcinomas. Staining was observed in 72% of the 190 breast carcinomas tested. No relationship with steroid receptor status, stage or node status was found. An association with grade was observed (chi 2 7.83, 2 degrees of freedom, P = 0.02) only when the negative cut-off level was raised from < 10% cells staining to < 25%. Antibody reactivity was always cytoplasmic. Immunoblotting shows the antibody is reactive with a component of M(r) 230,000 not detected by HMFG 2. A significant association was found between lack of reactivity and improved disease-free interval (0.005 > P > 0.001) and survival (0.02 > P > 0.01). Subdivision of cases on the basis of node status showed that staining could refine survival data. A decreased reactivity of LU-BCRU-G7 was observed after pretreatment with beta-galactosidase but not a sialidase or beta-N-acetylhexosaminodase indicating that non-reducing terminal galactose residues in beta 1-3 or beta 1-4 linkages may be involved in the antibody binding site. This approach has identified a useful and novel prognostic marker in early stage human breast carcinoma.