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.

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.

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.

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.

Immunohistochemical distribution of a breast cancer-associated glycoprotein.

The tissue distribution and specificity of a glycoprotein of M(r) 230,000 kDa which has previously been identified from breast carcinomas in culture and shown to be tumour-associated, has been assessed using a polyclonal antiserum. A wide range of tissues has been examined immunohistochemically. The tissue distribution of the glycoprotein show differences between normal, benign and malignant breast and other epithelial tissues, and are clearly specific for epithelial cells. This glycoprotein as detected by the polyclonal antiserum P5252-2, was either absent or showed a minimal presence in normal breast tissues. Evidence of the expression of the glycoprotein in hyperplastic breast was observed but was considerably less than that seen for carcinomas, for which 70% had greater than 50% of cells exhibiting reactivity with P5252-2. There was no relationship with grade or node status. Similar striking differences in glycoprotein expression between non-neoplastic and neoplastic tissue were observed for stomach, large intestine, thyroid and to lesser extent ovary. The differences in the expression of this glycoprotein between normal and malignant tissues is of obvious clinical and pathological potential.

The glycosylphosphatidylinositol-anchored lymphocyte antigen CDw52 is associated with the epididymal maturation of human spermatozoa.

The CAMPATH-1 (CDw52) antigen is a small glycosylphosphatidylinositol (GPI) anchored glycoprotein with a mature peptide comprising only 12 amino acids. It is abundantly expressed on human lymphocytes and is an unusually good target for complement-mediated cell lysis. The immunosuppressive and lymphocyte-depleting effects of CAMPATH-1 antibodies are being tested in a variety of diseases. Here we show that the antigen is also expressed at a high level in the male reproductive system, being found in the epididymis, seminal vesicle, seminal plasma and on the surface of mature (but not testicular) spermatozoa. Its possible transfer from epithelial cells in the epididymis to maturing sperm may represent a novel method of acquisition of cell surface antigens. In the presence of human complement, CAMPATH-1 antibodies inhibited the motility of washed sperm. However, seminal plasma blocks antibody binding and can protect sperm from this cytotoxic effect.

The desire to control terminal health care and attitudes toward living wills.

The desire for control over one's health care is defined as preferring to directly influence the nature of that care. We attempted to measure the following relevant constructs and to examine the relationships among them: (1) desire-for-control (DCON) in a specific health care setting--a hypothetical situation in which a person was facing imminent death; (2) attitude toward an action perceived as enhancing control in that situation--signing a living will; and (3) intention to exercise control by performing that action--actually signing a living will. An analysis of variance (DCON x attitude) of intention to sign a living will yielded statistically significant main effects for DCON and attitude, although DCON was in a direction counter to that predicted. Possible explanations for the DCON finding were explored.

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.

Analysis of glycoproteins released from benign and malignant human breast: changes in size and fucosylation with malignancy.

Radiolabelled glycoproteins released into media from benign and malignant human breast tissue after 48 h organ culture have been analysed using SDS polyacrylamide gel electrophoresis and fluorography. Major differences were identified between benign and malignant tissues: (i) a considerably higher incidence of glycoproteins in the molecular weight range 210-280 kDa detected from carcinomas than benign samples, with incidence being greater in poorly differentiated tumours; (ii) fucosylation of these glycoproteins released from carcinomas but not benign breast; (iii) heterogeneity, particularly of fucosylation, between the carcinomas but consistency amongst benign breast. A glycoprotein of MW 230 kDa was of particular interest since it was not detected from any benign samples but was present in 11 of 17 carcinomas, when it was almost always fucosylated. This could prove to be a useful tumour marker.

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?

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.

Instructions in learning skills: an integrated approach.

The transition from school to university education and a medical school environment can be difficult even for the very best students. However, little appears to be done to assist students in making this transition and in developing study skills during the early stages of their training. This article outlines a scheme which has been called supplemental instruction. Although developed for medical students in the United States, it is particularly well suited to developing essential study skills in first-year medical students in the United Kingdom. The scheme has been successfully introduced into some degree and diploma subjects in this country, with improvement in course grades and lower attrition rates, but has yet to be introduced into medical education. Evaluation data for non-medical courses show that student participation in supplemental instruction significantly improves overall course marks and could be of significant value in the medical curriculum.

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.

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.

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.

Immunohistochemical expression of tumour-associated glycoprotein and polymorphic epithelial mucin in the human endometrium during the menstrual cycle.

An epitope defined by a second generation murine monoclonal antibody (LU-BCRU-G7) produced against a breast tumour-associated fucosylated glycoprotein of M(r) 230,000 was found to exhibit phase specific reactivity in human endometrium during the menstrual cycle. Distribution and cycle related expression of the LU-BCRU-G7 determinant was different from the staining patterns observed with antibodies that react with the polymorphic epithelial mucins, HMFG 1 and NCRC 11, as determined by immunohistochemistry. Initial expression of the LU-BCRU-G7 determinant was associated with the peri-basal region of glandular epithelial cells with maximal staining during the mid-secretory phase. Diffuse cytoplasmic staining was also observed from day 10 to day 26, with a marked increase in the early secretory phase. Reactivity of NCRC 11 also showed maximal expression in the midsecretory phase with no reactivity detected in early and mid-proliferative phases. The HMFG 1 defined epitope exhibited the opposite pattern of expression with maximal reactivity in the proliferative phase and little or no reactivity in the secretory phase. These findings suggest that expression of the LU-BCRU-G7, HMFG 1 and NCRC 11 defined determinants in the human glandular epithelium of the endometrium is differentially hormonally regulated, and may be of value as markers for endometrial function.

Expression of the pS2 peptide in primary breast carcinomas: comparison of membrane and cytoplasmic staining patterns.

The pS2 protein is oestrogen-regulated in breast cancer cell lines. Previous studies have shown a relationship to oestrogen receptor in primary breast carcinomas. This study examined 178 breast carcinomas for pS2 using immunohistochemistry. A high frequency (77 per cent) of positive tumours was found, using a 10 per cent cut-off point to define a positive tumour. There was no relationship with menopausal status or node status, a significant association with differentiation, a weak association with oestrogen receptor, and no association with progesterone receptor or overall survival. Two patterns of cellular localization were observed: cytoplasmic and membrane. The former showed a stronger relationship with oestrogen receptor status, although there were oestrogen receptor-negative tumours with marked pS2 staining. Membrane staining showed a stronger relationship with differentiation, with a staining pattern similar to that observed for milk fat globule membrane. The staining patterns observed may support a role for pS2 in a secretory mechanism. However, the expression and function of pS2 in breast carcinomas remain complex, and are not simply related to oestrogen regulation.

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.