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Airway mucus is a major barrier to the delivery of lipid-based nanoparticles in chronic airway diseases such as cystic fibrosis (CF). Receptor-Targeted Nanocomplexes (RTN), comprise mixtures of cationic lipids and bifunctional peptides with receptor-targeting and nucleic acid packaging properties. The aim of this study is to improve the mucus-penetrating properties of cationic siRNA and mRNA RTNs by combining them with low molecular weight alginate oligosaccharides, OligoG and OligoM. Cationic RTNs formulated with either alginate become strongly anionic, while PEGylated messenger RNA (mRNA) and short interfering RNA (siRNA) RTNs remain cationic. Both alginates enhance mucus diffusion rates of cationic siRNA and mRNA RTNs in a static mucus barrier diffusion model, with OligoG particularly effective. PEGylation also enhance mucus diffusion rates of siRNA RTNs but not mRNA RTNs. Electron microscopy shows that RTNs remained intact after mucosal transit. The transfection efficiency of OligoM-coated mRNA RTNs is better than those coated with OligoG or PEG, and similar to cationic RTNs. In siRNA RTN transfections, OligoM is better than OligoG although 1% PEG is slightly better than both. The combination of cationic RTNs and alginate oligosaccharides represents a promising alternative to PEGylation for epithelial delivery of genetic therapies across the mucus barrier while retaining transfection efficiency., (© 2024 The Author(s). Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Oligosaccharides from uronic acid-containing polysaccharides can be produced either by chemical or enzymatic degradation. The benefit of using enzymes, called lyases, is their high specificity for various glycosidic linkages. Lyases cleave the polysaccharide chain by an β-elimination reaction, yielding oligosaccharides with an unsaturated sugar (4-deoxy-l-erythro-hex-4-enepyranosyluronate) at the non-reducing end. In this work we have systematically studied acid degradation of unsaturated uronic acid oligosaccharides. Based on these findings, a method for preparing saturated oligosaccharides by enzymatic degradation of uronic acid-containing polysaccharides was developed. This results in oligosaccharides with a pre-defined distribution and proportion of sugar residues compared to the products of chemical degradation, while maintaining the chemical structure of the non-reducing end. The described method was demonstrated for generating saturated oligosaccharides of alginate, heparin and polygalacturonic acid. In the case of alginate, the ratio of hydrolysis rate of Δ-G and Δ-M linkages to that of G-G and M-M linkages, respectively, was found to be approximately 65 and 43, at pH* 3.4, 90 °C. Finally, this method has been demonstrated to be superior in the production of α-l-guluronate oligosaccharides with a lower content of β-d-mannuronate residues compared to what can be achieved using chemical depolymerization alone., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2024. Published by Elsevier Ltd.)

Low molecular weight alginate oligosaccharides have been shown to exhibit anti-microbial activity against a range of multi-drug resistant bacteria, including Pseudomonas aeruginosa . Previous studies suggested that the disruption of calcium (Ca 2+ )-DNA binding within bacterial biofilms and dysregulation of quorum sensing (QS) were key factors in these observed effects. To further investigate the contribution of Ca 2+ binding, G-block (OligoG) and M-block alginate oligosaccharides (OligoM) with comparable average size DPn 19 but contrasting Ca 2+ binding properties were prepared. Fourier-transform infrared spectroscopy demonstrated prolonged binding of alginate oligosaccharides to the pseudomonal cell membrane even after hydrodynamic shear treatment. Molecular dynamics simulations and isothermal titration calorimetry revealed that OligoG exhibited stronger interactions with bacterial LPS than OligoM, although this difference was not mirrored by differential reductions in bacterial growth. While confocal laser scanning microscopy showed that both agents demonstrated similar dose-dependent reductions in biofilm formation, OligoG exhibited a stronger QS inhibitory effect and increased potentiation of the antibiotic azithromycin in minimum inhibitory concentration and biofilm assays. This study demonstrates that the anti-microbial effects of alginate oligosaccharides are not purely influenced by Ca 2+ -dependent processes but also by electrostatic interactions that are common to both G-block and M-block structures.

Background: The increasing prevalence of invasive fungal infections in immuno-compromised patients is a considerable cause of morbidity and mortality. With the rapid emergence of antifungal resistance and an inadequate pipeline of new therapies, novel treatment strategies are now urgently required., Methods: The antifungal activity of the alginate oligosaccharide OligoG in conjunction with nystatin was tested against a range of Candida spp. ( C. albicans , C. glabrata , C. parapsilosis , C. auris , C. tropicalis and C. dubliniensis ), in both planktonic and biofilm assays, to determine its potential clinical utility to enhance the treatment of candidal infections. The effect of OligoG (0-6%) ± nystatin on Candida spp. was examined in minimum inhibitory concentration (MIC) and growth curve assays. Antifungal effects of OligoG and nystatin treatment on biofilm formation and disruption were characterized using confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM) and ATP cellular viability assays. Effects on the cell membrane were determined using permeability assays and transmission electron microscopy (TEM)., Results: MIC and growth curve assays demonstrated the synergistic effects of OligoG (0-6%) with nystatin, resulting in an up to 32-fold reduction in MIC, and a significant reduction in the growth of C. parapsilosis and C. auris (minimum significant difference = 0.2 and 0.12 respectively). CLSM and SEM imaging demonstrated that the combination treatment of OligoG (4%) with nystatin (1 µg/ml) resulted in significant inhibition of candidal biofilm formation on glass and clinical grade silicone surfaces ( p < 0.001), with increased cell death ( p < 0.0001). The ATP biofilm disruption assay demonstrated a significant reduction in cell viability with OligoG (4%) alone and the combined OligoG/nystatin (MIC value) treatment ( p < 0.04) for all Candida strains tested. TEM studies revealed the combined OligoG/nystatin treatment induced structural reorganization of the Candida cell membrane, with increased permeability when compared to the untreated control ( p < 0.001)., Conclusions: Antimicrobial synergy between OligoG and nystatin against Candida spp. highlights the potential utility of this combination therapy in the prevention and topical treatment of candidal biofilm infections, to overcome the inherent tolerance of biofilm structures to antifungal agents., Competing Interests: DT and KH have received research funding from AlgiPharma AS. PR is the Chief Scientific Officer at AlgiPharma AS. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Powell, Adams, Quoraishi, Py, Oger, Gazze, Francis, von Ruhland, Owens, Rye, Hill, Pritchard and Thomas.)

OligoG has previously shown potentiation of aztreonam against Burkholderia cepacia complex (Bcc) through biofilm disruption. A randomized, double-blind, placebo-controlled cross-over design was used to evaluate safety and efficacy of inhaled OligoG as a therapy for Bcc-infected CF patients taking aztreonam. Subjects received OligoG (1050 mg daily) or matching placebo for 28-days. Of 14 subjects completing the study, 8 showed a mean decrease in total bacterial CFU's (0.82 log10) after OligoG treatment. There was a reduction in mean Bcc CFU's (2.19 log10) after OligoG treatment but this was not statistically significant. Rheology analysis showed improvements in phase-angle after OligoG, but there was no statistically significant improvement in lung function parameters. Six out of 12 QoL summary scores showed relative improvement after OligoG treatment compared to placebo. There was a favourable safety profile for OligoG. Potential for reducing Bcc warrants further investigation of OligoG for the treatment of infection in CF., Competing Interests: Declaration of Competing Interest RF and CS were investigators during the study and their institutions received standard clinical trial support from AlgiPharma. Investigators received support for travel to investigator meetings. No investigator received any personal funding to participate in the study. HF, and PDR hold stocks in AlgiPharma., (Copyright © 2022 European Cystic Fibrosis Society. All rights reserved.)

In a number of chronic respiratory diseases e.g. cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD), the production of viscous mucin reduces pulmonary function and represents an effective barrier to diffusion of inhaled therapies e.g. antibiotics. Here, a 2-compartment Transwell model was developed to study impaired diffusion of the antibiotic colistin across an artificial sputum (AS) matrix/medium and to quantify its antimicrobial activity against Pseudomonas aeruginosa NH57388A biofilms (alone and in combination with mucolytic therapy). High-performance liquid chromatography coupled with fluorescence detection (HPLC-FLD) revealed that the presence of AS medium significantly reduced the rate of colistin diffusion (> 85% at 48 h; p < 0.05). Addition of alginate oligosaccharide (OligoG CF-5/20) significantly improved colistin diffusion by 3.7 times through mucin-rich AS medium (at 48 h; p < 0.05). Increased diffusion of colistin with OligoG CF-5/20 was shown (using confocal laser scanning microscopy and COMSTAT image analysis) to be associated with significantly increased bacterial killing (p < 0.05). These data support the use of this model to study drug and small molecule delivery across clinically-relevant diffusion barriers. The findings indicate the significant loss of colistin and reduced effectiveness that occurs with mucin binding, and support the use of mucolytics to improve antimicrobial efficacy and lower antibiotic exposure., (© 2022. The Author(s).)

Culture-independent microbiota analysis is widely used in research and being increasingly used in translational studies. However, methods for standardisation and application of these analyses in clinical trials are limited. Here we report the microbiota analysis that accompanied two phase 2b clinical trials of the novel, non-antibiotic therapy OligoG CF-5/20 for cystic fibrosis (CF) lung infection. Standardised protocols (DNA extraction, PCR, qPCR and 16S rRNA gene sequencing analysis) were developed for application to the Pseudomonas aeruginosa (NCT02157922) and Burkholderia cepacia complex (NCT02453789) clinical trials involving 45 and 13 adult trial participants, respectively. Microbiota analysis identified that paired sputum samples from an individual participant, taken within 2 h of each other, had reproducible bacterial diversity profiles. Although culture microbiology had identified patients as either colonised by P. aeruginosa or B. cepacia complex species at recruitment, microbiota analysis revealed patient lung infection communities were not always dominated by these key CF pathogens. Microbiota profiles were patient-specific and remained stable over the course of both clinical trials (6 sampling points over the course of 140 days). Within the Burkholderia trial, participants were infected with B. cenocepacia (n = 4), B. multivorans (n = 6), or an undetermined species (n = 3). Colonisation with either B. cenocepacia or B. multivorans influenced the overall bacterial community structure in sputum. Overall, we have shown that sputum microbiota in adults with CF is stable over a 2 h time-frame, suggesting collection of a single sample on a collection day is sufficient to capture the microbiota diversity. Despite the uniform pathogen culture-positivity status at recruitment, trial participants were highly heterogeneous in their lung microbiota. Understanding the microbiota profiles of individuals with CF ahead of future clinical trials would be beneficial in the context of patient stratification and trial design., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Chronic Pseudomonas aeruginosa lung infections in cystic fibrosis (CF) evolve to generate environmentally adapted biofilm communities, leading to increased patient morbidity and mortality. OligoG CF-5/20, a low-molecular-weight inhaled alginate oligomer therapy, is currently in phase IIb/III clinical trials in CF patients. Experimental evolution of P. aeruginosa in response to OligoG CF-5/20 was assessed using a bead biofilm model allowing continuous passage (45 days; ∼245 generations). Mutants isolated after OligoG CF-5/20 treatment typically had a reduced biofilm-forming ability and altered motility profile. Genotypically, OligoG CF-5/20 provided no selective pressure on genomic mutations within morphotypes. Chronic exposure to azithromycin, a commonly prescribed antibiotic in CF patients, with or without OligoG CF-5/20 in the biofilm evolution model also had no effect on rates of resistance acquisition. Interestingly, however, cross-resistance to other antibiotics (e.g., aztreonam) was reduced in the presence of OligoG CF-5/20. Collectively, these findings show no apparent adverse effects from long-term exposure to OligoG CF-5/20, instead resulting in both fewer colonies with multidrug resistance (MDR)-associated phenotypes and improved antibiotic susceptibility of P. aeruginosa IMPORTANCE The emergence of multidrug-resistant (MDR) pathogens within biofilms in the cystic fibrosis lung results in increased morbidity. An inhalation therapy derived from alginate, OligoG CF-5/20, is currently in clinical trials for cystic fibrosis patients. OligoG CF-5/20 has been shown to alter sputum viscoelasticity, disrupt mucin polymer networks, and disrupt MDR pseudomonal biofilms. Long-term exposure to inhaled therapeutics may induce selective evolutionary pressures on bacteria within the lung biofilm. Here, a bead biofilm model with repeated exposure of P. aeruginosa to OligoG CF-5/20 (alone and in combination with azithromycin) was conducted to study these long-term effects and characterize the phenotypic and genotypic adaptations which result. These findings, over 6 weeks, show that long-term use of OligoG CF-5/20 does not lead to extensive mutational changes and may potentially decrease the pathogenicity of the bacterial biofilm and improve the susceptibility of P. aeruginosa to other classes of antibiotics., (Copyright © 2021 Oakley et al.)

Alginates are one of the major polysaccharide constituents of marine brown algae in commercial manufacturing. However, the content and composition of alginates differ according to the distinct parts of these macroalgae and have a direct impact on the concentration of guluronate and subsequent commercial value of the final product. The Azotobacter vinelandii mannuronan C-5 epimerases AlgE1 and AlgE4 were used to determine their potential value in tailoring the production of high guluronate low-molecular-weight alginates from two sources of high mannuronic acid alginates, the naturally occurring harvested brown algae ( Ascophyllum nodosum, Durvillea potatorum , Laminaria hyperborea and Lessonia nigrescens ) and a pure mannuronic acid alginate derived from fermented production of the mutant strain of Pseudomonas fluorescens NCIMB 10,525. The mannuronan C-5 epimerases used in this study increased the content of guluronate from 32% up to 81% in both the harvested seaweed and bacterial fermented alginate sources. The guluronate-rich alginate oligomers subsequently derived from these two different sources showed structural identity as determined by proton nuclear magnetic resonance ( 1 H NMR), high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and size-exclusion chromatography with online multi-angle static laser light scattering (SEC-MALS). Functional identity was determined by minimum inhibitory concentration (MIC) assays with selected bacteria and antibiotics using the previously documented low-molecular-weight guluronate enriched alginate OligoG CF-5/20 as a comparator. The alginates produced using either source showed similar antibiotic potentiation effects to the drug candidate OligoG CF-5/20 currently in development as a mucolytic and anti-biofilm agent. These findings clearly illustrate the value of using epimerases to provide an alternative production route for novel low-molecular-weight alginates.

The recent emergence of resistance to colistin, an antibiotic of last resort with dose-limiting toxicity, has highlighted the need for alternative approaches to combat infection. This study aimed to generate and characterise alginate oligosaccharide ("OligoG")-polymyxin (polymyxin B and E (colistin)) conjugates to improve the effectiveness of these antibiotics. OligoG-polymyxin conjugates (amide- or ester-linked), with molecular weights of 5200-12,800 g/mol and antibiotic loading of 6.1-12.9% w / w , were reproducibly synthesised. In vitro inflammatory cytokine production (tumour necrosis factor alpha (TNFα) ELISA) and cytotoxicity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) of colistin (2.2-9.3-fold) and polymyxin B (2.9-27.2-fold) were significantly decreased by OligoG conjugation. Antimicrobial susceptibility tests (minimum inhibitory concentration (MIC), growth curves) demonstrated similar antimicrobial efficacy of ester- and amide-linked conjugates to that of the parent antibiotic but with more sustained inhibition of bacterial growth. OligoG-polymyxin conjugates exhibited improved selectivity for Gram-negative bacteria in comparison to mammalian cells (approximately 2-4-fold). Both OligoG-colistin conjugates caused significant disruption of Pseudomonas aeruginosa biofilm formation and induced bacterial death (confocal laser scanning microscopy). When conjugates were tested in an in vitro "time-to-kill" (TTK) model using Acinetobacter baumannii , only ester-linked conjugates reduced viable bacterial counts (~2-fold) after 4 h. Bi-functional OligoG-polymyxin conjugates have potential therapeutic benefits in the treatment of multidrug-resistant (MDR) Gram-negative bacterial infections, directly reducing toxicity whilst retaining antimicrobial and antibiofilm activities.

Background: OligoG is a low molecular-weight alginate oligosaccharide that improves the viscoelastic properties of cystic fibrosis (CF) mucus and disrupts biofilms, thereby potentiating the activity of antimicrobial agents. The efficacy of inhaled OligoG was evaluated in adult patients with CF., Methods: A randomised, double-blind, placebo-controlled multicentre crossover study was used to demonstrate safety and efficacy of inhaled dry powder OligoG. Subjects were randomly allocated to receive OligoG 1050 mg per day (10 capsules three times daily) or matching placebo for 28 days, with 28-day washout periods following each treatment period. The primary end-point was absolute change in percentage predicted forced expiratory volume in 1 s (FEV 1 ) at the end of 28-day treatment. The intention-to-treat (ITT) population (n=65) was defined as randomised to treatment with at least one administration of study medication and post-dosing evaluation., Results: In this study, 90 adult subjects were screened and 65 were randomised. Statistically significant improvement in FEV 1 was not observed in the ITT population. Adverse events included nasopharyngitis, cough and pulmonary exacerbation. The number and proportions of patients with adverse events and serious adverse events were similar between OligoG and placebo group., Conclusions: Inhalation of OligoG-dry powder over 28 days was safe in adult CF subjects. Statistically significant improvement of FEV 1 was not reached. The planned analyses did not indicate a significant treatment benefit with OligoG compared to placebo. Post hoc exploratory analyses showed subgroup results that indicate that further studies of OligoG in this patient population are justified., Competing Interests: Conflict of interest: S. van Koningsbruggen-Rietschel reports grants from Horizon 2020 and personal fees from DZIF, outside the submitted work. Conflict of interest: J.C. Davies reports work on an advisory board and as a clinical trial lead for Algipharma AS, work as UK Lead Investigator and on an advisory board for Bayer AG, work on an advisory board for Boehringer Ingelheim Pharma GmbH & Co. KG, work on an advisory board and clinical trial leadership for Galapagos NV, advisory and clinical trial design assistance for ImevaX GmbH, work on an advisory board for Nivalis Therapeutics, Inc., work on an advisory board and clinical trial design advice for ProQR Therapeutics III B.V., advisory work and clinical trial leadership for Proteostasis Therapeutics, Inc., advisory work for Raptor Pharmaceuticals, Inc., work on an advisory board and National Co-ord/Global Co-I for Vertex Pharmaceuticals (Europe) Limited, work on advisory boards for Enterprise, Novartis, Pulmocide and Flatley, grants from the CF Trust, and educational activities for Teva, outside the submitted work. Conflict of interest: T. Pressler has nothing to disclose. Conflict of interest: R. Fischer has nothing to disclose. Conflict of interest: G. MacGregor has nothing to disclose. Conflict of interest: S.H. Donaldson reports grants from AlgiPharma during the conduct of the study; and grants from Vertex Pharmaceuticals, AstraZeneca and Proteostasis Therapeutics outside the submitted work. Conflict of interest: K. Smerud reports that his employer, Smerud Medical Research International AS, is a contract research organisation that delivered clinical trial management services (clinical trial management, clinical trial applications, data management, statistical planning and analysis, monitoring, and medical writing) to Algipharma and was remunerated for that work. Conflict of interest: N. Meland reports grants from AlgiPharma AS during the conduct of the study. Conflict of interest: J. Mortensen has nothing to disclose. Conflict of interest: M.Ø. Fosbøl has nothing to disclose. Conflict of interest: D.G. Downey reports grants and personal fees from Vertex, Proteostasis, Chiesi and Gilead, outside the submitted work. Conflict of interest: A.H. Myrset reports grants from Cystic Fibrosis Foundation during the conduct of the study, and holds stock in AlgiPharma AB outside the submitted work. Conflict of interest: H. Flaten reports grants from Cystic Fibrosis Foundation during the conduct of the study; and is a qualified person for Pharmacovigilance for AlgiPharma and holds stock in AlgiPharma AB, outside the submitted work. Conflict of interest: P.D. Rye reports grants from Cystic Fibrosis Foundation during the conduct of the study, and is Chief Scientific Officer at AlgiPharma and holds stock in AlgiPharma AB, outside the submitted work; in addition, he has patents WO 2015/128495 and WO 2016/151051 pending., (Copyright ©ERS 2020.)

Cellulose nanofibrils (CNFs) from wood pulp are a renewable material possessing advantages for biomedical applications because of their customizable porosity, mechanical strength, translucency, and environmental biodegradability. Here, we investigated the growth of multispecies wound biofilms on CNF formulated as aerogels and films incorporating the low-molecular-weight alginate oligosaccharide OligoG CF-5/20 to evaluate their structural and antimicrobial properties. Overnight microbial cultures were adjusted to 2.8 × 10 9 colony-forming units (cfu) mL -1 in Mueller Hinton broth and growth rates of Pseudomonas aeruginosa PAO1 and Staphylococcus aureus 1061A monitored for 24 h in CNF dispersions sterilized by γ-irradiation. Two CNF formulations were prepared (20 g m -2 ) with CNF as air-dried films or freeze-dried aerogels, with or without incorporation of an antimicrobial alginate oligosaccharide (OligoG CF-5/20) as a surface coating or bionanocomposite, respectively. The materials were structurally characterized by scanning electron microscopy (SEM) and laser profilometry (LP). The antimicrobial properties of the formulations were assessed using single- and mixed-species biofilms grown on the materials and analyzed using LIVE/DEAD staining with confocal laser scanning microscopy (CLSM) and COMSTAT software. OligoG-CNF suspensions significantly decreased the growth of both bacterial strains at OligoG concentrations >2.58% ( P < 0.05). SEM showed that aerogel-OligoG bionanocomposite formulations had a more open three-dimensional structure, whereas LP showed that film formulations coated with OligoG were significantly smoother than untreated films or films incorporating PEG400 as a plasticizer ( P < 0.05). CLSM of biofilms grown on films incorporating OligoG demonstrated altered biofilm architecture, with reduced biomass and decreased cell viability. The OligoG-CNF formulations as aerogels or films both inhibited pyocyanin production ( P < 0.05). These novel CNF formulations or bionanocomposites were able to modify bacterial growth, biofilm development, and virulence factor production in vitro. These data support the potential of OligoG and CNF bionanocomposites for use in biomedical applications where prevention of infection or biofilm growth is required.

Acquisition of a mucoid phenotype by Pseudomonas sp. in the lungs of cystic fibrosis (CF) patients, with subsequent over-production of extracellular polymeric substance (EPS), plays an important role in mediating the persistence of multi-drug resistant (MDR) infections. The ability of a low molecular weight (Mn = 3200 g mol -1 ) alginate oligomer (OligoG CF-5/20) to modify biofilm structure of mucoid Pseudomonas aeruginosa (NH57388A) was studied in vitro using scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM) with Texas Red (TxRd®)-labelled OligoG and EPS histochemical staining. Structural changes in treated biofilms were quantified using COMSTAT image-analysis software of CLSM z-stack images, and nanoparticle diffusion. Interactions between the oligomers, Ca 2+ and DNA were studied using molecular dynamics (MD) simulations, Fourier transform infrared spectroscopy (FTIR) and isothermal titration calorimetry (ITC). Imaging demonstrated that OligoG treatment (≥0.5%) inhibited biofilm formation, revealing a significant reduction in both biomass and biofilm height ( P  < 0.05). TxRd®-labelled oligomers readily diffused into established (24 h) biofilms. OligoG treatment (≥2%) induced alterations in the EPS of established biofilms; significantly reducing the structural quantities of EPS polysaccharides, and extracellular (e)DNA ( P  < 0.05) with a corresponding increase in nanoparticle diffusion ( P  < 0.05) and antibiotic efficacy against established biofilms. ITC demonstrated an absence of rapid complex formation between DNA and OligoG and confirmed the interactions of OligoG with Ca 2+ evident in FTIR and MD modelling. The ability of OligoG to diffuse into biofilms, potentiate antibiotic activity, disrupt DNA-Ca 2+ -DNA bridges and biofilm EPS matrix highlights its potential for the treatment of biofilm-related infections., Competing Interests: D.W.T. has a consultancy relationship and has, with K.E.H., received research funding from AlgiPharma AS. P.D.R. is a director/owner of AlgiPharma AS. The remaining authors declare no competing interests.

Pseudomonas aeruginosa plays a major role in many chronic infections. Its ability to readily form biofilms contributes to its success as an opportunistic pathogen and its resistance/tolerance to antimicrobial/antibiotic therapy. A low-molecular-weight alginate oligomer (OligoG CF-5/20) derived from marine algae has previously been shown to impair motility in P. aeruginosa biofilms and disrupt pseudomonal biofilm assembly. As these bacterial phenotypes are regulated by quorum sensing (QS), we hypothesized that OligoG CF-5/20 may induce alterations in QS signaling in P. aeruginosa QS regulation was studied by using Chromobacterium violaceum CV026 biosensor assays that showed a significant reduction in acyl homoserine lactone (AHL) production following OligoG CF-5/20 treatment (≥2%; P < 0.05). This effect was confirmed by liquid chromatography-mass spectrometry analysis of C 4 -AHL and 3-oxo-C 12 -AHL production (≥2%; P < 0.05). Moreover, quantitative PCR showed that reduced expression of both the las and rhl systems was induced following 24 h of treatment with OligoG CF-5/20 (≥0.2%; P < 0.05). Circular dichroism spectroscopy indicated that these alterations were not due to steric interaction between the AHL and OligoG CF-5/20. Confocal laser scanning microscopy (CLSM) and COMSTAT image analysis demonstrated that OligoG CF-5/20-treated biofilms had a dose-dependent decrease in biomass that was associated with inhibition of extracellular DNA synthesis (≥0.5%; P < 0.05). These changes correlated with alterations in the extracellular production of the pseudomonal virulence factors pyocyanin, rhamnolipids, elastase, and total protease ( P < 0.05). The ability of OligoG CF-5/20 to modify QS signaling in P. aeruginosa PAO1 may influence critical downstream functions such as virulence factor production and biofilm formation., (Copyright © 2018 American Society for Microbiology.)

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., (© 2017 The Society for Applied Microbiology.)

In chronic respiratory disease, the formation of dense, 3-dimensional "microcolonies" by Pseudomonas aeruginosa within the airway plays an important role in contributing to resistance to treatment. An in vitro biofilm model of pseudomonal microcolony formation using artificial-sputum (AS) medium was established to study the effects of low-molecular-weight alginate oligomers (OligoG CF-5/20) on pseudomonal growth, microcolony formation, and the efficacy of colistin. The studies employed clinical cystic fibrosis (CF) isolates ( n = 3) and reference nonmucoid and mucoid multidrug-resistant (MDR) CF isolates ( n = 7). Bacterial growth and biofilm development and disruption were studied using cell viability assays and image analysis with scanning electron and confocal laser scanning microscopy. Pseudomonal growth in AS medium was associated with increased ATP production ( P < 0.05) and the formation (at 48 h) of discrete (>10-μm) microcolonies. In conventional growth medium, colistin retained an ability to inhibit growth of planktonic bacteria, although the MIC was increased (0.1 to 0.4 μg/ml) in AS medium compared to Mueller-Hinton (MH) medium. In contrast, in an established-biofilm model in AS medium, the efficacy of colistin was decreased. OligoG CF-5/20 (≥2%) treatment, however, induced dose-dependent biofilm disruption ( P < 0.05) and led to colistin retaining its antimicrobial activity ( P < 0.05). While circular dichroism indicated that OligoG CF-5/20 did not change the orientation of the alginate carboxyl groups, mass spectrometry demonstrated that the oligomers induced dose-dependent (>0.2%; P < 0.05) reductions in pseudomonal quorum-sensing signaling. These findings reinforce the potential clinical significance of microcolony formation in the CF lung and highlight a novel approach to treat MDR pseudomonal infections., (Copyright © 2017 American Society for Microbiology.)

The goal of this study was to determine whether the guluronate (G) rich alginate OligoG CF-5/20 (OligoG) could detach cystic fibrosis (CF) mucus by calcium chelation, which is also required for normal mucin unfolding. Since bicarbonate secretion is impaired in CF, leading to insufficient mucin unfolding and thereby attached mucus, and since bicarbonate has the ability to bind calcium, we hypothesized that the calcium chelating property of OligoG would lead to detachment of CF mucus. Indeed, OligoG could compete with the N-terminus of the MUC2 mucin for calcium binding as shown by microscale thermophoresis. Further, effects on mucus thickness and attachment induced by OligoG and other alginate fractions of different length and composition were evaluated in explants of CF mouse ileum mounted in horizontal Ussing-type chambers. OligoG at 1.5% caused effective detachment of CF mucus and the most potent alginate fraction tested, the poly-G fraction of about 12 residues, had similar potency compared to OligoG whereas mannuronate-rich (M) polymers had minimal effect. In conclusion, OligoG binds calcium with appropriate affinity without any overt harmful effect on the tissue and can be exploited for treating mucus stagnation., (© 2017 John Wiley & Sons Australia, Ltd.)

Concerns about acquisition of antibiotic resistance have led to increasing demand for new antimicrobial therapies. OligoG CF-5/20 is an alginate oligosaccharide previously shown to have antimicrobial and antibiotic potentiating activity. We investigated the structural modification of the bacterial cell wall by OligoG CF-5/20 and its effect on membrane permeability. Binding of OligoG CF-5/20 to the bacterial cell surface was demonstrated in Gram-negative bacteria. Permeability assays revealed that OligoG CF-5/20 had virtually no membrane-perturbing effects. Lipopolysaccharide (LPS) surface charge and aggregation were unaltered in the presence of OligoG CF-5/20. Small angle neutron scattering and circular dichroism spectroscopy showed no substantial change to the structure of LPS in the presence of OligoG CF-5/20, however, isothermal titration calorimetry demonstrated a weak calcium-mediated interaction. Metabolomic analysis confirmed no change in cellular metabolic response to a range of osmolytes when treated with OligoG CF-5/20. This data shows that, although weak interactions occur between LPS and OligoG CF-5/20 in the presence of calcium, the antimicrobial effects of OligoG CF-5/20 are not related to the induction of structural alterations in the LPS or cell permeability. These results suggest a novel mechanism of action that may avoid the common route in acquisition of resistance via LPS structural modification.

Background: Cystic fibrosis (CF) patients experience intestinal complications characterized by the accumulation of thick viscous mucus. CF mice were utilized to determine if a novel guluronate oligomer, OligoG, may be a potential therapy in reducing intestinal mucus and subsequent CF-related intestinal manifestations., Methods: Intestinal transit, intestinal histology, survival and growth were examined in wildtype and CF mice on regular water and OligoG., Conclusions: OligoG improves intestinal transit and survival in CF mice by reducing the accumulation of intestinal mucus. OligoG's ability to directly bind mucin, disrupt mucin interaction and/or sequester calcium allowing for mucin expansion may explain the decrease in mucus accumulation., (Copyright © 2016 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Biofilm growth is a universal survival strategy for bacteria, providing an effective and resilient approach for survival in an otherwise hostile environment. In the context of an infection, a biofilm provides resistance and tolerance to host immune defenses and antibiotics, allowing the biofilm population to survive and thrive under conditions that would destroy their planktonic counterparts. Therefore, the disruption of the biofilm is a key step in eradicating persistent bacterial infections, as seen in many types of chronic disease. In these studies, we used both in vitro minimum biofilm eradication concentration (MBEC) assays and an in vivo model of chronic biofilm infection to demonstrate the biofilm-disrupting effects of an alginate oligomer, OligoG CF-5/20. Biofilm infections were established in mice by tracheal instillation of a mucoid clinical isolate of Pseudomonas aeruginosa embedded in alginate polymer beads. The disruption of the biofilm by OligoG CF-5/20 was observed in a dose-dependent manner over 24 h, with up to a 2.5-log reduction in CFU in the infected mouse lungs. Furthermore, in vitro assays showed that 5% OligoG CF-5/20 significantly reduced the MBEC for colistin from 512 μg/ml to 4 μg/ml after 8 h. These findings support the potential for OligoG CF-5/20 as a biofilm disruption agent which may have clinical value in reducing the microbial burden in chronic biofilm infections., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The host- and bacteria-derived extracellular polysaccharide coating of the lung is a considerable challenge in chronic respiratory disease and is a powerful barrier to effective drug delivery. A low molecular weight 12-15-mer alginate oligosaccharide (OligoG CF-5/20), derived from plant biopolymers, was shown to modulate the polyanionic components of this coating. Molecular modeling and Fourier transform infrared spectroscopy demonstrated binding between OligoG CF-5/20 and respiratory mucins. Ex vivo studies showed binding induced alterations in mucin surface charge and porosity of the three-dimensional mucin networks in cystic fibrosis (CF) sputum. Human studies showed that OligoG CF-5/20 is safe for inhalation in CF patients with effective lung deposition and modifies the viscoelasticity of CF-sputum. OligoG CF-5/20 is the first inhaled polymer therapy, represents a novel mechanism of action and therapeutic approach for the treatment of chronic respiratory disease, and is currently in Phase IIb clinical trials for the treatment of CF.

The oligosaccharide OligoG, an alginate derived from seaweed, has been shown to have anti-bacterial and anti-biofilm properties and potentiates the activity of selected antibiotics against multi-drug resistant bacteria. The ability of OligoG to perturb fungal growth and potentiate conventional antifungal agents was evaluated using a range of pathogenic fungal strains. Candida (n = 11) and Aspergillus (n = 3) spp. were tested using germ tube assays, LIVE/DEAD staining, scanning electron microscopy (SEM), atomic force microscopy (AFM) and high-throughput minimum inhibition concentration assays (MICs). In general, the strains tested showed a significant dose-dependent reduction in cell growth at ≥6% OligoG as measured by optical density (OD600; P<0.05). OligoG (>0.5%) also showed a significant inhibitory effect on hyphal growth in germ tube assays, although strain-dependent variations in efficacy were observed (P<0.05). SEM and AFM both showed that OligoG (≥2%) markedly disrupted fungal biofilm formation, both alone, and in combination with fluconazole. Cell surface roughness was also significantly increased by the combination treatment (P<0.001). High-throughput robotic MIC screening demonstrated the potentiating effects of OligoG (2, 6, 10%) with nystatin, amphotericin B, fluconazole, miconazole, voriconazole or terbinafine with the test strains. Potentiating effects were observed for the Aspergillus strains with all six antifungal agents, with an up to 16-fold (nystatin) reduction in MIC. Similarly, all the Candida spp. showed potentiation with nystatin (up to 16-fold) and fluconazole (up to 8-fold). These findings demonstrate the antifungal properties of OligoG and suggest a potential role in the management of fungal infections and possible reduction of antifungal toxicity.

Pseudomonas aeruginosa (PA) biofilm-associated infections are a common cause of morbidity in chronic respiratory disease and represent a therapeutic challenge. Recently, the ability of a novel alginate oligomer (OligoG) to potentiate the effect of antibiotics against gram-negative, multi-drug-resistant bacteria and inhibit biofilm formation in vitro has been described. Interaction of OligoG with the cell surface of PA was characterized at the nanoscale using atomic force microscopy (AFM), zeta potential measurement (surface charge), and sizing measurements (dynamic light scattering). The ability of OligoG to modify motility was studied in motility assays. AFM demonstrated binding of OligoG to the bacterial cell surface, which was irreversible after exposure to hydrodynamic shear (5,500 × g). Zeta potential analysis (pH 5-9; 0.1-0.001 M NaCl) demonstrated that binding was associated with marked changes in the bacterial surface charge (-30.9 ± 0.8 to -47.0 ± 2.3 mV; 0.01 M NaCl [pH 5]; P < 0.001). Sizing analysis demonstrated that alteration of surface charge was associated with cell aggregation with a 2- to 3-fold increase in mean particle size at OligoG concentrations greater than 2% (914 ± 284 to 2599 ± 472 nm; 0.01 M NaCl [pH 5]; P < 0.001). These changes were associated with marked dose-dependent inhibition in bacterial swarming motility in PA and Burkholderia spp. The ability of OligoG to bind to a bacterial surface, modulate surface charge, induce microbial aggregation, and inhibit motility represents important direct mechanisms by which antibiotic potentiation and biofilm disruption is affected. These results highlight the value of combining multiple nanoscale technologies to further our understanding of the mechanisms of action of novel antibacterial therapies.

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.

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., (Copyright 2004 S. Karger AG, Basel.)

The almost overwhelming volume of information and new technological developments that has demanded so much of our scientific attention over the last decade will shortly revolutionize clinical diagnostics. Some of these developments are already affecting the working lives of scientists and clinicians alike, but will eventually require a greater understanding and acceptance from a much wider audience. Therefore it is important in our current scientific endeavor and commercial enthusiasm for molecular diagnostics that we maintain some awareness of the significant obstacles that must be overcome if we are to see an appropriate, timely and widespread adoption of molecular diagnostic testing in oncology. This article presents a brief commentary on the current state of the art in molecular diagnostics in oncology and how this relates to a more personalized approach to treatment.

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., (Copyright 2003 S. Karger AG, Basel)

Tetraantennary peptides [glycine(n)-NHCH(2)](4)C can form stable noncovalent structures by self-assembly through intermolecular hydrogen bonding. The oligopeptide chains assemble as polyglycine II to yield submicron-sized, flat, one-molecule-thick sheets. Attachment of alpha-N-acetylneuraminic acid (Neu5Acalpha) to the terminal glycine residues gives rise to water-soluble assembled glycopeptides that are able to bind influenza virus multivalently and inhibit adhesion of the virus to cells 10(3)-fold more effectively than a monomeric glycoside of Neu5Acalpha. Another antiviral strategy based on virus-promoted assembly of the glycopeptides was also demonstrated. Consequently, the self-assembly principle offers new perspectives on the design of multivalent antivirals.

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., (Copyright 2001 S. Karger AG, Basel)

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., (Copyright 2001 S. Karger AG, Basel)

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?, (Copyright 2001 S. Karger AG, Basel)

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.

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.

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.

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.

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.

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.

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.

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.

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)

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.

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.