There is an urgent need for new high-quality markers for the early detection of hepatocellular carcinoma (HCC). angstrom strom et al. suggested that S2-bound alpha 1-acid glycoprotein (AGP) might be a promising marker. Consequently, we evaluated the predictive advantage of S2-bound AGP in the early detection of HCC. In a retrospective case-control study of patients chronically infected with hepatitis C virus (HCV) and treated with direct-acting antiviral agents (n = 93), we measured S2-bound AGP using the HepaCheC (R) ELISA kit (Glycobond AB, Linkoping, SE) at the start of treatment, end of treatment and follow-up (maximum: 78 months). Patients were retrospectively propensity score matched (1:2). Thirty-one patients chronically infected with HCV developed HCC after a sustained virological response, while 62 did not. In addition, samples of patients with chronic hepatitis B virus infection, metabolic dysfunction-associated steatotic liver disease and HCC of different etiologies were analysed. S2-bound AGP elevation in HCC patients was confirmed. However, we did not observe a predictive advantage of S2-bound AGP for the early detection of HCC during treatment and follow-up. Interestingly, S2-bound AGP levels correlated with aspartate aminotransferase (rho = .56, p = 9.5x10-15) and liver elastography (rho = .67, p = 2.2x10-16). Of note, S2-bound AGP decreased in patients chronically infected with HCV after treatment-induced HCV clearance. Fucosylated S2-bound AGP levels were elevated in patients with chronic HCV and HCC. The potential role of S2-bound AGP as a novel tumour marker requires further investigation.

Fucoidans are sulfated fucose-based polysaccharides that activate platelets and have pro- and anticoagulant effects; thus, they may have therapeutic value. In the present study, we show that 2 synthetic sulfated a-l-fucoside-pendant glycopolymers (with average monomeric units of 13 and 329) and natural fucoidans activate human platelets through a Src- and phosphatidylinositol 3-kinase (PI3K)-dependent and Syk-independent signaling cascade downstream of the platelet endothelial aggregation receptor 1 (PEAR1). Synthetic glycopolymers and natural fucoidan stimulate marked phosphorylation of PEAR1 and Akt, but not Syk. Platelet aggregation and Akt phosphorylation induced by natural fucoidan and synthetic glycopolymers are blocked by a monoclonal antibody to PEAR1. Direct binding of sulfated glycopolymers to epidermal like growth factor (EGF)-like repeat 13 of PEAR1 was shown by avidity-based extracellular protein interaction screen technology. In contrast, synthetic glycopolymers and natural fucoidans activate mouse platelets through a Src- and Syk-dependent pathway regulated by C-type lectin-like receptor 2 (CLEC-2) with only a minor role for PEAR1. Mouse platelets lacking the extracellular domain of GPIba and human platelets treated with GPIba-blocking antibodies display a reduced aggregation response to synthetic glycopolymers. We found that synthetic sulfated glycopolymers bind directly to GPIba, substantiating that GPIba facilitates the interaction of synthetic glycopolymers with CLEC-2 or PEAR1. Our results establish PEAR1 as the major signaling receptor for natural fucose-based polysaccharides and synthetic glycopolymers in human, but not in mouse, platelets. Sulfated a-l-fucoside-pendant glycopolymers are unique tools for further investigation of the physiological role of PEAR1 in platelets and beyond.

Gold nanoparticles coated with fucoidan-mimetic glycopolymers were synthesized that displayed good colloidal stability and promising anti-cancer properties. Fucoidan mimetic glycopolymers on their own were nontoxic, while glycopolymer coated gold nanoparticles displayed selective cytotoxicity to human colon cancer cell lines (HCT116) while it was non-toxic to mouse fibroblast cells (NIH3T3).

The sulfated marine polysaccharide fucoidan has been reported to have health benefits ranging from antivirus and anticancer properties to modulation of high blood pressure. Hence, they could enhance the biological function of materials for biomedical applications. However, the incorporation of fucoidan into biomaterials has been difficult, possibly due to its complex structure and lack of suitable functional groups for covalent anchoring to biomaterials. We have developed an approach for a rapid synthesis of fucoidanmimetic glycopolymer chains through cyanoxyl-mediated free-radical polymerization, a method suitable for chain-end functionalizing and subsequent linkage to biomaterials. The resulting sulfated and nonsulfated methacrylamido alpha-L-fucoside glycopolymers fucoidan-mimetic properties were studied in HSV-1 infection and platelet activation assays. The sulfated glycopolymer showed similar properties to natural fucoidan in inducing platelet activation and inhibiting HSV-1 binding and entry to cells, thus indicating successful syntheses of fucoidan-mimetic glycopolymers.

Background

Carbohydrate deficient transferrin (CDT) is used for detection of alcohol abuse and follow-up. High performance liquid chromatography (HPLC) of transferrin glycoforms is highly specific for identification of alcohol abuse, but unresolved disialo- and trisialotransferrin glycoforms sometimes makes interpretation difficult. The cause of this phenomenon is unknown, cannot be explained by genetic variants of transferrin, but seems to be associated with liver disease.

Methods

Nineteen serum samples showing di–tri bridging when analyzed by HPLC were collected. Transferrin was purified by affinity chromatography, and N-linked oligosaccharides were released enzymatically. The N-glycans were further analyzed by high performance anion-exchange chromatography with pulsed amperometric detection and MALDI-TOF mass spectrometry.

Results

The HPLC-analysis showed three different types of glycoform patterns. The N-glycans from fifteen samples showed patterns with increased number of triantennary structures containing one or two fucose residues. One sample contained an increased amount of triantennary glycans without fucose. Three samples showed a glycosylation pattern similar to normal transferrin.

Conclusions

The di–tri bridging phenomenon was associated with alterations in transferrin glycosylation in the majority of cases. Transferrin contained a higher extent of triantennary and often fucosylated N-linked oligosaccharides. These results may be important in future diagnostic approaches to liver diseases.

Lab-on-a-chip detection of fluorescence transduced chemical stimuli is demonstrated using fluidics and optical coupling disposable elements in a configuration compatible with distributed diagnosis.

Disposable optical elements are designed to separate excitation by total internal reflection using regular glass slides as optical light guide and fluidics support, while high dynamic range image acquisition with consumer cameras complement the platform to support a broad range of responses with a same configuration. Complementary tone mapping procedures are introduced to systematically double the sensitivity for selected range intervals.

Chemical sensitization to free fucose, a diagnostic marker for liver cirrhosis and several cancer forms, illustrates the platform capabilities for diagnosis targets.

Aberrant glycosylation is connected to several pathological conditions and lectins are useful tools to characterize glycosylated biomarkers. The Aleuria aurantia lectin (AAL) is of special interest since it interacts with all types of fucosylated saccharides. AAL has been expressed in Escherichia coil as a fully functional recombinant protein. Engineered variants of AAL have been developed with the aim of creating monovalent lectins with more homogenous binding characteristics. Four different forms of AAL were studied in the present work: native AAL purified from A. aurantia mushrooms, recombinant AAL dimer, recombinant AAL monomer and recombinant AAL site 2 (S2-AAL). The affinities of these AAL forms toward a number of saccharides were determined with weak affinity chromatography (WAC). Disaccharides with fucose linked alpha 1-3 to GIcNAc interacted with higher affinity compared to fucose linked alpha 1-6 or alpha 1-4 and the obtained dissociation constants (K-d) were in the range of 10 mu M for all AAL forms. Tetra- and pentasaccharides with fucose in alpha 1-2, alpha 1-3 or alpha 1-4 had K-d values ranging from 0.1 to 7 mM while a large alpha 1-6 fucosylated oligosaccharide had a K-d of about 20 mu M. The recombinant multivalent AAL forms and native AAL exhibited similar affinities toward all saccharides, but S2-AAL had a lower affinity especially regarding a sialic acid containing fucosylated saccharide. It was demonstrated that WAC is a valuable technique in determining the detailed binding profile of the lectins. Specific advantages with WAC include a low consumption of non-labeled saccharides, possibility to analyze mixtures and a simple procedure using standard HPLC equipment.

Background. Systemic corticosteroid treatment has been shown to exert some protection against renal deterioration in IgA nephropathy (IgAN) but is not commonly recommended for long-term use due to the well-known systemic side effects. In this study, we investigated the efficacy and safety of a new enteric formulation of the locally acting glucocorticoid budesonide (Nefecon (R)), designed to release the active compound in the ileocecal region. The primary objective was to evaluate the efficacy of targeted release budesonide on albuminuria. less thanbrgreater than less thanbrgreater thanMethods. Budesonide 8 mg/day was given to 16 patients with IgAN for 6 months, followed by a 3-month follow-up period. The efficacy was measured as change in 24-h urine albumin excretion, serum creatinine and estimated glomerular filtration rate (eGFR). less thanbrgreater than less thanbrgreater thanResults. The median relative reduction in urinary albumin excretion was 23% during the treatment period (interquartile range: -0.36 to -0.04, P = 0.04) with pretreatment values ranging from 0.3 to 6 g/24 h (median: 1.5 g/24 h). The median reduction in urine albumin peaked at 40% (interquartile range: -0.58 to -0.15) 2 months after treatment discontinuation. Serum creatinine was reduced by 6% (interquartile range: -0.12 to -0.02; P = 0.003), and eGFR [Modification of Diet in Renal Disease (MDRD)] increased similar to 8% (interquartile range: 0.02-0.16, P = 0.003) during treatment. No major corticosteroid-related side effects were observed. less thanbrgreater than less thanbrgreater thanConclusions. In the present pilot study, enteric budesonide targeted to the ileocecal region had a significant effect on urine albumin excretion, accompanied by a minor reduction of serum creatinine and a modest increase of eGFR calculated by the MDRD equation, while eGFR calculated from Cockcroft-Gault equation and cystatin C was not changed. Enteric budesonide may represent a new treatment of IgAN warranting further investigation.

Lectins have been widely used in structural and functional studies of complex carbohydrates. Lectins usually bind carbohydrates with relatively low affinity but compensate for this by multivalency. When using lectins in different biological and analytical assays the multivalent nature of lectins can sometimes produce unwanted reactions such as agglutination or precipitation of target glycoproteins. The mushroom lectin Aleuria aurantia binds to fucose-containing oligosaccharides. It is composed of two identical subunits where each subunit contains five binding sites for fucose. In the present study two forms of recombinant AAL were produced using site-directed mutagenesis. A monomeric form of AAL was produced by exchange of Tyr6 to Arg6, and a monovalent fragment of AAL was produced by insertion of a NdeI restriction enzyme cleavage site and a stop codon in the coding sequence. The AAL forms were expressed as His-tagged proteins in E.coli and purified by affinity chromatography. Binding properties of the two AAL forms were performed using hemagglutination assay, surface plasmon resonance and enzyme-linked lectin assay analyses. Both the monomeric AAL form (mAAL) and the monovalent AAL form (S2-AAL) retained their capacity to bind fucosylated oligosaccharides. However, both constructs exhibited properties that differed from the intact recombinant AAL (rAAL). Monomeric AAL showed similar binding affinities to fucosylated oligosaccharides compared to rAAL but had less hemagglutinating capacity. S2-AAL showed a lower binding affinity to fucosylated oligosaccharides and, in contrast to rAAL and mAAL, S2-AAL did not bind to sialylated fuco-oligosaccharides such as sialyl-Lex. The study shows that molecular engineering techniques may be a tool for producing lectins with more defined properties such as decreased valency and defined specificities and affinities. This may be very valuable for development of reliable diagnostic and biological assays for carbohydrate analysis.

We have recently shown that terminal sialic acid residues are essential for alpha(1)-acid glycoprotein (AGP)-induced Ca2+ mobilization in neutrophils. The aim of the present study was to establish the importance of sialic acid residues on AGP in modulating human neutrophil functions, with emphasis on the generation of reactive oxygen species (ROS). ROS were measured by luminol-enhanced chemiluminescence in isolated human neutrophils. We found that AGP did not provoke ROS generation in resting or L-selectin presensitized neutrophils. Moreover, AGP did not affect the N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced ROS generation, but it slightly suppressed opsonized zymosan-induced responses. However, when the neutrophils were prestimulated with fMLP, the subsequent addition of AGP provoked a marked ROS response. Dose-response studies and time studies revealed that the ROS generating capacity of AGP was highest at a concentration of 0.05 mg/ml and when given 3-10 min after addition of fMLP. A desialylated form of AGP or pretreatment of neutrophils with 3- and 6-sialyllactose caused a substantially lower ROS response in neutrophils prestimulated with fMLP. Our data show that AGP can stimulate a second ROS response in fMLP preactivated neutrophils and that terminal sialic acid residues on AGP play a crucial role in this regard.