Synthesis of the 6-deoxytalose-containing tri- and hexasaccharides of the O-antigen polysaccharide from Mesorhizobium huakuii IFO15243T

The synthesis of a trisaccharide and a hexasaccharide, the monomer and dimer of the repeating unit of O-antigen polysaccharide from Mesorhizobium huakuii IFO15243, has been accomplished through suitable protecting group manipulations and stereoselective glycosylation reactions starting from commercially available l-rhamnose. The target oligosaccharides in the form of their p-methoxyphenyl glycosides are suitable for further glycoconjugate formation via selective cleavage of this group.     

Synthesis of the Lewis b hexasaccharide and squarate acid-HSA conjugates thereof with various saccharide loadings

The Lewis b hexasaccharide, alpha-L-Fucp-(1 --> 2)-beta-D-Galp-(1 --> 3)-[alpha-L-Fucp-(1 --> 4)]-beta-D-GlcpNAc-(1 --> 3)-beta-D-Galp-(l --> 4)-beta-D-Glcp, has been synthesised using a convergent synthesis. Starting from ethyl 4,6-O-benzylidene-2-deoxy-2-phthalimido-1-thio-beta-D-glucopyranoside, a thioglycoside tetrasaccharide donor block, was constructed through two orthogonal glycosylations with glycosyl bromide donors. First, the galactose moiety was introduced using silver triflate as a promoter and then the two fucose residues under halide-assisted conditions. Finally, this tetrasaccharide was linked to a spacer-equipped 3I,4I-diol lactose acceptor in a DMTST-promoted coupling to give, after deprotection, the Lewis b hexasaccharide as its 2-aminoethyl spacer-equipped derivative. This was coupled to human serum albumin (HSA), using the squarate ester methodology, in various saccharide-protein ratios, to give neoglycoconjugates with different saccharide loadings in about 50%) efficiency.     

Synthesis of the Lewis b hexasaccharide and HSA-conjugates thereof

An efficient and short route has been elaborated for the aminopropyl spacer equipped Leb hexasaccharide. For the preparation of HSA-conjugates of this oligosaccharide, the use of disuccinimidyl suberate (DSS) and disuccinimidyl glutarate (DSG) as cross-linker reagents has been evaluated. This conjugation method emerged as being faster and easier to monitor by standard MALDI-TOF spectrometry than squarate ester based conjugations of similar efficiency if DSS is used as cross-linker.     

Chemoenzymatic synthesis of immunogenic meningococcal group C polysialic acid-tetanus Hc fragment glycoconjugates

Vaccination with meningococcal glycoconjugate vaccines has decreased the incidence of invasive meningitis worldwide. These vaccines contain purified capsular polysaccharides attached to a carrier protein. Because of derivatization chemistries used in the process, conjugation of polysaccharide to protein often results in heterogeneous mixtures. Well-defined vaccines are needed to determine the relationship between vaccine structure and generated immune response. Here, we describe efforts to produce well-defined vaccine candidates by chemoenzymatic synthesis. Chemically synthesized lactosides were substrates for recombinant sialyltransferase enzymes from Camplyobacter jejuni and Neisseria meningitidis serogroup C. These resulting oligosialic acids have the same α(2-9) sialic acid repeat structure as Neisseria polysaccharide capsule with the addition of a conjugatable azide aglycon. The degree of polymerization (DP) of carbohydrate products was controlled by inclusion of the inhibitor CMP-9-deoxy-NeuNAc. Polymers with estimated DP?<?47 (median DP 25) and DP?<?100 (median DP 51) were produced. The receptor binding domain of the tetanus toxin protein (TetHc) was coupled as a carrier to the enzymatically synthesized oligosialic acids. Recombinant TetHc was derivatized with an alkyne squarate. Protein modification sites were determined by trypsin proteolysis followed by LC/MS-MS^E analysis of peptides. Oligosialic acid azides were conjugated to modified TetHc via click chemistry. These chemoenzymatically prepared glycoconjugates were reactive in immunoassays with specific antibodies against either group C polysaccharide or TetHc. Sera of mice immunized with oligosialic acid-TetHc glycoconjugates contained much greater levels of polysaccharide-reactive IgG than the sera of control mice receiving unconjugated oligosialic acids. There was no apparent difference between glycoconjugates containing oligosaccharides of DP?<?47 and DP?<?100. These results suggest that chemoenzymatic synthesis may provide a viable method for making defined meningococcal vaccine candidates.     

Hyaluronan Breakdown Contributes to Immune Defense against Group A Streptococcus

Group A Streptococcus (GAS) commonly infects human skin and occasionally causes severe and life-threatening invasive diseases. The hyaluronan (HA) capsule of GAS has been proposed to protect GAS from host defense by mimicking endogenous HA, a large and abundant glycosaminoglycan in the skin. However, HA is degraded during tissue injury, and the functions of short-chain HA that is generated during infection have not been studied. To examine the impact of the molecular mass of HA on GAS infection, we established infection models in vitro and in vivo in which the size of HA was defined by enzymatic digestion or custom synthesis. We discovered that conversion of high molecular mass HA to low molecular mass HA facilitated GAS phagocytosis by macrophages and limited the severity of infection in mice. In contrast, native high molecular mass HA significantly impaired internalization by macrophages and increased GAS survival in murine blood. Thus, our data demonstrate that GAS virulence can be influenced by the size of HA derived from both the bacterium and host and suggest that high molecular mass HA facilitates GAS deep tissue infections, whereas the generation of short-chain HA can be protective.     

Long-chain noncoding RNA-GAS5/hsa-miR-138-5p attenuates high glucose-induced cardiomyocyte damage by targeting CYP11B2

Objective: Diabetic cardiomyopathy (DCM) is one of the complications experienced by patients with diabetes. In recent years, long noncoding RNAs (lncRNAs) have been investigated because of their role in the progression of various diseases, including DCM. The purpose of the present study was to explore the role of lncRNA GAS5 in high glucose (HG)-induced cardiomyocyte injury and apoptosis.Materials and methods: We constructed HG-induced AC16 cardiomyocytes and a streptozotocin (STZ)-induced rat diabetes model. GAS5 was overexpressed and knocked out at the cellular level, and GAS5 was knocked down by lentiviruses at the animal level to observe its effect on myocardial injury. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of GAS5. Cell proliferation and apoptosis after GAS5 knockout were detected by CCK-8, TUNEL, and flow cytometry assays. ELISA was used to detect the changes in myocardial enzyme content in cells and animal myocardial tissues during the action of GAS5 on myocardial injury.Results: GAS5 expression was up-regulated in HG-treated AC16 cardiomyocytes and the rat diabetic myocardial injury model. The down-regulation of GAS5 could inhibit HG-induced myocardial damage. This work proved that the down-regulation of GAS5 could reverse cardiomyocyte injury and apoptosis by targeting miR-138 to down-regulate CYP11B2.Conclusion: We confirmed for the first time that the down-regulation of GAS5 could reverse CYP11B2 via the miR-138 axis to reverse HG-induced cardiomyocyte injury. This research might provide a new direction for explaining the developmental mechanism of DCM and potential targets for the treatment of myocardial injury.     

Development of a microbioreactor for glycoconjugate synthesis

A microbioreactor immobilized with a synthase-type mutant enzyme, Endo-M-N175Q (glycosynthase) of endo-β-N-acetylglucosaminidase derived from Mucor hiemalis (Endo-M), was constructed and used for glycoconjugate synthesis. The transglycosylation was performed with a reaction mixture containing an oxazoline derivative of sialo complex-type glycoside (SG), which was prepared from a sialo complex-type glycopeptide SGP derived from hen egg yolk, as a glycosyl donor and N-Fmoc-N-acetylglucosaminyl-l-asparagine [Fmoc-Asn(GlcNAc)-OH] as an acceptor. The reaction mixture was injected into a glycosynthase microbioreactor at a constant flow rate. Highly efficient and nearly stoichiometric transglycosylation occurred in the microbioreactor, and the transglycosylation product was eluted from the other end of the reactor. The glycosynthase microbioreactor was stable and could be used repeatedly for a long time.     

A Conserved UDP-Glucose Dehydrogenase Encoded outside the hasABC Operon Contributes to Capsule Biogenesis in Group A Streptococcus

Group A Streptococcus (GAS) is a human-specific bacterial pathogen responsible for serious morbidity and mortality worldwide. The hyaluronic acid (HA) capsule of GAS is a major virulence factor, contributing to bloodstream survival through resistance to neutrophil and antimicrobial peptide killing and to in vivo pathogenicity. Capsule biosynthesis has been exclusively attributed to the ubiquitous hasABC hyaluronan synthase operon, which is highly conserved across GAS serotypes. Previous reports indicate that hasA, encoding hyaluronan synthase, and hasB, encoding UDP-glucose 6-dehydrogenase, are essential for capsule production in GAS. Here, we report that precise allelic exchange mutagenesis of hasB in GAS strain 5448, a representative of the globally disseminated M1T1 serotype, did not abolish HA capsule synthesis. In silico whole-genome screening identified a putative HasB paralog, designated HasB2, with 45% amino acid identity to HasB at a distant location in the GAS chromosome. In vitro enzymatic assays demonstrated that recombinant HasB2 is a functional UDP-glucose 6-dehydrogenase enzyme. Mutagenesis of hasB2 alone slightly decreased capsule abundance; however, a ΔhasB ΔhasB2 double mutant became completely acapsular. We conclude that HasB is not essential for M1T1 GAS capsule biogenesis due to the presence of a newly identified HasB paralog, HasB2, which most likely resulted from gene duplication. The identification of redundant UDP-glucose 6-dehydrogenases underscores the importance of HA capsule expression for M1T1 GAS pathogenicity and survival in the human host.     

NAD+-Glycohydrolase Promotes Intracellular Survival of Group A Streptococcus

A global increase in invasive infections due to group A Streptococcus (S. pyogenes or GAS) has been observed since the 1980s, associated with emergence of a clonal group of strains of the M1T1 serotype. Among other virulence attributes, the M1T1 clone secretes NAD⁺-glycohydrolase (NADase). When GAS binds to epithelial cells in vitro, NADase is translocated into the cytosol in a process mediated by streptolysin O (SLO), and expression of these two toxins is associated with enhanced GAS intracellular survival. Because SLO is required for NADase translocation, it has been difficult to distinguish pathogenic effects of NADase from those of SLO. To resolve the effects of the two proteins, we made use of anthrax toxin as an alternative means to deliver NADase to host cells, independently of SLO. We developed a novel method for purification of enzymatically active NADase fused to an amino-terminal fragment of anthrax toxin lethal factor (LFn-NADase) that exploits the avid, reversible binding of NADase to its endogenous inhibitor. LFn-NADase was translocated across a synthetic lipid bilayer in vitro in the presence of anthrax toxin protective antigen in a pH-dependent manner. Exposure of human oropharyngeal keratinocytes to LFn-NADase in the presence of protective antigen resulted in cytosolic delivery of NADase activity, inhibition of protein synthesis, and cell death, whereas a similar construct of an enzymatically inactive point mutant had no effect. Anthrax toxin-mediated delivery of NADase in an amount comparable to that observed during in vitro infection with live GAS rescued the defective intracellular survival of NADase-deficient GAS and increased the survival of SLO-deficient GAS. Confocal microscopy demonstrated that delivery of LFn-NADase prevented intracellular trafficking of NADase-deficient GAS to lysosomes. We conclude that NADase mediates cytotoxicity and promotes intracellular survival of GAS in host cells.     

Glycoconjugate vaccines against Salmonella enterica serovars and Shigella species: existing and emerging methods for their analysis

The global spread of enteric disease, the increasingly limited options for antimicrobial treatment and the need for effective eradication programs have resulted in an increased demand for glycoconjugate enteric vaccines, made with carbohydrate-based membrane components of the pathogen, and their precise characterisation. A set of physico-chemical and immunological tests are employed for complete vaccine characterisation and to ensure their consistency, potency, safety and stability, following the relevant World Health Organization and Pharmacopoeia guidelines. Variable requirements for analytical methods are linked to conjugate structure, carrier protein nature and size and O-acetyl content of polysaccharide. We investigated a key stability-indicating method which measures the percent free saccharide of Salmonella enterica subspecies enterica serovar Typhi capsular polysaccharide, by detergent precipitation, depolymerisation and HPAEC-PAD quantitation. Together with modern computational approaches, a more precise design of glycoconjugates is possible, allowing for improvements in solubility, structural conformation and stability, and immunogenicity of antigens, which may be applicable to a broad spectrum of vaccines. More validation experiments are required to establish the most effective and suitable methods for glycoconjugate analysis to bring uniformity to the existing protocols, although the need for product-specific approaches will apply, especially for the more complex vaccines. An overview of current and emerging analytical approaches for the characterisation of vaccines against Salmonella Typhi and Shigella species is described in this paper. This study should aid the development and licensing of new glycoconjugate vaccines aimed at the prevention of enteric diseases.     

Bivalency and epitope specificity of a high-affinity IgG3 monoclonal antibody to the Streptococcus group A carbohydrate antigen. Molecular modeling of a Fv fragment

The binding of Strep 9, a mouse monoclonal antibody (mAb) of the IgG3 subclass directed against the cell-wall polysaccharide of Group A Streptococcus (GAS), has been characterized. The intact antibody and proteolytic fragments of Strep 9 bind differently to GAS: the intact mAb and F(ab)2' have greater affinity for the carbohydrate epitope than the monomeric Fab or F(ab)'. A mode of binding in which Strep 9 binds bivalently to portions of the polysaccharide on adjacent chains on GAS is proposed. A competitive ELISA protocol using a panel of carbohydrate inhibitors shows that the branched trisaccharide, beta-D-GlcpNAc-(1-->3)-[alpha-L-Rhap-(1-->2)]-alpha-L-Rhap, and an extended surface are key components of the epitope recognized by Strep 9. Microcalorimetry measurements with the mAb and two synthetic haptens, a tetrasaccharide and a hexasaccharide, show enthalpy-entropy compensation as seen in other oligosaccharide-protein interactions. Molecular modeling of the antibody variable region by homology modeling techniques indicates a groove-shaped combining site that can readily accommodate extended surfaces. Visual docking of an oligosaccharide corresponding to the cell-wall polysaccharide into the site provides a putative model for the complex, in which a heptasaccharide unit occupies the site and the GlcpNAc residues of two adjacent branched trisaccharide units occupy binding pockets within the groove-shaped binding site.     

The macro nutrient removal efficiencies of a vertical flow constructed wetland fed with demineralized cheese whey powder solution

This study aims to remove the macro-sized nutrients that are present in the cheese whey powder solution through the use of constructed wetland systems. For this purpose, 70% and 40% demineralized solutions of cheese whey powder were used. For both concentrations, control reactors are run in parallel with Typha angustifolia planted reactors for the duration of a 92 day period. Zeolite and gravel were used as the filling material. The planted reactor, which was fed with the 70% solution, was named as Cheese Whey Powder Solution (CWPS) 1 and its unplanted control was named CWPS 2 while the reactor, which was fed with the 40% solution, was named as CWPS 3 and its unplanted control was named CWPS 4. The removal of COD, PO4-P and NH4-N were obtained as 37.47%, 45.62%, and 68.88% in CWPS 1; 24.89%, 35.74%, and 63.15% in CWPS 2; 51.15%, 54.96%, and 64.13% in CWPS 3; and 28.35%, 23.99%, and 65.92% in CWPS 4, respectively.     

A dual-adjuvanting strategy for peptide-based subunit vaccines against group A Streptococcus: Lipidation and polyelectrolyte complexes

In order to improve the immunogenicity of peptide-based vaccines against group A Streptococcus (GAS), lipid moieties (C16 lipoamino acid and cholic acid) were conjugated with peptide antigen (P25-J8) and further modified with α-poly(glutamic acid) (α-PGA). Thus, positively charged lipopeptide vaccine candidates LCP-1 (P25-K(J8)-SS-C16-C16) and LCP-2 (P25-K(J8)-SS-K(cholic acid)) were synthesized. Negatively charged LCP-3 (P25-K(PGA-J8)-SS-K(cholic acid)) was also produced by attaching α-PGA to the J8 N-terminus of LCP-2. Polyelectrolyte complex (PEC) nanoparticles were formulated with heparin and/or trimethyl chitosan (TMC) for delivery of the lipopeptide vaccine candidates. The ability of the antigen-loaded nanoparticles to induce humoral immune responses was examined in outbred female Swiss mice following intranasal immunization. The antibodies produced were opsonic against all clinical GAS isolates tested.     

On limited platinum chains. Synthesis and crystal structure of a green tetranuclear platinum compound,cis-diammine tetraplatinum squarate,Pt4(NH3)8(C404)4· 4H20. A structural analogue of platinum blues

From the reaction of the cis-dichlorodiammine hydrolysis product with squaric acid, green crystals of cis-diammine tetraplatinum squarate are obtained. A single crystal X-ray diffraction analysis shows that the compound consists of a tetranuclear chain, whose adjacent platinum atoms are bridged by squaric ligands.     

Strain Selection for Generation of O-Antigen-Based Glycoconjugate Vaccines against Invasive Nontyphoidal Salmonella Disease

Nontyphoidal Salmonellae, principally S. Typhimurium and S. Enteritidis, are a major cause of invasive bloodstream infections in sub-Saharan Africa with no vaccine currently available. Conjugation of lipopolysaccharide O-antigen to a carrier protein constitutes a promising vaccination strategy. Here we describe a rational process to select the most appropriate isolates of Salmonella as source of O-antigen for developing a bivalent glycoconjugate vaccine. We screened a library of 30 S. Typhimurium and 21 S. Enteritidis in order to identify the most suitable strains for large scale O-antigen production and generation of conjugate vaccines. Initial screening was based on growth characteristics, safety profile of the isolates, O-antigen production, and O-antigen characteristics in terms of molecular size, O-acetylation and glucosylation level and position, as determined by phenol sulfuric assay, NMR, HPLC-SEC and HPAEC-PAD. Three animal isolates for each serovar were identified and used to synthesize candidate glycoconjugate vaccines, using CRM₁₉₇ as carrier protein. The immunogenicity of these conjugates and the functional activity of the induced antibodies was investigated by ELISA, serum bactericidal assay and flow cytometry. S. Typhimurium O-antigen showed high structural diversity, including O-acetylation of rhamnose in a Malawian invasive strain generating a specific immunodominant epitope. S. Typhimurium conjugates provoked an anti-O-antigen response primarily against the O:5 determinant. O-antigen from S. Enteritidis was structurally more homogeneous than from S. Typhimurium, and no idiosyncratic antibody responses were detected for the S. Enteritidis conjugates. Of the three initially selected isolates, two S. Typhimurium (1418 and 2189) and two S. Enteritidis (502 and 618) strains generated glycoconjugates able to induce high specific antibody levels with high breadth of serovar-specific strain coverage, and were selected for use in vaccine production. The strain selection approach described is potentially applicable to the development of glycoconjugate vaccines against other bacterial pathogens.     

Convergent synthesis of higher-order oligosaccharides corresponding to the cell-wall polysaccharide of the beta-hemolytic Streptococci group A. A branched hexasaccharide hapten.

A convergent synthesis of a hexasaccharide corresponding to the cell-wall polysaccharide of the beta-hemolytic Streptococci Group A is described. The strategy relies on the preparation of a key branched trisaccharide unit alpha-L-Rhap-(1----2)-[beta-D-GlcpNAc-(1----3)]-alpha-L-Rhap which functions both as a glycosyl acceptor and donor. The hexasaccharide is obtained after only three glycosylation reactions. This fully functionalized unit can serve, in turn, as a glycosyl acceptor or donor for the synthesis of higher-order structures. Deprotection gives a hexasaccharide for use as a hapten in immunochemical studies. The characterization of all compounds by high resolution 1H- and 13C-n.m.r. spectroscopy is also described.     

Development of a new vaccine formulation that enhances the immunogenicity of tumor-associated antigen CaMBr1

Aberrant glycosylation is one of the most constant traits of malignant cells. The CaMBr1 hexasaccharide antigen, originally defined on the human breast carcinoma cell line MCF7, is expressed on some normal tissues but overexpressed in a high percentage of human breast, ovary, prostate and lung carcinomas. CaMBr1 overexpression is associated with poor prognosis. The epitope consists of the tetrasaccharide Fuc(α1-2)Gal(β1-3)GalNAc(β1-3)Galα-O-spacer, which has recently become available as a synthetic oligosaccharide. Here we report the CaMBr1 tetrasaccharide conjugation to two different carrier proteins (CRM197 and KLH) and the evaluation of conjugate immunogenicity in mice following their administration in various vaccine formulations with two adjuvants (MPL-SE and Detox-PC). Radioimmunoassay to determine the level and isotype of anti-tetrasaccharide antibodies in mouse sera, and cytofluorimetric analysis and ⁵¹Cr-release assay on human tumor cells, to evaluate specificity of binding and complement-dependent lysis respectively, identified CaMBr1-CRM197, in association with the MPL-SE adjuvant, as the best vaccine formulation. This combination induced (1) production of tetrasaccharide-specific antibodies, with negligible side-effects; (2) antibodies with complement-mediated cytotoxic activity on human CaMBr1-positive cells and (3) a high titer of IgG1 detected in sera obtained 3 months after the first injection, indicating that the anti-tetrasaccharide antibody response was mediated by T cell activation. The availability of CaMBr1-glycoconjugate in the minimal and functional antigenic structure and the identification of an efficacious vaccine formulation opens the way to exploring the activity of this glycoconjugate in a clinical setting. Received: 20 January 2000 / Accepted: 16 March 2000     

The tumor promoter, 12-O-tetradecanoylphorbol-13-acetate accelerates keratinocyte differentiation and stimulates growth of an unidentified cell type in cultured human epidermis

Studies were conducted to determine the effects of the mouse skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on cultured human epidermal cells for comparison with known effects on mouse keratinocytes. In contrast to its effect on mouse cells, TPA did not stimulate human epidermal cell DNA synthesis. TPA stimulated differentiation in human keratinocytes resulting in sloughing of many cells by the 3rd day after exposure. Quantitative assays revealed that 50% of the TPA-exposed population was composed of cornified cells as opposed to 8% in untreated controls. A morphologically distinct cell type (TT cell) emerged after TPA treatment which was triangular in shape, did not stratify, appeared to proliferate rapidly and at most TPA concentrations became the predominant cell type within 1–2 weeks. Cultures composed predominantly of TT cells formed few cornified envelopes, grew well in the absence of TPA and formed colonies at low cell input. In contrast to its effect on keratinocytes, TPA enhanced TT colony formation 3–4-fold and decreased the doubling time of TT cells. Studies were performed to determine the origin of TT cells. Immunofluorescent staining indicated that TT cells lacked the keratinocyte antigens keratin, pemphigus and pemphigoid. Tonofilaments and desmosomes were not seen by electron microscopy. The lack of both melanosomes and standard histochemical DOPA oxidase staining indicated that TT cells were probably not of melanocyte origin. Tests used to identify Langerhans cells were negative. Whereas TT cells, as well as dermal fibroblasts, yielded positive immunofluorescence with antibodies to vimentin, TT cells gave a weak histochemical leucine aminopeptidase reaction, while the reaction of fibroblasts exposed to TPA was strong. Treatment of human dermal fibroblasts with TPA did not yield TT cells. The endothelial cell antigen factor VIII-associated protein was absent by immunofluorescence. These results suggest that the primary effect of TPA on cultured human epidermis is to accelerate terminal differentiation in the keratinocyte population and to stimulate growth of an as yet unidentified cell type.