A generic protocol to immobilize lipopolysaccharides on microbeads for multiplex analysis.

Bead-based multiplex serodiagnostics enables simultaneous analysis of antibodies against several antigens. Binding of the antigens onto the surface of the bead, preserving the antigenicity of the antigen is a pivotal step to ensure high sensitivity and selectivity of the assay. Here, a generic method for immobilization of lipopolysaccharide (LPS) antigens from different Gram-negative bacteria to microbeads using non-covalent conjugation has been developed and tested. The method involves coupling of N,N-diethylethylenediamine (DEDA) and derivatives to microbeads. This enhances non-covalent interactions so that LPS is easily immobilized. LPS antigens from the Gram-negative bacteria Actinobacillus pleuropneumoniae (APP) and Salmonella enterica serogroup B (Sal. B) were immobilized on the DEDA-coupled microbeads. In parallel, the same LPS antigens were coupled to beads using two previously reported methods. The performance of microbeads coupled with antigen using the different methods was compared by measuring antibodies in positive and negative serum samples from pigs. DEDA-beads coupled with LPS detected pathogen specific serum antibodies with equal or higher sensitivity and specificity compared to the other coupling methods used in this study. Furthermore, derivatives of DEDA, where the tertiary amine was alkylated with a methyl (m-DEDA) and ethyl group (e-DEDA) to give a positively charged tetraalkylammonium group, were compared with DEDA for the binding of LPS antigens. Here, it was concluded that the DEDA-modified bead was most efficient in the binding of LPS antigens from two Actinobacillus pleuropneumoniae serovars and Salmonella enterica serogroup B.

Phosphocholine-Decorated PPI-Dendrimers Mimic Cell Membrane Phosphocholine Clusters and Tune the Innate Immune Activity of C-Reactive Protein.

C-reactive protein (CRP) is widely used as biomarkers of infection and inflammation. It has a well-described ability to bind phosphocholine (PC), as well as PC-clusters from compromised and inflamed cell membranes and tissues. The binding of PC-clusters to CRP is of interest as this binding determines subsequent innate immune activity. We investigated PC-decorated dendrimers as mimics for PC-clusters. Five generations of poly(propylene imine) (PPI) dendrimers were modified with PC surface groups via a three-step synthetic sequence obtaining the PC-decorated dendrimers in high purity. The dendrimers were analyzed by NMR and infrared spectroscopy as well as HPLC. We developed immunoassays to show that dendrimer-PC binding to CRP was Ca2+-dependent with an apparent overall Kd of 11.9 nM for first generation (G1) PPI-PC, while G2-PPI-PC and G3-PPI-PC had slightly higher affinities, and G4-PPI-PC and G5-PPI-PC had slightly lower affinities. For all PC-dendrimers, the affinity was orders of magnitude higher than the affinity of free phosphocholine (PC), indicating a PC-cluster effect. Next, we investigated the binding of CRP:PPI-PC complexes to complement component C1q. C1q binding to CRP was dependent on the generation of PPI-PC bound to CRP, with second and third generation PPI-PCs leading to the highest affinity. The dendrimer-based approach to PC-cluster mimics and the simple binding assays presented here hold promise as tools to screen PC-compounds for their abilities to tune the innate immune activity of CRP.

Antiparasitic activity of chicory (Cichorium intybus) and its natural bioactive compounds in livestock: a review.

Increasing drug resistance in gastrointestinal (GI) parasites of livestock and concerns about chemical residues in animal products and the environment are driving the development of alternative control strategies that are less reliant on the use of synthetic drugs. An increasingly investigated approach is the use of bioactive forages with antiparasitic properties as part of the animal's diet (nutraceuticals) or as potential sources of novel, natural parasiticides. Chicory (Cichorium intybus) is a multi-purpose crop and one of the most promising bioactive forages in temperate regions, and numerous in vivo trials have explored its potential against parasitic nematodes in livestock. However, it is unclear whether chicory can induce a direct and broad activity against various GI parasites in different livestock species, and the levels of chicory in the diet that are required to exert an efficient antiparasitic effect. Moreover, the mechanisms leading to the reported parasiticidal activity of chicory are still largely unknown, and its bioactive phytochemicals have only recently been investigated. In this review, we summarise the progress in the study of the antiparasitic activity of chicory and its natural bioactive compounds against GI parasites in livestock, through examination of the published literature. The available evidence indicates that feeding chicory can reduce faecal egg counts and/or worm burdens of abomasal nematodes, but not infections with intestinal worms, in ruminants. Highly chicory-rich diets (≥ 70% of chicory dry matter in the diet) may be necessary to directly affect abomasal parasitism. Chicory is known to synthesise several bioactive compounds with potential antiparasitic activity, but most research has been devoted to the role of sesquiterpene lactones (SL). Recent in vitro studies have confirmed direct and potent activity of SL-rich extracts from chicory against different GI helminths of livestock. Chicory SL have also been reported to exhibit antimalarial properties and its potential antiprotozoal activity in livestock remains to be evaluated. Furthermore, the detailed identification of the main antiparasitic metabolites of chicory and their pharmacokinetics need further confirmation. Research gaps and perspectives on the potential use of chicory as a nutraceutical forage and a source of bioactive compounds for parasite control in livestock are discussed.

Simultaneous detection of antibodies to five Actinobacillus pleuropneumoniae serovars using bead-based multiplex analysis.

We developed and made a preliminary validation of a bead-based multiplexed immunoassay for simultaneous detection of porcine serum antibodies to Actinobacillus pleuropneumoniae serovars 1, 2, 6, 7, and 12. Magnetic fluorescent beads were coupled with A. pleuropneumoniae antigens and tested with a panel of serum samples from experimentally infected pigs and with serum samples from uninfected and naturally infected pigs. The multiplex assay was compared to in-house ELISAs and complement fixation (CF) tests, which have been used for decades as tools for herd classification in the Danish Specific Pathogen Free system. Assay specificities and sensitivities as well as the corresponding cutoff values were determined using receiver operating characteristic (ROC) curve analysis, and the A. pleuropneumoniae multiplex assay showed good correlation with the in-house ELISAs and CF tests with areas under ROC curves ≥ 0.988. Benefits of multiplexed assays compared to ELISAs and CF tests include reduced serum sample volumes needed for analysis, less labor, and shorter assay time.

Anthelmintic activity of chicory (Cichorium intybus): in vitro effects on swine nematodes and relationship to sesquiterpene lactone composition.

Chicory is a perennial crop that has been investigated as a forage source for outdoor-reared ruminants and pigs, and has been reported to have anthelmintic properties. Here, we investigated in vitro anthelmintic effects of forage chicory-extracts against the highly prevalent swine parasites Ascaris suum and Oesophagostomum dentatum. Methanol extracts were prepared and purified from two different cultivars of chicory (Spadona and Puna II). Marked differences were observed between the anthelmintic activity of extracts from the two cultivars. Spadona extracts had potent activity against A. suum third (L3) and fourth (L4) - stage larvae, as well as O. dentatum L4 and adults, whereas Puna II extracts had less activity against A. suum and no activity towards O. dentatum L4. Transmission-electron microscopy of A. suum L4 exposed to Spadona extracts revealed only subtle changes, perhaps indicative of a specific anthelmintic effect rather than generalized toxicity. Ultra-high liquid chromatography-mass spectrometry analysis revealed that the purified extracts were rich in sesquiterpene lactones (SL), and that the SL profile differed significantly between cultivars. This is the first report of anthelmintic activity of forage chicory towards swine nematodes. Our results indicate a significant anthelmintic effect, which may possibly be related to SL composition.

Urea and thiourea modified polypropyleneimine dendrimers clear intracellular α-synuclein aggregates in a human cell line.

Synucleinopathies are neurodegenerative pathologies in which disease progression is closely correlated to brain accumulation of insoluble α-synuclein, a small protein abundantly expressed in neural tissue. Here, two types of modified polypropyleneimine (PPI) dendrimers having either urea or methylthiourea (MTU) surface functional groups were investigated in a cellular model of synucleinopathy. Dendrimers are synthetic macromolecules that may be produced in a range of well-defined molecular sizes. Using cellomics array scan high-content screening, we show that both types of dendrimers are able to significantly reduce intracellular levels of α-synuclein aggregates dependent on the concentration, the type and molecular size of the dendrimer with the bigger size MTU-dendrimers having the highest potency. The intracellular clearance of α-synuclein aggregates by dendrimers was achieved at noncytotoxic concentrations.

Sesquiterpene lactone containing extracts from two cultivars of forage chicory (Cichorium intybus) show distinctive chemical profiles and in vitro activity against Ostertagia ostertagi.

The study investigated direct anthelmintic effects of sesquiterpene lactones (SL)-containing extracts from forage chicory against free-living and parasitic stages of Ostertagia ostertagi. Freeze-dried leaves from chicory cultivars 'Spadona' and 'Puna II' were extracted using methanol/water. Total SL were further fractionated by solid-phase extraction and resulting extracts were characterised by high-performance liquid chromatography (HPLC). O. ostertagi eggs from faeces of mono-infected calves were hatched and L1 were used in a larval feeding inhibition assay (LFIA), while cultured L3 were used in a larval exsheathment inhibition assay (LEIA). Adult worms were immediately recovered after slaughter and used for motility inhibition assays (AMIA). Electron microscopy (EM) was performed on adult O. ostertagi exposed to 1000 µg extract mL(-1) of both chicory cultivars. In all assays, decreasing concentrations of SL-containing extracts in PBS (1% DMSO) were tested in replicates with 1% DMSO in PBS as negative controls. HPLC demonstrated similar concentrations of most SL in both extracts. However, Spadona-extract contained significantly higher concentrations of 11, 13-dihydro-8-deoxylactucin (P = 0.01), while Puna II-extract had increased levels of 11, 13-dihydrolactucin (P < 0.0001). In the LFIA, both extracts reduced larval feeding at increasing concentrations, but Spadona-extract showed higher potency confirmed by significantly lower EC50 (P < 0.0001). In the LEIA, neither of the two extracts interfered with the exsheathment of L3 (P > 0.05). In the AMIA, both SL-containing extracts induced a dose-dependent effect but Spadona-extract showed greater activity and exerted faster worm paralysis than Puna II-extract with significantly lower EC50 (P < 0.0001). No cuticular damage was observed by EM in worms exposed to any of the extracts. We have demonstrated that SL-containing extracts from forage chicory can inhibit feeding of free-living larvae and exert direct effects against parasitic stages of O. ostertagi. Our results may contribute to the identification of natural anti-parasitic compounds and to interpret the in vivo anthelmintic effects of forage chicory.

Color test for selective detection of secondary amines on resin and in solution.

Resins for solid-phase synthesis give orange to red-brown resin beads selectively when secondary amines are present on the resin when treated with a solution of acetaldehyde and an Fmoc-amino acid in NMP. The method shows good specificity and gives colorless beads when exposed to a variety of other functional groups. Furthermore, the acetaldehyde/Fmoc amino acid method can be used as a selective colorimetric test for secondary amines in solution.

Method to conjugate polysaccharide antigens to surfaces for the detection of antibodies.

A new generic method for the conjugation of lipopolysaccharide (LPS)-derived polysaccharide antigens from gram-negative bacteria has been developed using Salmonella as a model. After removal of lipid A from the LPS by mild acidolysis, the polysaccharide antigen was conjugated to polystyrene microbeads modified with N-alkyl hydroxylamine and N-alkyl-O-methyl hydroxylamine surface groups by incubation of antigen and beads for 16 h at 40 °C without the need for coupling agents. The efficiency of the new method was evaluated by flow cytometry in model samples and serum samples containing antibodies against Salmonella typhimurium and Salmonella dublin. The presented method was compared with a similar method for conjugation of Salmonella polysaccharide antigens to surfaces. Here, the new method showed higher antigen coupling efficiency by detecting low concentrations of antibodies. Furthermore, the polysaccharide-conjugated beads showed preserved bioactivity after 1 year of use.

Enhancement of muramyldipeptide (MDP) immunostimulatory activity by controlled multimerization on dendrimers.

Peptidoglycan is a widespread bacterial PAMP molecule and a powerful initiator of innate immune responses. It consists of repeating units of MDP, which as a monomer is only weakly immunostimulatory. Here, MDP-coupled dendrimers were prepared and investigated for stimulation of pig blood mononuclear cells. Compared to monomeric MDP, MDP-dendrimers induced a markedly enhanced production of IL-12 p40, IL-1ß and IL-6 and completely down-regulated surface expression of B7 and MHC class II. These results suggest a possible novel strategy based on controlled multimerization of minimal PAMP motifs on dendrimers for preparing molecularly defined immunostimulators with predictable bioactivities.

Dendrimers for vaccine and immunostimulatory uses. A review.

Dendrimers are well-defined (monodisperse) synthetic globular polymers with a range of interesting chemical and biological properties. Chemical properties include the presence of multiple accessible surface functional groups that can be used for coupling biologically relevant molecules and methods that allow for precise heterofunctionalization of surface groups. Biologically, dendrimers are highly biocompatible and have predictable biodistribution and cell membrane interacting characteristics determined by their size and surface charge. Dendrimers have optimal characteristics to fill the need for efficient immunostimulating compounds (adjuvants) that can increase the efficiency of vaccines, as dendrimers can provide molecularly defined multivalent scaffolds to produce highly defined conjugates with small molecule immunostimulators and/or antigens. The review gives an overview on the use of dendrimers as molecularly defined carriers/presenters of small antigens, including constructs that have built-in immunostimulatory (adjuvant) properties, and as stand-alone adjuvants that can be mixed with antigens to provide efficient vaccine formulations. These approaches allow the preparation of molecularly defined vaccines with highly predictable and specific properties and enable knowledge-based vaccine design substituting the traditional empirically based approaches for vaccine development and production.

A robust quantitative solid phase immunoassay for the acute phase protein C-reactive protein (CRP) based on cytidine 5'-diphosphocholine coupled dendrimers.

C-reactive protein (CRP) is an important acute phase protein, being used as a sensitive indicator of inflammation and infection and is also associated with the risk of cardiovascular problems. The present paper describes a robust and sensitive ELISA for CRP, based on the affinity of CRP for phosphocholine. In this design synthetic globular polymers (dendrimers) are used as scaffolds for the multivalent display of phosphocholine molecules. CRP present in a sample binds to the phosphocholine moiety presented at high density in the coating layer and is detectable by specific antibodies. The ELISA was applied to determination of pig and human CRP using commercially available antibodies against human CRP. The assay was shown to be more sensitive than previously published immunoassays employing albumin-coupled cytidine diphosphocholine. The coating was stable for at least 30 days at room temperature and the assay showed high intra- and interassay reproducibility. Results were compared with an immunoturbidimetric method and with a commercial ELISA kit and there was very good agreement with the immunoturbidimetric method, however not with the commercial assay, probably due to a calibration discrepancy. The assay is applicable to other species by providing an adequate detection antibody having the desired species specificity.

THAL, a sterically unhindered linker for the solid-phase synthesis of acid-sensitive protected peptide acids.

The 5-(4-hydroxyphenyl)-3,4-ethylenedioxythienyl alcohol (THAL, Thiophene Acid Labile) is described as a new linker for the solid-phase synthesis of peptide carboxylic acids. It is based on the electron-rich 3,4-ethylenedioxythenyl (EDOTn) moiety and allows the obtention of free and tert-butyl-protected peptides by cleavage with 90% and 0.5% TFA, respectively. This very high acid lability makes it useful for the synthesis of sensitive peptides. Free and tert-butyl-protected Leu-enkephalins have been synthesized as models to demonstrate the utility of the linker.

Dendrimers destabilize proteins in a generation-dependent manner involving electrostatic interactions.

Dendrimers are well-defined chemical polymers with a characteristic branching pattern that gives rise to attractive features such as antibacterial and antitumor activities as well as drug delivery properties. In addition, dendrimers can solubilize prion protein aggregates at very low concentrations, but their mode of action is unclear. We show that poly(propylene imine) dendrimers based on di-aminobutane (DAB) and modified with guanidinium surface groups reduce insulin thermostability and solubility considerably at microgram per microliter concentrations, while urea-modified groups have hardly any effect. Destabilization is markedly generation-dependent and is most pronounced for generation 3, which is also the most efficient at precipitating insulin. This suggests that proteins can interact with both dendrimer surface and interior. The pH-dependence reveals that interactions are mainly mediated by electrostatics, confirmed by studies on four other proteins. Ability to precipitate and destabilize are positively correlated, in contrast to conventional small-molecule denaturants and stabilizers, indicating that surface immobilization of denaturing groups profoundly affects its interactions with proteins.

Guanidino- and urea-modified dendrimers as potent solubilizers of misfolded prion protein aggregates under non-cytotoxic conditions. dependence on dendrimer generation and surface charge.

Amino-terminated dendrimers are well-defined synthetic hyperbranched polymers and have previously been shown to destabilize aggregates of the misfolded, pathogenic, and partially protease-resistant form of the prion protein (PrPSc), transforming it into a partially dissociated, protease-sensitive form with strongly reduced infectivity. The mechanism behind this is not known, but a low pH, creating multiple positively charged primary amines on the dendrimer surface, increases the efficiency of the reaction. In the present study, surface amines of the dendrimers were modified to yield either guanidino surface groups (being positively charged at neutral pH) or urea groups (uncharged). The ability of several generations of modified dendrimers and unmodified amino-terminated dendrimers to deplete PrPSc from persistently PrPSc-infected cells in culture (SMB cells) was studied. It was found that destabilization correlated with both the generation number of the dendrimer, with higher generations being more efficient, and the charge density of the surface groups. Urea-decorated dendrimers having an uncharged surface were less efficient than positively charged unmodified- (amino) and guanidino-modified dendrimers. The most efficient dendrimers (generation 4 (G4) and G5-unmodified and guanidino dendrimers) cleared PrPSc completely by incubation for 4 days at less than 50 nM. In contrast to both unmodified and guanidine-modified dendrimers, the uncharged urea dendrimers showed much lower cytotoxicity toward noninfected SMB cells. Therapeutic uses of modified dendrimers are indicated by the low concentrations of dendrimers needed.

Dendrimer effects on peptide and protein fibrillation.

Dendrimers are synthetic, symmetrically branched polymers that can be manufactured to a high degree of definition and therefore present themselves as monodisperse entities. Flexible and globular in shape and compartementalized into a partly inaccessible interior and a highly exposed surface, they offer numerous possibilities for interactions with and responses to biological macromolecules and biostructures including cell membranes and proteins. By way of their multiple functional surface groups, they allow the design of surfaces carrying a multitude of biological motifs and/or charges giving rise to quite significant biological and physico-chemical effects. Here we describe the surprising ability of dendrimers to interact with and perturb polypeptide conformations, particularly efficiently towards amyloid structures; that is, the structures of highly insoluble polypeptide aggregates involved in a range of serious and irreversibly progressive pathological conditions (protein-misfolding diseases). Interesting as this may be, the interaction of dendrimers with such generic peptidic aggregates also offers a new perspective on the molecular mechanisms governing assembly and disassembly of amyloid structures and thereby on determinants of protein and peptide folding. Despite the potent disaggregative nature of various dendrimers, they have variable effects on the stability of different proteins, suggesting that they do not act as generic denaturants, but rather exert their effects via specific interactions with individual parts of each protein.

Carbocations in action. Design, synthesis, and evaluation of a highly acid-sensitive naphthalene-based backbone amide linker for solid-phase synthesis.

The design, synthesis, and properties of an extremely acid-labile backbone amide linker based on a regiospecifically substituted tetraalkoxy naphthaldehyde core are presented. This handle enables cleavage of peptide backbone amides (secondary amides) off a solid support using as little as 0.5% TFA in CH2Cl2. This proceeds without cleavage of tert-butyl ethers and tert-butyl esters. The design is based on a DFT study that predicted the most stabile alkoxy-substituted methyl naphthyl carbocation. [structure: see text]

Thiophene backbone amide linkers, a new class of easily prepared and highly acid-labile linkers for solid-phase synthesis.

Solid-phase synthesis is of tremendous importance for small-molecule and biopolymer synthesis. Linkers (handles) that release amide-containing products after completion of solid-phase synthesis are widely used. Here we present a new class of highly acid-labile backbone amide linkers (BAL handles) based on 3,4-ethylenedioxythiophene (EDOT), which we have termed T-BAL. These thiophene linkers are synthesized in three convenient steps from commercially available EDOT. In the linker design, the spacer was introduced to the EDOT core either via a carbon-carbon bond or via a thioether linkage. Introduction of the spacer via a C-C bond was performed by a chemoselective Negishi coupling without transient protection of the aldehyde group to provide the T-BAL1 handle. Introduction via a thioether linkage was performed by a facile nucleophilic aromatic substitution between the brominated EDOT aldehyde and unprotected mercapto acids to provide T-BAL2 and T-BAL3 handles. The minimal use of protecting groups gave the corresponding linker molecules in few synthetic steps and in good yields. After anchoring of the linker to a polymeric support, introduction of the first amino acid was achieved by reductive amination, giving a secondary amine. A following acylation of the secondary amine with a symmetrical amino acid anhydride resulted in a backbone amide linkage between the handle and the growing substrate (e.g., peptide chain). After solid-phase synthesis, the substrates could be released from the resin by either low acid conditions using 1% TFA in CH2Cl2 or high acid conditions such as 50% TFA in CH2Cl2. Peptide thioesters could be released from the T-BAL1 handle under very mild conditions using aqueous acetic acid. Tert-butyl based protecting groups, tert-butyl esters, tert-butyl ethers, and Boc groups, as well as dimethyl acetals were relatively stable to these mild conditions for release of the peptides.

Dendrimer based anti-infective and anti-inflammatory drugs.

Alternatives to traditional antibiotics and to antiviral and anti-inflammatory drugs are much in need and the molecular design and development of anti-infective compounds constitute a pivotal area in modern medicinal research. Dendrimers are a relatively new class of structurally well-defined, i.e. monodisperse, synthetic polymers with hyperbranched structures which enable a given molecular motif to be presented in a highly multivalent fashion. Several types of dendrimers with various structural elements and molecular dimensions are commercially available at an affordable price. The surface of dendrimers can be modified relatively easily and, depending on the surface motif, the pharmacological properties of the dendrimer such as cytotoxicity, bacteriocidal and virucidal effect, biodistribution and biopermeability may be modulated to fit a specific medicinal purpose. Dendrimers are thus highly suitable tools in drug discovery and they allow the synthesis of molecules with high and specific binding affinities to a wide variety of receptors, viruses and bacteria. Hence the use of dendrimers for the development of antiviral or antibacterial drugs, destroying the infective agent or disrupting multivalent binding interactions between the infective agent and cells of the host organism has become a highly active research field. The wide range of applications reported for the use of dendrimers as anti-infective and anti-inflammatory drugs in the patent literature demonstrates the general applicability of these molecules as drug candidates. The present review will briefly treat the intrinsic properties of dendrimers in biological systems, as well as general concerns regarding the treatment of infective diseases. The use of dendrimers as anti-infective and anti-inflammatory drugs will be based on a thorough review of the recent patent literature.